Differentiation is accompanied by a progressive loss in transcriptional memory.

BACKGROUND: Cell differentiation requires the integration of two opposite processes, a stabilizing cellular memory, especially at the transcriptional scale, and a burst of gene expression variability which follows the differentiation induction. Therefore, the actual capacity of a cell to undergo phenotypic change during a differentiation process relies upon a modification in this balance which favors change-inducing gene expression variability. However, there are no experimental data providing insight on how fast the transcriptomes of identical cells would diverge on the scale of the very first two cell divisions during the differentiation process. RESULTS: In order to quantitatively address this question, we developed different experimental methods to recover the transcriptomes of related cells, after one and two divisions, while preserving the information about their lineage at the scale of a single cell division. We analyzed the transcriptomes of related cells from two differentiation biological systems (human CD34+ cells and T2EC chicken primary erythrocytic progenitors) using two different single-cell transcriptomics technologies (scRT-qPCR and scRNA-seq). CONCLUSIONS: We identified that the gene transcription profiles of differentiating sister cells are more similar to each other than to those of non-related cells of the same type, sharing the same environment and undergoing similar biological processes. More importantly, we observed greater discrepancies between differentiating sister cells than between self-renewing sister cells. Furthermore, a progressive increase in this divergence from first generation to second generation was observed when comparing differentiating cousin cells to self renewing cousin cells. Our results are in favor of a gradual erasure of transcriptional memory during the differentiation process.

scAN1.0: A reproducible and standardized pipeline for processing 10X single cell RNAseq data.

Single cell transcriptomics has recently seen a surge in popularity, leading to the need for data analysis pipelines that are reproducible, modular, and interoperable across different systems and institutions.To meet this demand, we introduce scAN1.0, a processing pipeline for analyzing 10X single cell RNA sequencing data. scAN1.0 is built using the Nextflow DSL2 and can be run on most computational systems. The modular design of Nextflow pipelines enables easy integration and evaluation of different blocks for specific analysis steps.We demonstrate the usefulness of scAN1.0 by showing its ability to examine the impact of the mapping step during the analysis of two datasets: (i) a 10X scRNAseq of a human pituitary gonadotroph tumor dataset and (ii) a murine 10X scRNAseq acquired on CD8 T cells during an immune response.

Dimeric G-quadruplex motifs-induced NFRs determine strong replication origins in vertebrates.

Replication of vertebrate genomes is tightly regulated to ensure accurate duplication, but our understanding of the interplay between genetic and epigenetic factors in this regulation remains incomplete. Here, we investigated the involvement of three elements enriched at gene promoters and replication origins: guanine-rich motifs potentially forming G-quadruplexes (pG4s), nucleosome-free regions (NFRs), and the histone variant H2A.Z, in the firing of origins of replication in vertebrates. We show that two pG4s on the same DNA strand (dimeric pG4s) are sufficient to induce the assembly of an efficient minimal replication origin without inducing transcription in avian DT40 cells. Dimeric pG4s in replication origins are associated with formation of an NFR next to precisely-positioned nucleosomes enriched in H2A.Z on this minimal origin and genome-wide. Thus, our data suggest that dimeric pG4s are important for the organization and duplication of vertebrate genomes. It supports the hypothesis that a nucleosome close to an NFR is a shared signal for the formation of replication origins in eukaryotes.

LC-MS/MS Bioanalysis of Radioligand Therapeutic Drug Candidate for Preclinical Toxicokinetic Assessment.

