Effects of water-aging for 6 months on the durability of a novel antimicrobial and protein-repellent dental bonding agent

Biofilms at the tooth-restoration bonded interface can produce acids and cause recurrent caries. Recurrent caries is a primary reason for restoration failures. The objectives of this study were to synthesize a novel bioactive dental bonding agent containing dimethylaminohexadecyl methacrylate(DMAHDM) and 2-methacryloyloxyethyl phosphorylcholine(MPC) to inhibit biofilm formation at the tooth-restoration margin and to investigate the effects of water-aging for 6 months on the dentin bond strength and protein-repellent and antibacterial durability. A protein-repellent agent(MPC) and antibacterial agent(DMAHDM) were added to a Scotchbond multi-purpose(SBMP) primer and adhesive. Specimens were stored in water at 37 °C for 1, 30, 90, or 180 days(d).At the end of each time period, the dentin bond strength and protein-repellent and antibacterial properties were evaluated. Protein attachment onto resin specimens was measured by the micro-bicinchoninic acid approach. A dental plaque microcosm biofilm model was used to test the biofilm response. The SBMP + MPC + DMAHDM group showed no decline in dentin bond strength after water-aging for 6 months, which was significantly higher than that of the control(P < 0.05). The SBMP + MPC + DMAHDM group had protein adhesion that was only 1/20 of that of the SBMP control(P < 0.05). Incorporation of MPC and DMAHDM into SBMP provided a synergistic effect on biofilm reduction. The antibacterial effect and resistance to protein adsorption exhibited no decrease from 1 to 180 d(P > 0.1). In conclusion, a bonding agent with MPC and DMAHDM achieved a durable dentin bond strength and long-term resistance to proteins and oral bacteria. The novel dental bonding agent is promising for applications in preventive and restorative dentistry to reduce biofilm formation at the tooth-restoration margin.

EZH2 identifies the precursors of human natural killer cells with trained immunity

Objective:Trained immunity of natural killer(NK)cells has shown great potential in the treatment of cancers by eliciting enhanced effector responses to restimulation by cytokines or cancer cells for long time periods after preactivation.However,the human NK cells responsible for the generation and maintenance of trained immunity are largely unknown.We hypothesized that heterogeneous human NK cells would respond differentially to stimulation with a combination of IL-12,IL-15,and IL-18,and that an NK cell subset might exist that is mainly responsible for the induction of trained immunity.On the basis of our hypothesis,we aimed to identify the subset from which cytokine-trained human NK cells originate and to explore possible regulatory targets for drug intervention.Methods:Flow cytometry assays were performed to analyze the functions of cytokine-trained NK cells and examine cell division and protein expression in NK cell subsets.Single-cell RNA sequencing(scRNA-seq)plus TotalSeq™technology was used to track the heterogeneity of NK cells during the induction of trained immunity.Results:Traditional developmental markers for peripheral NK cells were unable to identify the precursors of human NK cells with trained immunity.Therefore,we used scRNA-seq plus TotalSeq™technology to track the heterogeneity of NK cells during the induction of trained immunity and identified a unique cluster of CD57−NKG2A+EZH2+IFNG+MKI67+IL12R+IL15R+IL18R+NK cells.Enrichment and pseudotime trajectory analyses suggested that this cluster of NK cells contained the precursor of trained NK cells.We then used flow cytometry to further investigate the role of EZH2 in trained NK precursors and found that CD57−NKG2A+EZH2+NK cells had faster cell cycles and an enhanced trained phenotype,and EZH2 inhibition significantly impaired the induction of trained immunity in NK cells.These results suggested that EZH2 is a unique epigenetic marker of precursors of human NK cells with trained immunity.Conclusions:Our work revealed human NK heterogeneity in the induction of trained immunity,identified the precursor subset for trained NK cells,and demonstrated the critical role of EZH2 in the induction of trained immunity in human NK cells.

p21/Zbtb18 repress the expression of cKit to regulate the self-renewal of hematopoietic stem cells

