High-Performance Bioinspired Microspheres for Boosting Dental Adhesion.

Dental adhesives are widely used in daily practice for minimally invasive restorative dentistry but suffer from bond degradation and biofilm attack. Bio-inspired by marine mussels having excellent surface-adhesion capability and high chemical affinity of polydopamine (PDA) to metal ions, herein, experimental zinc (Zn)-containing polydopamine-based adhesive formulation, further being referred to as "Zn-PDA@SiO2 "-incorporated adhesive is proposed as a novel dental adhesive. Different Zn contents (5 and 10 mm) of Zn-PDA@SiO2 are prepared. Considering the synergistic effect of Zn and PDA, Zn-PDA@SiO2 not only presents excellent antibacterial potential and notably inhibits enzymatic activity (soluble and matrix-bound proteases), but also exhibits superior biocompatibility and biosafety in vitro/vivo. The long-term bond stability is substantially improved by adding 5 wt% 5 mm Zn-PDA@SiO2 to the primer. The aged bond strength of the experimentally formulated dental adhesives applied in self-etch (SE) bonding mode is 1.9 times higher than that of the SE gold-standard adhesive. Molecular dynamics calculations indicate the stable formation of covalent bonds, Zn-assisted coordinative bonds, and hydrogen bonds between PDA and collagen. Overall, this bioinspired dental adhesive provides an avenue technology for innovative biomedical applications and has already revealed promising perspectives for dental restorative dentistry.

Gestational weight gain and neonatal outcomes in different zygosity twins: a cohort study in Wuhan, China.

OBJECTIVE: To evaluate whether twin zygosity influences the association between neonatal outcomes and gestational weight gain (GWG) based on the Chinese guidelines in twin-pregnancy women. DESIGN: A retrospective cohort study. And it is not a clinical trial. SETTING: Women with twin pregnancies living in Wuhan, China. PARTICIPANTS: A total of 5140 women who delivered live and non-malformed twins from 1 January 2011 to 31 August 2017 were included in this study. MAIN OUTCOME MEASURE: The primary neonatal outcomes included paired small for gestational age (SGA, <10 th percentile birth weight for gestational age and sex), low birth weight (LBW, <2500 g) and gestational age (<33 weeks and <37 weeks). The association between GWG and neonatal outcomes was examined by Logistic regression analyses. RESULTS: A total of 5140 women were included, of whom 22.24%, 54.78% and 22.98% were below, within and above the Chinese guidelines, respectively. Among the including 10 280 infants, 26.28% of them were monozygotic (MZ) twins and 73.72% of them were dizygotic (DZ) twins. Women with low GWG had a significantly higher proportion of LBW/LBW and LBW/NBW infants, a greater likelihood of SGA/SGA and SGA/appropriate for gestational age (AGA) infants and a higher incidence of preterm birth. The associations persisted both in MZ and DZ twins, and twin zygosity influenced the degree of association between GWG and SGA, LBW and preterm birth. High GWG was associated with significant risk reductions in SGA/AGA pairs, LBW/LBW or LBW/NBW pairs, and less than 33 gestational weeks. However, high GWG was only associated with reduced risk of LBW/LBW pairs both in MZ and DZ twins. CONCLUSIONS: GWG below the Chinese recommendations increased the risk of SGA, LBW and preterm birth in both MZ and DZ twins. The effect was more pronounced in MZ twins than that in DZ twin pairs. A high GWG only reduced the risk of LBW/LBW pairs both in MZ and DZ twins.

Antigen-specific TCR-T cells from Rag2 gene-deleted pluripotent stem cells impede solid tumour growth in a mouse model.

The technology of adoptive transfer of T-cell receptor (TCR) engineered T cells is wildly investigated as it has the potential to treat solid cancers. However, the therapeutic application of TCR-T cells is hampered by the poor quality derived mainly from patients' peripheral blood, as well as heterogeneous TCRs caused by the mismatch between transgenic and endogenous TCRs. To improve the homogeneity, antigen-specificity and reduce possible autoreactivity, here we developed a technique to generate antigen-specific T cells from Rag2 gene-deleted pluripotent stem cells (PSCs) and further measured their anti-tumour efficacy. PSCs were first targeted with OT1 TCR into the Rag2 locus to prevent TCR rearrangement during T-cell development. The engineered PSCs were then differentiated through a two-step strategy, in vitro generation of haematopoietic progenitor cells, and in vivo development and maturation of TCR-T cells. Finally, the response to tumour cells was assessed in vitro and in vivo. The regenerated OT1-iT displayed monoclonal antigen-specific TCR expression, and phonotypic normalities in the spleen and lymph node tissues. Importantly, the OT1-iT cells eliminated tumour cells while releasing specific cytokines in vitro. Furthermore, adoptive transfer of OT1-iT cells suppresses solid tumour growth in tumour-bearing animals. Our study presents a novel and straightforward strategy for producing antigen-specific TCR-T cells in vivo from PSCs, allowing for allogeneic transplantation and therapy of solid tumours.

