Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis.

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9-13. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA12, and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.

Performance Optimization for a Class of Petri Nets.

Petri nets (PNs) are widely used to model flexible manufacturing systems (FMSs). This paper deals with the performance optimization of FMSs modeled by Petri nets that aim to maximize the system's performance under a given budget by optimizing both quantities and types of resources, such as sensors and devices. Such an optimization problem is challenging since it is nonlinear; hence, a globally optimal solution is hard to achieve. Here, we developed a genetic algorithm combined with mixed-integer linear programming (MILP) to solve the problem. In this approach, a set of candidate resource allocation strategies, i.e., the choices of the number of resources, are first generated by using MILP. Then, the choices of the type and the cycle time of the resources are evaluated by MILP; the promising ones are used to spawn the next generation of candidate strategies. The effectiveness and efficiency of the developed methodology are illustrated by simulation studies.

Event-Triggered Bounded Consensus Tracking for Second-Order Nonlinear Multi-Agent Systems with Uncertainties.

This paper is concerned with event-triggered bounded consensus tracking for a class of second-order nonlinear multi-agent systems with uncertainties (MASs). Remarkably, the considered MASs allow multiple uncertainties, including unknown control coefficients, parameterized unknown nonlinearities, uncertain external disturbances, and the leader's control input being unknown. In this context, a new estimate-based adaptive control protocol with a triggering mechanism is proposed. We rule out Zeno behavior by testifying that the lower bound on the interval between two consecutive events is positive. It is shown that under the designed protocol, all signals caused by the closed-loop systems are bounded globally uniformly and tracking errors ultimately converge to a bounded set. The effectiveness of the devised control protocol is demonstrated through a simulation example.

Fixed-Time Leader-Following Consensus Tracking Control for Nonliear Multi-Agent Systems under Jointly Connected Graph.

This paper researches the fixed-time leader-following consensus problem for nonlinear multi-agent systems (MASs) affected by unknown disturbances under the jointly connected graph. In order to achieve control goal, this paper designs a fixed-time consensus protocol, which can offset the unknown disturbances and the nonlinear item under the jointly connected graph, simultaneously. In this paper, the states of multiple followers can converge to the state of the leader within a fixed time regardless of the initial conditions rather than just converging to a small neighborhood near the leader state. Finally, a simulation example is given to illustrate the theoretical result.

Predictive values of carotid high-resolution magnetic resonance imaging for large embolus shedding in carotid artery stenting.

BACKGROUND: Embolus shedding is one of the important complications in carotid artery stenting (CAS). Carotid high-resolution magnetic resonance imaging (HR-MRI) is often used to directly reflect important biological characteristics, such as plaque size and composition, as well as the structure of the carotid artery wall. The aim of this study was to investigate the predictive values of carotid HR-MRI for large embolus shedding in CAS. METHODS: In total, 195 patients with carotid stenosis were enrolled. Preoperative carotid HR-MRI was performed to define the nature of the carotid plaques. CAS was performed in all patients, and intraoperative embolic protection devices were used to collect the shed emboli. According to the diameter and number of shed emboli, the patients were divided into the small-embolus group (group X) and largeembolus group (group Y). Logistic regression analysis was used to analyze the risk factors of large embolus shedding. RESULTS: Group Y included 58 patients, and group X included 137 patients. Age, stenosis length, smoking, and ≥3 transient cerebral ischemic attacks were risk factors for large embolusshedding. Two cases of shed large emboli developed from stable plaques, and 56 cases of large emboli developed from vulnerable plaques. When vulnerable plaques were associated with more risk factors, the incidences of large embolus shedding in cases with vulnerable plaques combined with 0, 1, 2, 3, and 4 risk factors were 44 % (4/9), 68.1% (15/22), 72.2% (13/18), 76.5% (13/17), and 84.6% (11/13), respectively. DISCUSSION: Carotid HR-MRI can predict the incidence of large embolus shedding in CAS.

DNA Methylation Landscape Reflects the Spatial Organization of Chromatin in Different Cells.