Radioligand therapy (RLT) has gained significant momentum in recent years in the diagnosis, treatment, and monitoring of cancers. In preclinical development, the safety profile of RLT drug candidate(s) is investigated at relatively low dose levels using the cold (non-radioactive, e.g., 175Lu) ligand as a surrogate of the hot (radioactive, e.g., 177Lu) one in the "ligand-linker-chelator" complex. The formulation of the test article used in preclinical safety studies contains a mixture of free ligand (i.e., ligand-linker-chelator without metal) and cold ligand (i.e., ligand-linker-chelator with non-radioactive metal) in a similar molar ratio as seen under the manufacturing conditions for the RLT drug for clinical use, where only a fraction of free ligand molecules chelate the radioactive metal to form a hot ligand. In this very first report of LC-MS/MS bioanalysis of RLT molecules in support of a regulated preclinical safety assessment study, a highly selective and sensitive LC-MS/MS bioanalytical method was developed for the simultaneous determination of free ligand (NVS001) and cold ligand (175Lu-NVS001) in rat and dog plasma. Several unexpected technical challenges in relation to LC-MS/MS of RLT molecules were successfully addressed. The challenges include poor assay sensitivity of the free ligand NVS001, formation of the free ligand (NVS001) with endogenous metal (e.g., potassium), Ga loss from the Ga-chelated internal standard during sample extraction and analysis, "instability" of the analytes at low concentrations, and inconsistent IS response in the extracted plasma samples. The methods were validated according to the current regulatory requirements in a dynamic range of 0.5-250 ng/mL for both the free and cold ligands using a 25 µL sample volume. The validated method was successfully implemented in sample analysis in support of regulated safety studies, with very good results from incurred sample reanalysis. The current LC-MS/MS workflow can be expanded to quantitative analysis of other RLTs in support of preclinical RLT drug development.

Non-regulated LC-MS/MS bioanalysis in support of early drug development: a Novartis perspective.

Scientifically qualified LC-MS/MS methods are essential for the determination of small molecule drug candidates and/or their metabolite(s) in support of various non-regulated safety assessment and in vivo absorption, distribution, metabolism and excretion studies in preclinical development. This article outlines an effective method development workflow to fit for this purpose. The workflow features a 'universal' protein precipitation solvent for efficient sample extraction, a mobile phase additive for managing chromatographic resolution and addressing carryover and an internal standard cocktail to select the best analogue internal standard to track the analyte of interest in LC-MS/MS. In addition, good practices are recommended to prevent bioanalytical pitfalls due to instability, non-specific binding and dosing vehicle-induced matrix effect. Proper handling of non-liquid matrix is also discussed.

Toxicokinetics in preclinical drug development of small-molecule new chemical entities.

Toxicokinetics (TK) is an integral part of nonclinical (preclinical) safety assessment of small-molecule new chemical entities in drug development. It is employed to describe the systemic exposure of a drug candidate and/or its important metabolite(s) achieved in study animals and elucidate the relationship (proportional, over-proportional, or under-proportional) between systemic exposure and dose administered and the associated differences/similarities between male and female animals along with the possible accumulation/induction. TK data and the derived parameters are employed to propose safe starting doses for clinical use of the new drug candidate through proper extrapolation of findings in study animals to humans. This review has attempted to highlight the health authority expectations on TK assessment in supporting preclinical safety profiling of new chemical entities. A robust TK assessment requires good understanding of absorption, distribution, metabolism, and elimination processes of drug candidate, adequate TK sampling (e.g., controls where relevant), implementation of fit-for-purpose bioanalytical methods (validated or scientifically qualified) along with necessary measures to prevent mis-dosing or ex vivo contamination, and establishment of stability of the drug candidate and/or its metabolite(s) in the intended species matrix to ensure the reliability of bioanalytical and TK data. The latter provides a vital link between animal experiments and human safety.

Evidence for close molecular proximity between reverting and undifferentiated cells.