Biomineralization-inspired sandwich dentin desensitization strategy based on multifunctional nanocomposite with yolk-shell structure.

Dentin hypersensitivity (DH) treatment is far from being unequivocal in providing a superior strategy that combines immediate and long-term efficiency of dentinal tubule (DT) occlusion and clinical applicability. In order to achieve this aim, a type of multifunctional yolk-shell nanocomposite with acid resistance, mechanical resistance and biomineralization properties was developed in this study, which consists of a silica/mesoporous titanium-zirconium nanocarrier (STZ) and poly(allylamine hydrochloride) (PAH)-stabilized amorphous calcium phosphate (ACP) liquid precursor. First, the nanocomposite, named as PSTZ, immediately occluded DTs and demonstrated outstanding acid and mechanical resistance. Second, the PSTZ nanocomposite induced intrafibrillar mineralization of single-layer collagen fibrils and remineralization of demineralized dentin matrix. Finally, PSTZ promoted the odontogenic differentiation of dental pulp stem cells by releasing ACP and silicon ions. The reconstruction of the dentin-mimicking hierarchical structure and the introduction of newly formed minerals in the upper, middle and lower segments of DTs, defined as sandwich-like structures, markedly reduced the permeability and achieved superior long-term sealing effects. The nanocomposite material based on mesoporous yolk-shell carriers and liquid-phase mineralized precursors developed in this study represents a versatile biomimetic sandwich desensitization strategy and offers fresh insight into the clinical management of DH.

Lateral plate mesoderm cell-based organoid system for NK cell regeneration from human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-induced NK (iNK) cells are a source of off-the-shelf cell products for universal immune therapy. Conventional methods for iNK cell regeneration from hPSCs include embryoid body (EB) formation and feeder-based expansion steps, which are time-consuming and cause instability and high costs of manufacturing. Here, we develop an EB-free, organoid aggregate method for NK cell regeneration from hPSCs. In a short time-window of 27-day induction, millions of hPSC input can output over billions of iNK cells without the necessity of NK cell expansion feeders. The iNK cells highly express classical toxic granule proteins, apoptosis-inducing ligands, as well as abundant activating and inhibitory receptors. Functionally, the iNK cells eradicate human tumor cells via mechanisms of direct cytotoxicity, apoptosis, and antibody-dependent cellular cytotoxicity. This study provides a reliable scale-up method for regenerating human NK cells from hPSCs, which promotes the universal availability of NK cell products for immune therapy.

IRX5 promotes adipogenesis of hMSCs by repressing glycolysis.

Iroquois homeobox transcription factor 5 (IRX5) plays a pivotal role in extramedullary adipogenesis, but little is known about the effects of IRX5 on adipogenesis of human bone marrow-derived mesenchymal stem cells (hMSCs). In this study, we aimed to determine the effect of IRX5 on hMSCs adipogenesis. By means of qPCR analysis, we determined that IRX5 expression was elevated during adipogenic commitment of hMSCs. The biologic role of IRX5 was further investigated by employing a gain/loss-of-function strategy using an in vitro lentivirus-based system. IRX5 overexpression promoted adipogenesis whereas IRX5 knockdown reduced the adipogenic phenotype. RNA-seq and metabolomics revealed that IRX5 overexpression repressed glycolysis. Dual-luciferase assay results showed that IRX5 overexpression transcriptionally activates peroxisome proliferator-activated receptor gamma coactivator (PGC-1α). Metformin and PGC-1α inhibitor reversed IRX5-induced adipogenesis and glycolytic inhibition. Collectively, IRX5 facilitates adipogenic differentiation of hMSCs by transcriptionally regulating PGC-1α and inhibiting glycolysis, revealing a potential target to control bone marrow-derived mesenchymal stem cells (BMSCs) fate decision and bone homeostasis.

Hematopoietic lineage-converted T cells carrying tumor-associated antigen-recognizing TCRs effectively kill tumor cells.