The relationship between DNA methylation and chromatin structure is still largely unknown. By analyzing a large set of published sequencing data, we observed a long-range power law correlation of DNA methylation with cell class-specific scaling exponents in the range of tens of kilobases. We showed that such cell class-specific scaling exponents are caused by different patchiness of DNA methylation in different cells. By modeling the chromatin structure using high-resolution chromosome conformation capture data and mapping the methylation level onto the modeled structure, we demonstrated that the patchiness of DNA methylation is related to chromatin structure. The scaling exponents of the power law correlation are thus a display of the spatial organization of chromatin. Besides the long-range correlation, we also showed that the local correlation of DNA methylation is associated with nucleosome positioning. The local correlation of partially methylated domains is different from that of nonpartially methylated domains, suggesting that their chromatin structures differ at the scale of several hundred base pairs (covering a few nucleosomes). Our study provides a novel, to our knowledge, view of the spatial organization of chromatin structure from a perspective of DNA methylation, in which both long-range and local correlations of DNA methylation along the genome reflect the spatial organization of chromatin.

Dynamics of spontaneous flipping of a mismatched base in DNA duplex.

DNA base flipping is a fundamental theme in DNA biophysics. The dynamics for a B-DNA base to spontaneously flip out of the double helix has significant implications in various DNA-protein interactions but are still poorly understood. The spontaneous base-flipping rate obtained previously via the imino proton exchange assay is most likely the rate of base wobbling instead of flipping. Using the diffusion-decelerated fluorescence correlation spectroscopy together with molecular dynamics simulations, we show that a base of a single mismatched base pair (T-G, T-T, or T-C) in a double-stranded DNA can spontaneously flip out of the DNA duplex. The extrahelical lifetimes are on the order of 10 ms, whereas the intrahelical lifetimes range from 0.3 to 20 s depending on the stability of the base pairs. These findings provide detailed understanding on the dynamics of DNA base flipping and lay down foundation to fully understand how exactly the repair proteins search and locate the target mismatched base among a vast excess of matched DNA bases.

Trehalose facilitates DNA melting: a single-molecule optical tweezers study.

Using optical tweezers, here we show that the overstretching transition force of double-stranded DNA (dsDNA) is lowered significantly by the addition of the disaccharide trehalose as well as certain polyol osmolytes. This effect is found to depend linearly on the logarithm of the trehalose concentration. We propose an entropic driving mechanism for the experimentally observed destabilization of dsDNA that is rooted in the higher affinity of the DNA bases for trehalose than for water, which promotes base exposure and DNA melting. Molecular dynamics simulation reveals the direct interaction of trehalose with nucleobases. Experiments with other osmolytes confirm that the extent of dsDNA destabilization is governed by the ratio between polar and apolar fractions of an osmolyte.

Auto-suppression of Tet dioxygenases protects the mouse oocyte genome from oxidative demethylation.

DNA cytosine methylation plays a vital role in repressing retrotransposons, and such derepression is linked with developmental failure, tumorigenesis and aging. DNA methylation patterns are formed by precisely regulated actions of DNA methylation writers (DNA methyltransferases) and erasers (TET, ten-eleven translocation dioxygenases). However, the mechanisms underlying target-specific oxidation of 5mC by TET dioxygenases remain largely unexplored. Here we show that a large low-complexity domain (LCD), located in the catalytic part of Tet enzymes, negatively regulates the dioxygenase activity. Recombinant Tet3 lacking LCD is shown to be hyperactive in converting 5mC into oxidized species in vitro. Endogenous expression of the hyperactive Tet3 mutant in mouse oocytes results in genome-wide 5mC oxidation. Notably, the occurrence of aberrant 5mC oxidation correlates with a consequent loss of the repressive histone mark H3K9me3 at ERVK retrotransposons. The erosion of both 5mC and H3K9me3 causes ERVK derepression along with upregulation of their neighboring genes, potentially leading to the impairment of oocyte development. These findings suggest that Tet dioxygenases use an intrinsic auto-regulatory mechanism to tightly regulate their enzymatic activity, thus achieving spatiotemporal specificity of methylome reprogramming, and highlight the importance of methylome integrity for development.

Dynamics of DNA hydroxymethylation and methylation during mouse embryonic and germline development.