The maintenance of hematopoietic stem cells(HSCs)is a complex process involving numerous cell-extrinsic and-intrinsic regulators.The first member of the cyclin-dependent kinase family of inhibitors to be identified,p21,has been reported to perform a wide range of critical biological functions,including cell cycle regulation,transcription,differentiation,and so on.Given the previous inconsistent results regarding the functions of p21 in HSCs in a p21-knockout mouse model,we employed p21-tdTomato(tdT)mice to further elucidate its role in HSCs during homeostasis.The results showed that p21-tdT+HSCs exhibited increased self-renewal capacity compared to p21-tdT−HSCs.Zbtb18,a transcriptional repressor,was upregulated in p21-tdT+HSCs,and its knockdown significantly impaired the reconstitution capability of HSCs.Furthermore,p21 interacted with ZBTB18 to co-repress the expression of cKit in HSCs and thus regulated the self-renewal of HSCs.Our data provide novel insights into the physiological role and mechanisms of p21 in HSCs during homeostasis independent of its conventional role as a cell cycle inhibitor.

Probing the fate of transplanted hematopoietic stem cells: is the combinational approach “FIT” for purpose?

Hematopoietic stem cells (HSCs)—with their self-renewal and multi-lineage differentiation potential—are top of the hematopoietic cell hierarchy. Stress conditions activate the quiescent HSCs, which in turn enter cell cycle to produce their progenies (Sawai et al., 2016).

New paradigms on hematopoietic stem cell differentiation

Ever since hematopoietic stem cells(HSCs)were first identified half a century ago,their differentiation roadmap has been extensively studied.The classical model of hematopoiesis has long held as a dogma that HSCs reside at the top of a hierarchy in which HSCs possess self-renewal capacity and can progressively give rise to all blood lineage cells.However,over the past several years,with advances in single cell technologies,this developmental scheme has been challenged.In this review,we discuss the evidence supporting heterogeneity within HSC and progenitor populations as well as the hierarchical models revised by novel approaches mainly in mouse system.These evolving views provide further understanding of hematopoiesis and highlight the complexity of hematopoietic differentiation.

Understanding the“SMART”features of hematopoietic stem cells and beyond

Since the huge success of bone marrow transplantation technology in clinical practice,hematopoietic stem cells(HSCs)have become the gold standard for defining the properties of adult stem cells(ASCs).Here,we describe the“self-renewal,multilineage differentiation,apoptosis,rest,and trafficking”or“SMART”model,which has been developed based on data derived from studies of HSCs as the most well-characterized stem cell type.Given the potential therapeutic applications of ASCs,we delineate the key characteristics of HSCs using this model and speculate on the physiological relevance of stem cells identified in other tissues.Great strides are being made in understanding the biology of ASCs,and efforts are now underway to develop safe and effective ASC-based therapies in this emerging area.

Differentiation of embryonic stem cells in adult bone marrow

Embryonic stem cells （ESCs） are a potential source of generating transplantable hematopoietic stem and progenitor cells,which in turn can serve as ＂seed＂ cells for hematopoietic regeneration.In this study,we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow （BM）.To this end,we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein （GFP） and then injected the ESCs into syngeneic BM via intra-tibia.The progeny of the transplanted ESCs were then analyzed at different time points after transplantation.Notably,however,most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers.This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment,thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs.

Single-cell analysis of transcription factor regulatory networks reveals molecular basis for subtype-specific dysregulation in acute myeloid leukemia

Highly heterogeneous acute myeloid leukemia(AML)exhibits dysregulated transcriptional programs.Transcription factor(TF)regulatory networks underlying AML subtypes have not been elucidated at single-cell resolution.Here,we comprehensively mapped malignancy-related TFs activated in different AML subtypes by analyzing single-cell RNA sequencing data from AMLs and healthy donors.We first identified six modules of regulatory networks which were prevalently dysregulated in all AML patients.AML subtypes featured with different malignant cellular composition possessed subtype-specific regulatory TFs associated with differentiation suppression or immune modulation.At last,we validated that ERF was crucial for the development of hematopoietic stem/progenitor cells by performing loss-and gain-of-function experiments in zebrafish embryos.Collectively,our work thoroughly documents an abnormal spectrum of transcriptional regulatory networks in AML and reveals subtype-specific dysregulation basis,which provides a prospective view to AML pathogenesis and potential targets for both diagnosis and therapy.