Tumor-associated antigen (TAA) T-cell receptor (TCR) gene-engineered T cells exhibit great potential in antitumor immunotherapy. Considering the high costs and low availability of patient-derived peripheral blood T cells, substantial efforts have been made to explore alternatives to natural T cells. We previously reported that enforced expression of Hoxb5 converted B cells into induced T (iT) cells in vivo Here, we successfully regenerated naive OT1 (major histocompatibility complex I restricted ovalbumin antigen) iT cells (OT1-iT) in vivo by expressing Hoxb5 in pro-pre-B cells in the OT1 transgenic mouse. The OT1-iT cells can be activated and expanded in vitro in the presence of tumor cells. Particularly, these regenerated OT1-iT cells effectively eradicated tumor cells expressing the TAA (ovalbumin) both in vitro and in vivo This study provides insights into the translational applications of blood lineage-transdifferentiated T cells in immunotherapy.

NUP98-HOXA10hd fusion protein sustains multi-lineage haematopoiesis of lineage-committed progenitors in transplant setting.

OBJECTIVES: Exploring approaches of extending the haematopoiesis time window of MPPs and lineage-committed progenitors might produce promising therapeutic effects. NUP98-HOXA10hd (NA) fusion protein can expand long-term haematopoietic stem cells (HSCs) and promote engraftment competitiveness without causing obvious oncogenesis. Our objectives were to investigate the roles of NA fusion protein in MPP and downstream lineage-committed progenitor context. MATERIAL AND METHODS: 300 sorted MPPs (Lin- CD48- c-kit+ Sca1+ CD135+ CD150- ) were mixed with 5 × 105 total BM helper/competitor cells and injected into irradiated recipients. For secondary transplantation, 5 × 106 total BM cells from primary recipient mice were injected into lethally irradiated recipients. NA-MPP recipient mice were sacrified for flow cytometric analysis of bone marrow progenitors at indicated time points. Sorted MPPs and myeloid progenitors were used for RNA-seq library preparation. RESULTS: We showed that NA-expressing MPPs achieved significantly longer multi-lineage haematopoiesis (>44-week) than natural MPPs (20-week). NA upregulated essential genes regulating long-term haematopoiesis, cell cycle, epigenetic regulation and responses to stress in MPPs. These molecular traits are associated with the earlier appearance of a Sca1- c-kit+ myeloid progenitor population, and more abundant cellularity of lineage-committed progenitor as well as bone marrow nucleated cells. Further, the NA-derived primary bone marrow cells, which lack NA-LSK cells, successfully repopulated secondary multi-lineage haematopoiesis over 20 weeks. CONCLUSIONS: This study unveiled that NA fusion protein promotes MPP and lineage-committed progenitor engraftment via extending long-term multi-lineage haematopoiesis.

T cell regeneration: an update on progress and challenges.

T cells play essential roles in antitumor therapy. Via gene engineering technique to enhance tumor-antigen specificity, patient peripheral blood-derived T cells (PBT) show encouraging clinical outcomes in treating certain blood malignancies. However, the high costs, functionality exhaustion, and disease-condition-dependent availability of PBT prompt the attempts of exploring alternative T cell sources. Theoretically, induced T cells from pluripotent stem cells (PSC) are ideal candidates that integrate plenty of advantages that primary T cells lack, including unlimited off-the-shelf cell source and precision gene editing feasibility. However, researchers are still struggling with developing a straightforward protocol to induce functional and immunocompetent human T cells from PSC. Based on stromal cell-expressing or biomaterial-presenting Notch ligands DLL1 or DLL4, natural and induced blood progenitors can differentiate further toward T lineage commitment. However, none of the reported T induction protocols has yet translated into any clinical application, signaling the existence of numerous technical barriers for regenerating T cells functionally matching their natural PBT counterparts. Alternatively, new approaches have been developed to repopulate induced T lymphopoiesis via in vivo reprogramming or transplanting induced T cell precursors. Here, we review the most recent progress in the T cell regeneration field, and the remaining challenges dragging their clinical applications.

Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors.

Achievement of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells (PSCs) is a central aim in T cell regenerative medicine. To date, preferentially reconstituting T lymphopoiesis in vivo from PSCs remains a practical challenge. Here we documented that synergistic and transient expression of Runx1 and Hoxa9 restricted in the time window of endothelial-to-hematopoietic transition and hematopoietic maturation stages in a PSC differentiation scheme (iR9-PSC) in vitro induced preferential generation of engraftable hematopoietic progenitors capable of homing to thymus and developing into mature T cells in primary and secondary immunodeficient recipients. Single-cell transcriptome and functional analyses illustrated the cellular trajectory of T lineage induction from PSCs, unveiling the T-lineage specification determined at as early as hemogenic endothelial cell stage and identifying the bona fide pre-thymic progenitors. The induced T cells distributed normally in central and peripheral lymphoid organs and exhibited abundant TCRαß repertoire. The regenerative T lymphopoiesis restored immune surveillance in immunodeficient mice. Furthermore, gene-edited iR9-PSCs produced tumor-specific T cells in vivo that effectively eradicated tumor cells. This study provides insight into universal generation of functional and therapeutic T cells from the unlimited and editable PSC source.