In mammals, DNA 5-hydroxymethylcytosine (5hmC) is involved in methylation reprogramming during early embryonic development. Yet, to what extent 5hmC participates in genome-wide methylation reprogramming remains largely unknown. Here, we characterize the 5hmC landscapes in mouse early embryos and germ cells with parental allele specificity. DNA hydroxymethylation was most strongly correlated with DNA demethylation as compared with de novo or maintenance methylation in zygotes, while 5hmC was targeted to particular de novo methylated sites in postimplantation epiblasts. Surprisingly, DNA replication was also required for 5hmC generation, especially in the female pronucleus. More strikingly, aberrant nuclear localization of Dnmt1/Uhrf1 in mouse zygotes due to maternal deficiency of Nlrp14 led to defects in DNA-replication-coupled passive demethylation and impaired 5hmC deposition, revealing the divergency between genome-wide 5-methylcytosine (5mC) maintenance and Tet-mediated oxidation. In summary, our work provides insights and a valuable resource for the study of epigenetic regulation in early embryo development.

Chemical Pretreatment Activated a Plastic State Amenable to Direct Lineage Reprogramming.

Somatic cells can be chemically reprogrammed into a pluripotent stem cell (CiPSC) state, mediated by an extraembryonic endoderm- (XEN-) like state. We found that the chemical cocktail applied in CiPSC generation initially activated a plastic state in mouse fibroblasts before transitioning into XEN-like cells. The plastic state was characterized by broadly activated expression of development-associated transcription factors (TFs), such as Sox17, Ascl1, Tbx3, and Nkx6-1, with a more accessible chromatin state indicating an enhanced capability of cell fate conversion. Intriguingly, introducing such a plastic state remarkably improved the efficiency of chemical reprogramming from fibroblasts to functional neuron-like cells with electrophysiological activity or beating skeletal muscles. Furthermore, the generation of chemically induced neuron-like cells or skeletal muscles from mouse fibroblasts was independent of the intermediate XEN-like state or the pluripotency state. In summary, our findings revealed a plastic chemically activated multi-lineage priming (CaMP) state at the onset of chemical reprogramming. This state enhanced the cells' potential to adapt to other cell fates. It provides a general approach to empowering chemical reprogramming methods to obtain functional cell types bypassing inducing pluripotent stem cells.

Obesity modulates cell-cell interactions during ovarian folliculogenesis.

Obesity is known to affect female reproduction, as evidenced by obese patients suffering from subfertility and abnormal oogenesis. However, the underlying mechanisms by which obesity impairs folliculogenesis are poorly documented. Here, we performed comprehensive single-cell transcriptome analysis in both regular diet (RD) and obese mouse models to systematically uncover how obesity affects ovarian follicle cells and their interactions. We found an increased proportion of Inhbb highly expressed granulosa cells (GCs) among all the GC subpopulations in obese mice. Under obese conditions, excessive androgen secreted from endocrine theca cells (ETCs) may contribute to the imbalanced change of GC subtypes through ETCs-GCs interactions. This is alleviated by enzalutamide, an androgen receptor antagonist. We also identified and confirmed typical GC markers, such as Marcks and Prkar2b, for sensitive evaluation of female fertility in obesity. These data represent a resource for studying transcriptional networks and cell-cell interactions during folliculogenesis under physiological and pathological conditions.

Decoding dynamic epigenetic landscapes in human oocytes using single-cell multi-omics sequencing.

Developing female human germ cells undergo genome-wide epigenetic reprogramming, but de novo DNA methylation dynamics and their interplay with chromatin states and transcriptional activation in developing oocytes is poorly understood. Here, we developed a single-cell multi-omics sequencing method, scChaRM-seq, that enables simultaneous profiling of the DNA methylome, transcriptome, and chromatin accessibility in single human oocytes and ovarian somatic cells. We observed a global increase in DNA methylation during human oocyte growth that correlates with chromatin accessibility, whereas increases of DNA methylation at specific features were associated with active transcription. Integrated analyses of multi-omics data from humans and mice revealed species-specific gene expression, and promoter accessibility contributes to gene body methylation programs. Alu elements retained low DNA methylation levels and high accessibility in early growing oocytes and were located near developmental genes in humans and mice. Together, these findings show how scChaRM-seq can provide insight into DNA methylation pattern establishment.