PUMA facilitates EMI 1-promoted cytoplasmic Rad51 ubiquitination and inhibits DNA repair in stem and progenitor cells

Maintenance of genetic stability via proper DNA repair in stem and progenitor cells is essential for the tissue repair and regeneration,while preventing cell transformation after damage.Loss of PUMA dramatically increases the survival of mice after exposure to a lethal dose of ionizing radiation(IR),while without promoting tumorigenesis in the long-term survivors.This finding suggests that PUMA(p53 upregulated modulator of apoptosis)may have a function other than regulates apoptosis.Here,we identify a novel role of PUMA in regulation of DNA repair in embryonic or induced pluripotent stem cells(PSCs)and immortalized hematopoietic progenitor cells(HPCs)after IR.We found that PUMA-deficient PSCs and HPCs exhibited a significant higher doublestrand break(DSB)DNA repair activity via Rad51-mediated homologous recombination(HR).This is because PUMA can be associated with early mitotic Inhibitor 1(EMI1)and Rad51 in the cytoplasm to facilitate EMI1-mediated cytoplasmic Rad51 ubiquitination and degradation,thereby inhibiting Rad51 nuclear translocation and HR DNA repair.Our data demonstrate that PUMA acts as a repressor for DSB DNA repair and thus offers a new rationale for therapeutic targeting of PUMA in regenerative cells in the context of DNA damage.

Preleukemic stem cells:leave it or not?

Acute myeloid leukemia(AML)has been shown to undergo multiple acquired mutations in hematopoietic cell lineages over years before becoming clinically apparent.The early stage of AML(before it becomes clinically recognizable)may be characterized by acquisition of some,but not all,leukemia-related somatic mutations in hematopoietic stem cells(HSCs).The physiological roles of these mutations remain puzzling.These HSCs have been termed as preleukemic HSCs.However,those frequent acquired somatic mutations are also found in healthy aging adults,namely,“age-related clonal hematopoiesis.”Multiple studies have demonstrated that the preleukemic HSCs survive through chemotherapy and then contribute to the relapse and the development of de novo AML.Whether preleukemic HSCs should be targeted or whether a preventive therapy should be considered for those individuals remains to be determined.This article aims to shed light on this special subject and to discuss the roles of preleukemic HSCs in leukemogenesis.

SIEVE: identifying robust single cell variable genes for single-cell RNA sequencing data

Single-cell RNA-seq data analysis generally requires quality control,normalization,highly variable genes screening,dimensionality reduction and clustering.Among these processes,downstream analysis including dimensionality reduction and clustering are sensitive to the selection of highly variable genes.Though increasing number of tools for selecting the highly variable genes have been developed,an evaluation of theirperformances and a general strategy are lack.Here,wecompare the performance of nine commonly usedmethods for screening variable genes by using single-cell RNA-seq data from hematopoietic stem/progenitor cells and mature blood cells,and find that SCHS outperforms other methods regarding to reproducibility and accuracy.However,this method prefers the selection of highly expressed genes.We further propose a new strategy SIEVE(SIngle-cEll Variable gEnes)bymultiple rounds of randomsampling,therefore minimizing the stochastic noise and identifying a robust set of variable genes.Moreover,SIEVE recovers lowly expressed genes as variable genes and substantially improves the accuracy of single cell classification,especially for the methods with lower reproducibility.The SIEVE software is freely available at https://github.com/YinanZhang522/SIEVE.

Function of FEZF1 during early neural differentiation of human embryonic stem cells

The understanding of the mechanism underlying human neural development has been hampered due to lack of a cellular system and complicated ethical issues. Human embryonic stem cells (hESCs) provide an invaluable model for dissecting human development because of unlimited self-renewal and the capacity to differentiate into nearly all cell types in the human body. In this study,using a chemical defined neural induction protocol and molecular profiling, we identified Fez family zinc finger 1 (FEZF1) as a potential regulator of early human neural development. FEZF1 is rapidly up-regulated during neural differentiation in hESCs and expressed before PAX6, a well-established marker of early human neural induction. We generated FEZF1-knockout H1 hESC lines using CRISPR-CAS9 technology and found that depletion of FEZF1 abrogates neural differentiation of hESCs. Moreover,loss of FEZF1 impairs the pluripotency exit of hESCs during neural specification, which partially explains the neural induction defect caused by FEZF1 deletion. However, enforced expression of FEZF1 itself fails to drive neural differentiation in hESCs,suggesting that FEZF1 is necessary but not sufficient for neural differentiation from hESCs. Taken together, our findings identify one of the earliest regulators expressed upon neural induction and provide insight into early neural development in human.