BACKGROUND: According to Waddington's epigenetic landscape concept, the differentiation process can be illustrated by a cell akin to a ball rolling down from the top of a hill (proliferation state) and crossing furrows before stopping in basins or "attractor states" to reach its stable differentiated state. However, it is now clear that some committed cells can retain a certain degree of plasticity and reacquire phenotypical characteristics of a more pluripotent cell state. In line with this dynamic model, we have previously shown that differentiating cells (chicken erythrocytic progenitors (T2EC)) retain for 24 h the ability to self-renew when transferred back in self-renewal conditions. Despite those intriguing and promising results, the underlying molecular state of those "reverting" cells remains unexplored. The aim of the present study was therefore to molecularly characterize the T2EC reversion process by combining advanced statistical tools to make the most of single-cell transcriptomic data. For this purpose, T2EC, initially maintained in a self-renewal medium (0H), were induced to differentiate for 24H (24H differentiating cells); then, a part of these cells was transferred back to the self-renewal medium (48H reverting cells) and the other part was maintained in the differentiation medium for another 24H (48H differentiating cells). For each time point, cell transcriptomes were generated using scRT-qPCR and scRNAseq. RESULTS: Our results showed a strong overlap between 0H and 48H reverting cells when applying dimensional reduction. Moreover, the statistical comparison of cell distributions and differential expression analysis indicated no significant differences between these two cell groups. Interestingly, gene pattern distributions highlighted that, while 48H reverting cells have gene expression pattern more similar to 0H cells, they are not completely identical, which suggest that for some genes a longer delay may be required for the cells to fully recover. Finally, sparse PLS (sparse partial least square) analysis showed that only the expression of 3 genes discriminates 48H reverting and 0H cells. CONCLUSIONS: Altogether, we show that reverting cells return to an earlier molecular state almost identical to undifferentiated cells and demonstrate a previously undocumented physiological and molecular plasticity during the differentiation process, which most likely results from the dynamic behavior of the underlying molecular network.

Sensitive LC-MS/MS quantification of unconjugated maytansinoid DM4 and its metabolite S-methyl-DM4 in human plasma.

Aim: To report the development and validation of an LC-MS/MS method for the simultaneous determination of unconjugated payload DM4 and its metabolite S-methyl-DM4 in human plasma. Methodology: A workflow of protein precipitation followed by reduction and solid phase extraction was employed to remove antibody-maytansinoid conjugates from plasma matrix, release DM4 from endogenous conjugates, and generate a clean sample extract for analysis, respectively. Sodium adduct species of both analytes were selected for multiple reaction monitoring to meet the assay sensitivity requirement in liquid chromatography with tandem mass spectrometry. Conclusion: The method was fully validated for a dynamic range of 0.100-50.0 ng/ml for both analytes along with desired stability and acceptable incurred sample reanalysis.

A derived ZW chromosome system in Amborella trichopoda, representing the sister lineage to all other extant flowering plants.

The genetic basis and evolution of sex determination in dioecious plants is emerging as an active area of research with exciting advances in genome sequencing and analysis technologies. As the sole species within the sister lineage to all other extant flowering plants, Amborella trichopoda is an important model for understanding the evolution and development of flowers. Plants typically produce only male or female flowers, but sex determination mechanisms are unknown for the species. Sequence data derived from plants of natural origin and an F1 mapping population were used to identify sex-linked genes and the nonrecombining region. Amborella trichopoda has a ZW sex determination system. Analysis of genes in a 4 Mb nonrecombining sex-determination region reveals recent divergence of Z and W gametologs, and few Z- and W-specific genes. The sex chromosomes of A. trichopoda evolved less than 16.5 Myr ago, long after the divergence of the extant angiosperms.

Divergent clonal differentiation trajectories establish CD8+ memory T cell heterogeneity during acute viral infections in humans.

The CD8+ T cell response to an antigen is composed of many T cell clones with unique T cell receptors, together forming a heterogeneous repertoire of effector and memory cells. How individual T cell clones contribute to this heterogeneity throughout immune responses remains largely unknown. In this study, we longitudinally track human CD8+ T cell clones expanding in response to yellow fever virus (YFV) vaccination at the single-cell level. We observed a drop in clonal diversity in blood from the acute to memory phase, suggesting that clonal selection shapes the circulating memory repertoire. Clones in the memory phase display biased differentiation trajectories along a gradient from stem cell to terminally differentiated effector memory fates. In secondary responses, YFV- and influenza-specific CD8+ T cell clones are poised to recapitulate skewed differentiation trajectories. Collectively, we show that the sum of distinct clonal phenotypes results in the multifaceted human T cell response to acute viral infections.