Unveiling the heterogeneity of NKT cells in the liver through single cell RNA sequencing.

CD1d-dependent type I NKT cells, which are activated by lipid antigen, are known to play important roles in innate and adaptive immunity, as are a portion of type II NKT cells. However, the heterogeneity of NKT cells, especially NKT-like cells, remains largely unknown. Here, we report the profiling of NKT (NK1.1+CD3e+) cells in livers from wild type (WT), Jα18-deficient and CD1d-deficient mice by single-cell RNA sequencing. Unbiased transcriptional clustering revealed distinct cell subsets. The transcriptomic profiles identified the well-known CD1d-dependent NKT cells and defined two CD1d-independent NKT cell subsets. In addition, validation of marker genes revealed the differential organ distribution and landscape of NKT cell subsets during liver tumor progression. More importantly, we found that CD1d-independent Sca-1-CD62L+ NKT cells showed a strong ability to secrete IFN-γ after costimulation with IL-2, IL-12 and IL-18 in vitro. Collectively, our findings provide a comprehensive characterization of NKT cell heterogeneity and unveil a previously undefined functional NKT cell subset.

Impact of Insurance Status on Diagnostic Stage in Hodgkin's Lymphoma in the United States: Implications for Detection and Outcomes.

Introduction and objective Hodgkin's lymphoma (HL) is a form of cancer originating from white blood cells that presents upon diagnosis with well-characterized symptoms (palpable lymph nodes, fever, night sweats, weight loss). HL is currently one of the most treatable cancers, with a successful treatment rate of 75% worldwide. The objective of this study is to evaluate the association between insurance status and the stage of diagnosis of HL in the United States from the years 2007 to 2016. Methods A cross-sectional study using secondary data from the Surveillance, Epidemiology, and End Results (SEER) program database was used. Insurance status of each patient was defined as uninsured (not insured or self-pay), any Medicaid (includes Indian/public health service), insured (private insurance, managed care, Health Maintenance Organization (HMO), Preferred Provider Organization (PPO), or Medicare) and insured not specified. Staging was dictated via the SEER combined/American Joint Committee on Cancer (AJCC) cancer staging guidelines. We divided the stages into early-stage (localized) and late-stage (regional by direct extension, involving distant sites/nodes). We used univariate descriptive analysis to determine baseline characteristics, bivariate analysis to evaluate potential confounding, and binary logistic regression to compute unadjusted and adjusted odd ratios and corresponding 95% confidence intervals.  Results  Approximately 77% of insured individuals presented with a late-stage diagnosis, compared with 78.1% for insured not specified, 82% for any Medicaid, and 84.9% for uninsured. After adjusting for age, sex, race and marital status, insurance status had a significant impact on the stage of diagnosis of Hodgkin's lymphoma. The odds ratio (OR) for advanced stage diagnosis of HL in uninsured patients compared to insured patients was 1.72 (95% CI 1.03-2.86, p=0.037); for any Medicaid, the OR was 1.37 (95% CI 1.02-1.83, p=0.036), and for insured not specified, 1.09 (95% CI 0.83-1.44, p=0.522). Conclusions Uninsured patients are significantly more likely to have a later stage diagnosis of HL compared to those that are insured. The findings of this study coincide with the associations found in previous studies involving other cancers such as breast, cervical, prostate, colorectal, hepatocellular, bladder and kidney cancers outcomes and insurance status.

Chemicals orchestrate reprogramming with hierarchical activation of master transcription factors primed by endogenous Sox17 activation.