Evidences for mutations in the histone modifying gene SETD2 as critical drivers in leukemia development

Cancer is thought to be largely caused by genetic mutations that alter nucleotide sequences of DNA, yet epigenetic fac- tors are increasingly recognized to also play causative roles in cancer development. The idea of ＂epimutations＂ involves aberrations in chemical modification of the chromatin or DNA that do not change nucleotide sequence. While it is widely accepted that aberrations of DNA methylation con- tribute to carcinogenesis, the functional importance of his- tone modifications and other chromatin features in the de- velopment of human cancer remains to be elucidated. Re- cent efforts in large-scale sequencing of cancer genomes have revealed mutations in numerous chromatin regulatory genes, and thus demand further investigations on the epige- netic mechanisms as drivin~ forces in cancer development.

Single-cell transcriptomes of peripheral blood cells indicate and elucidate severity of COVID-19

The blood and immune system of coronavirus disease 2019(COVID-19)infected patients are dysfunctional,and numerous studies have been conducted to resolve their characteristics and pathogenic mechanisms.Nevertheless,the variations of immune responses along with disease severity have not been comprehensively documented.Here,we profiled the single-cell transcriptomes of 96,313 peripheral blood mononuclear cells(PBMCs)derived from 12 COVID-19 patients(including four moderate,four severe and four critical cases)and three healthy donors.We showed that proliferative CD8 effector T cells with declined immune functions and cytotoxicity accumulated in the critical stage.By contrast,the quantity of natural killer(NK)cells was significantly reduced,while they exhibited enhanced immune activities.Notably,a gradually attenuated responseto COVID-19 along with disease severity was observed in monocytes,in terms of cellular composition,transcriptional discrepancy and transcription factor regulatory network.Furthermore,we identified immune cell-type dependent cytokine signatures distinguishing the severity of COVID-19 patients.In addition,cell interactions between CD8 effector T/NK cells and monocytes mediated by inflammatory cytokines were enhanced in moderate and severe stages,but weakened in critical cases.Collectively,our work uncovers the cellular and molecular players underlying the disordered and heterogeneous immune responses associated with COVID-19 severity,which could provide valuable insights for the treatment of critical COVID-19 patients.

Global transcriptome analysis for identification of interactions between coding and noncoding RNAs during human erythroid differentiation

Studies on coding genes, miRNAs, and lncRNAs during erythroid development have been performed in recent years. However, analysis focusing on the integration of the three RNA types has yet to be done. In the present study, we compared the dynamics of coding genes, miRNA, and IncRNA expression profiles. To explore dynamic changes in erythropoiesis and potential mechanisms that control these changes in the transcriptome level, we took advantage of high throughput sequencing technologies to obtain transcriptome data from cord blood hematopoietic stem cells and the following four erythroid differentiation stages, as well as from mature red blood cells. Results indicated that lncRNAs were promising cell marker candidates for erythroid differentiation. Clustering analysis classified the differentially expressed genes into four subtypes that corresponded to dynamic changes during sternness maintenance, mid-differentiation, and maturation. Integrated analysis revealed that noncoding RNAs potentially participated in controlling blood cell maturation, and especially associated with heine metabolism and responses to oxygen species and DNA damage. These regulatory interactions were displayed in a comprehensive network, thereby inferring correlations between RNAs and their associated functions. These data provided a substantial resource for the study of normal erythropoiesis, which will permit further investigation and understanding of erythroid development and acquired erythroid disorders.

NK cells play a significant role in immunosurveillance at the early stage of MLL-AF9 acute myeloid leukemia via CD226/CD155 interactions