Detecting sex-linked genes using genotyped individuals sampled in natural populations.

We propose a method, SDpop, able to infer sex-linkage caused by recombination suppression typical of sex chromosomes. The method is based on the modeling of the allele and genotype frequencies of individuals of known sex in natural populations. It is implemented in a hierarchical probabilistic framework, accounting for different sources of error. It allows statistical testing for the presence or absence of sex chromosomes, and detection of sex-linked genes based on the posterior probabilities in the model. Furthermore, for gametologous sequences, the haplotype and level of nucleotide polymorphism of each copy can be inferred, as well as the divergence between them. We test the method using simulated data, as well as data from both a relatively recent and an old sex chromosome system (the plant Silene latifolia and humans) and show that, for most cases, robust predictions are obtained with 5 to 10 individuals per sex.

Clustering of strong replicators associated with active promoters is sufficient to establish an early-replicating domain.

Vertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into A/B compartments. Until now, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late-replicating region for earlier replication. The two origins overlap with a constitutive or a silent tissue-specific promoter. When inserted side-by-side, these modules advance replication timing over a 250 kb region through the cooperation with one endogenous origin located 30 kb away. Moreover, when inserted at two chromosomal sites separated by 30 kb, these two modules come into close physical proximity and form an early-replicating domain establishing more contacts with active A compartments. The synergy depends on the presence of the active promoter/origin. Our results show that clustering of strong origins located at active promoters can establish early-replicating domains.

The wild grape genome sequence provides insights into the transition from dioecy to hermaphroditism during grape domestication.

BACKGROUND: A key step in domestication of the grapevine was the transition from separate sexes (dioecy) in wild Vitis vinifera ssp. sylvestris (V. sylvestris) to hermaphroditism in cultivated Vitis vinifera ssp. sativa (V. vinifera). It is known that V. sylvestris has an XY system and V. vinifera a modified Y haplotype (Yh) and that the sex locus is small, but it has not previously been precisely characterized. RESULTS: We generate a high-quality de novo reference genome for V. sylvestris, onto which we map whole-genome re-sequencing data of a cross to locate the sex locus. Assembly of the full X, Y, and Yh haplotypes of V. sylvestris and V. vinifera sex locus and examining their gene content and expression profiles during flower development in wild and cultivated accessions show that truncation and deletion of tapetum and pollen development genes on the X haplotype likely causes male sterility, while the upregulation of a Y allele of a cytokinin regulator (APRT3) may cause female sterility. The downregulation of this cytokinin regulator in the Yh haplotype may be sufficient to trigger reversal to hermaphroditism. Molecular dating of X and Y haplotypes is consistent with the sex locus being as old as the Vitis genus, but the mechanism by which recombination was suppressed remains undetermined. CONCLUSIONS: We describe the genomic and evolutionary characterization of the sex locus of cultivated and wild grapevine, providing a coherent model of sex determination in the latter and for transition from dioecy to hermaphroditism during domestication.

Development and validation of an LC-MS/MS method for the quantitative analysis of the adenosine A2a receptor antagonist NIR178 and its monohydroxy metabolite in human plasma: Application to clinical pharmacokinetics.

We report a selective LC-MS/MS method for the simultaneous quantitative determinations of the adenosine A2a receptor antagonist NIR178 (NIR178) and its major metabolite NJI765 in human plasma. Sample preparation steps involved protein precipitation, sample evaporation and reconstitution using a plasma sample volume of 0.1 ml plasma. Separation was achieved in 10 min on an Acquity UPLC BEH C18 1.7 µm, 2.1 × 50 mm column heated at 60°C with a gradient elution at 0.6 ml/min mobile phase made of water and acetonitrile both acidified with 0.1% formic acid. The detection was performed in positive ion mode and quantification based on multiple reaction monitoring. The linear response range was 1.00-1,000 ng/ml using a 1/x2 weighting factor. The intra- and inter-day accuracies (bias %) and intra- and inter-day precisions (CV, %) obtained for NIR178 and NJI765 were within the acceptance criteria. The normalized NIR178 and NJI765 matrix factor calculated from six lots of normal, lipemic and hemolyzed plasmas ranged from 0.97 to 1.05. The normalized recoveries of both NIR178 and NJI765 compared with their internal standards were consistent and reproducible with a CV ≤8.0. This method was successfully applied to support pharmacokinetic studies in adult patients with cancer.