Mouse somatic cells can be chemically reprogrammed into pluripotent stem cells (CiPSCs) through an intermediate extraembryonic endoderm (XEN)-like state. However, it is elusive how the chemicals orchestrate the cell fate alteration. In this study, we analyze molecular dynamics in chemical reprogramming from fibroblasts to a XEN-like state. We find that Sox17 is initially activated by the chemical cocktails, and XEN cell fate specialization is subsequently mediated by Sox17 activated expression of other XEN master genes, such as Sall4 and Gata4. Furthermore, this stepwise process is differentially regulated. The core reprogramming chemicals CHIR99021, 616452 and Forskolin are all necessary for Sox17 activation, while differently required for Gata4 and Sall4 expression. The addition of chemical boosters in different phases further improves the generation efficiency of XEN-like cells. Taken together, our work demonstrates that chemical reprogramming is regulated in 3 distinct "prime-specify-transit" phases initiated with endogenous Sox17 activation, providing a new framework to understand cell fate determination.

Integrative single-cell analysis of transcriptome, DNA methylome and chromatin accessibility in mouse oocytes.

Oocyte growth is a key step in forming mature eggs that are ready to be fertilized. The states and modifications of chromatin represent critical sources of information for this process. However, the dynamics and interrelations of these chromatin characteristics remain elusive. In this study, we developed an improved scCOOL-seq technique (iscCOOL-seq), which is a multi-omics, single-cell and single-base resolution method with high mapping rates, and explored the chromatin accessibility landscape and its relationship to DNA methylation in growing mouse oocytes. The most dramatic change in chromatin accessibility occurs during oocyte growth initiation, accompanied with prominent transcriptome alterations and an elevated variation in DNA methylation levels among individual oocytes. Unlike CpG islands (CGIs), partially methylated domains (PMDs) are associated with a low density of nucleosome-depleted regions (NDRs) during the whole maturation period. Surprisingly, highly expressed genes are usually associated with NDRs at their transcriptional end sites (TESs). In addition, genes with de novo methylated gene bodies during oocyte maturation are already open at their promoters before oocyte growth initiation. Furthermore, epigenetic and transcription factors that might be involved in oocyte maturation are identified. Our work paves the way for dissecting the complex, yet highly coordinated, epigenetic alterations during mouse oocyte growth and the establishment of totipotency.

Med23 serves as a gatekeeper of the myeloid potential of hematopoietic stem cells.

In response to myeloablative stresses, HSCs are rapidly activated to replenish myeloid progenitors, while maintaining full potential of self-renewal to ensure life-long hematopoiesis. However, the key factors that orchestrate HSC activities during physiological stresses remain largely unknown. Here we report that Med23 controls the myeloid potential of activated HSCs. Ablation of Med23 in hematopoietic system leads to lymphocytopenia. Med23-deficient HSCs undergo myeloid-biased differentiation and lose the self-renewal capacity. Interestingly, Med23-deficient HSCs are much easier to be activated in response to physiological stresses. Mechanistically, Med23 plays essential roles in maintaining stemness genes expression and suppressing myeloid lineage genes expression. Med23 is downregulated in HSCs and Med23 deletion results in better survival under myeloablative stress. Altogether, our findings identify Med23 as a gatekeeper of myeloid potential of HSCs, thus providing unique insights into the relationship among Med23-mediated transcriptional regulations, the myeloid potential of HSCs and HSC activation upon stresses.

The sequence preference of DNA methylation variation in mammalians.

Methylation of cytosine at the 5 position of the pyrimidine ring is the most prevalent and significant epigenetic modifications in mammalian DNA. The CpG methylation level shows a bimodal distribution but the bimodality can be overestimated due to the heterogeneity of per-base depth. Here, we developed an algorithm to eliminate the effect of per-base depth inhomogeneity on the bimodality and obtained a random CpG methylation distribution. By quantifying the deviation of the observed methylation distribution and the random one using the information formula, we find that in tetranucleotides 5'-N5CGN3-3' (N5, N3 = A, C, G or T), GCGN3 and CCGN3 show less apparent deviation than ACGN3 and TCGN3, indicating that GCGN3 and CCGN3 are less variant in their level of methylation. The methylation variation of N5CGN3 are conserved among different cells, tissues and species, implying common features in the mechanisms of methylation and demethylation, presumably mediated by DNMTs and TETs in mammalians, respectively. Sequence dependence of DNA methylation variation also relates to gene regulatory and promotes the reexamination of the role of DNA sequence in fundamental biological processes.

The effects of cytosine methylation on general transcription factors.

DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.