Acute myeloid leukemia(AML) is an aggressive hematological malignancy, and the mechanism underlying immune system involvement in leukemia development is unclear. In the present study, we utilized a myeloid/lymphoid or mixed-lineage leukemia; translocated to, 3(MLLT3/MLL-AF9)-induced AML mouse model with or without exposure to irradiation. We found that the leukemia cells could survive and expand in hosts with intact immune systems, whereas leukemia progression was accelerated in mice with impaired immune systems. Moreover, the leukemia cells escaped from host immunosurveillance via editing their immunogenicity, including the up-regulation of an inhibitory antigen(i.e., CD47) and the down-regulation of active antigens(i.e., CD86, CD54, retinoic acid early transcript(RAE), histocompatibility 2, D region locus b(H2-Db) and H2-Dd). Natural killer(NK) cells were activated in the early phase of AML progression, whereas T cells were stimulated in the late phase. Furthermore, NK cell depletion showed that NK cells were necessary for the elimination of leukemia cells in our AML mouse model. Notably, CD155/CD226 primarily mediated the interaction between NK cells and leukemia cells and contributed to the antitumor effects of NK cells during the early phase of AML. Clinical data from patients with diverse hematological malignancies showed that CD155 expression was decreased in hematological malignancies. Taken together, our results demonstrate that NK cells play a pivotal role in immunosurveillance against leukemia cells during the early stage of AML primarily through the CD226/CD155 interaction; however, NK cells are not sufficient to eliminate leukemia cells.

Identification and characterization of human hematopoietic mesoderm

The embryonic mesoderm comprises heterogeneous cell subpopulations with distinct lineage biases.It is unclear whether a bias for the human hematopoietic lineage emerges at this early developmental stage.In this study,we integrated single-cell transcriptomic analyses of human mesoderm cells from embryonic stem cells and embryos,enabling us to identify and define the molecular features of human hematopoietic mesoderm(HM)cells biased towards hematopoietic lineages.We discovered that BMP4 plays an essential role in HM specification and can serve as a marker for HM cells.Mechanistically,BMP4 acts as a downstream target of HDAC1,which modulates the expression of BMP4 by deacetylating its enhancer.Inhibition of HDAC significantly enhances HM specification and promotes subsequent hematopoietic cell differentiation.In conclusion,our study identifies human HM cells and describes new mechanisms for human hematopoietic development.

HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs

Genome-edited human induced pluripotent stem cells(iPSCs)hold great promise for therapeutic applications.However,low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair(HDR)-edited iPSC lines,particularly for silent genes.This is partially due to chromatin compaction,inevitably limiting Cas9 access to the target DNA.Among the six HDAC inhibitors we examined,vorinostat,or suberoylanilide hydroxamic acid(SAHA),led to the highest HDR efficiency at both open and closed loci,with acceptable toxicity.HDAC inhibitors equally increased non-homologous end joining(NHEJ)editing efficiencies(~50%)at both open and closed loci,due to the considerable HDAC inhibitor-mediated increase in Cas9 and sgRNA expression.However,we observed more substantial HDR efficiency improvement at closed loci relative to open chromatin(2.8 vs.1.7-fold change).These studies provide a new strategy for HDRediting of silent genes in iPSCs.

Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing

A series of clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR associated protein 9(Cas9)systems have been engineered for genome editing.The most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus.However,a comparison of their detailed gene editing outcomes is still lacking.By characterizing the editing outcomes of 11 sites in human induced pluripotent stem cells(iPSCs)and K562 cells,we found that SaCas9 could edit the genome with greater efficiencies than SpCas9.We also compared the effects of spacer lengths of single-guide RNAs(sgRNAs;18–21 nt for SpCas9 and 19–23 nt for SaCas9)and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9,respectively.However,the optimal spacer length for a particular sgRNA was 18–21 nt for SpCas9 and 21–22 nt for SaCas9.Furthermore,SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining(NHEJ)+1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif(PAM),indicating a characteristic of a staggered cut.Accordingly,editing with SaCas9 led to higher efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide(dsODN)insertion or homology-directed repair(HDR)-mediated adeno-associated virus serotype 6(AAV6)donor knock-in.Finally,GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with SpCas9.Our work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.

Advances in measuring DNA methylation

DNA methylation is one of the most important components of epigenetics,which plays essential roles in maintaining genome stability and regulating gene expression.In recent years,DNA methylation measuring methods have been continuously optimized.Combined with next generation sequencing technologies,these approaches have enabled the detection of genome-wide cytosine methylation at single-base resolution.In this paper,we review the development of 5-methylcytosine and its oxidized derivatives measuring methods,and recent advancement of single-cell epigenome sequencing technologies,offering more referable information for the selection and optimization of DNA methylation sequencing technologies and related research.