Evaluation, identification and impact assessment of abnormal internal standard response variability in regulated LC-MS bioanalysis.

Internal standard (IS) plays an important role in LC-MS bioanalysis by compensating for the variability of the analyte of interest in bioanalytical workflow. Due to the complexity of biological sample compositions and bioanalytical processes, a certain level of IS response variability across a run or a study is anticipated. However, an extensive variability may raise doubts to the accuracy of the measured results and also suggest nonoptimal analytical method. In this current paper, recent publications and guidelines regarding IS response in LC-MS bioanalysis were thoroughly reviewed with focus on the evaluation, identification and impact assessment of 'abnormal' IS response variability. A systematic decision tree was proposed to facilitate investigation into abnormal IS response variability after each run.

Evolution of replication origins in vertebrate genomes: rapid turnover despite selective constraints.

The replication program of vertebrate genomes is driven by the chromosomal distribution and timing of activation of tens of thousands of replication origins. Genome-wide studies have shown the association of origins with promoters and CpG islands, and their enrichment in G-quadruplex motifs (G4). However, the genetic determinants driving their activity remain poorly understood. To gain insight on the constraints operating on origins, we conducted the first evolutionary comparison of origins across vertebrates. We generated a genome-wide map of chicken origins (the first of a bird genome), and performed a comparison with human and mouse maps. The analysis of intra-species polymorphism revealed a strong depletion of genetic diversity at the core of replication initiation loci. This depletion is not linked to the presence of G4 motifs, promoters or CpG islands. In contrast, we show that origins experienced a rapid turnover during vertebrate evolution, since pairwise comparisons of origin maps revealed that <24% of them are conserved among vertebrates. This study unravels the existence of a novel determinant of origins, the precise functional role of which remains to be determined. Despite the importance of replication initiation for the fitness of organisms, the distribution of origins along vertebrate chromosomes is highly flexible.

Probabilistic count matrix factorization for single cell expression data analysis.

MOTIVATION: The development of high-throughput single-cell sequencing technologies now allows the investigation of the population diversity of cellular transcriptomes. The expression dynamics (gene-to-gene variability) can be quantified more accurately, thanks to the measurement of lowly expressed genes. In addition, the cell-to-cell variability is high, with a low proportion of cells expressing the same genes at the same time/level. Those emerging patterns appear to be very challenging from the statistical point of view, especially to represent a summarized view of single-cell expression data. Principal component analysis (PCA) is a most powerful tool for high dimensional data representation, by searching for latent directions catching the most variability in the data. Unfortunately, classical PCA is based on Euclidean distance and projections that poorly work in presence of over-dispersed count data with dropout events like single-cell expression data. RESULTS: We propose a probabilistic Count Matrix Factorization (pCMF) approach for single-cell expression data analysis that relies on a sparse Gamma-Poisson factor model. This hierarchical model is inferred using a variational EM algorithm. It is able to jointly build a low dimensional representation of cells and genes. We show how this probabilistic framework induces a geometry that is suitable for single-cell data visualization, and produces a compression of the data that is very powerful for clustering purposes. Our method is competed against other standard representation methods like t-SNE, and we illustrate its performance for the representation of single-cell expression data. AVAILABILITY AND IMPLEMENTATION: Our work is implemented in the pCMF R-package (https://github.com/gdurif/pCMF). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genomic imprinting mediates dosage compensation in a young plant XY system.

Sex chromosomes have repeatedly evolved from a pair of autosomes. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced expression and eventual loss of Y genes1. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals2-4. However, the early steps in the evolution of dosage compensation remain unknown, and dosage compensation is poorly understood in plants5. Here, we describe a dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males, but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno6 for the evolution of X inactivation in mammals.