Synergy of NUP98-HOXA10 Fusion Gene and NrasG12D Mutation Preserves the Stemness of Hematopoietic Stem Cells on Culture Condition.

Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.

Publisher Correction: Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes.

In the version of this article initially published, some identification of the supplementary information was incorrect. The items originally called Supplementary Tables 1, 2, 3, 4 and 5 should be Source Data Figures 1, 2, 4, 5 and 7, respectively; those originally called Supplementary Tables 6, 7 and 8 should be Supplementary Tables 1, 2 and 3, respectively; and those originally called Source Data Figures 1, 2, 4, 5 and 7 should be Supplementary Tables 4, 5, 6, 7 and 8, respectively. The errors have been corrected in the HTML version of the article.

Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes.

Deletion of master regulators of the B cell lineage reprograms B cells into T cells. Here we found that the transcription factor Hoxb5, which is expressed in uncommitted hematopoietic progenitor cells but is not present in cells committed to the B cell or T cell lineage, was able to reprogram pro-pre-B cells into functional early T cell lineage progenitors. This reprogramming started in the bone marrow and was completed in the thymus and gave rise to T lymphocytes with transcriptomes, hierarchical differentiation, tissue distribution and immunological functions that closely resembled those of their natural counterparts. Hoxb5 repressed B cell 'master genes', activated regulators of T cells and regulated crucial chromatin modifiers in pro-pre-B cells and ultimately drove the B cell fate-to-T cell fate conversion. Our results provide a de novo paradigm for the generation of functional T cells through reprogramming in vivo.

Anti-herpes simplex virus type 1 activity of Houttuynoid A, a flavonoid from Houttuynia cordata Thunb.

Early events in herpes simplex virus type 1 (HSV-1) infection reactivate latent human immunodeficiency virus, Epstein-Barr virus, and human papillomavirus in the presence of acyclovir (ACV). The common use of nucleoside analog medications, such as ACV and pencyclovir, has resulted in the emergence of drug-resistant HSV-1 strains in clinical therapy. Therefore, new antiherpetics that can inhibit early events in HSV-1 infection should be developed. An example of this treatment is Houttuynia cordata Thunb. water extract, which can inhibit HSV-1 infection through multiple mechanisms. In this study, the anti-HSV-1 activity of Houttuynoid A, a new type of flavonoid isolated from H. cordata, was investigated. Three different assays confirmed that this compound could exhibit strong in vitro anti-HSV-1 activity. One assay verified that this compound could inhibit HSV-1 multiplication and prevent lesion formation in a HSV-1 infection mouse model. Mechanism analysis revealed that this compound could inactivate HSV-1 infectivity by blocking viral membrane fusion. Moreover, Houttuynoid A exhibited antiviral activities against other alpha herpes viruses, such as HSV-2 and varicella zoster virus (VZV). In conclusion, Houttuynoid A may be a useful antiviral agent for HSV-1.

Pyrimidoindole derivative UM171 enhances derivation of hematopoietic progenitor cells from human pluripotent stem cells.

In the field of hematopoietic regeneration, deriving hematopoietic stem cells (HSCs) from pluripotent stem cells with engraftment potential is the central mission. Unstable hematopoietic differentiation protocol due to variation factors such as serums and feeder cells, remains a major technical issue impeding the screening of key factors for the derivation of HSCs. In combination with hematopoietic cytokines, UM171 has the capacity to facilitate the maintenance and expansion of human primary HSCs in vitro. Here, using a serum-free, feeder-free, and chemically defined induction protocol, we observed that UM171 enhanced hematopoietic derivation through the entire process of hematopoietic induction in vitro. UM171 facilitated generation of robust CD34+CD45+ derivatives that formed more and larger sized CFU-GM as well as larger sized CFU-Mix. In our protocol, the derived hematopoietic progenitors failed to engraft in NOG mice, indicating the absence of long-term HSC from these progenitors. In combination with other factors and protocols, UM171 might be broadly used for hematopoietic derivation from human pluripotent stem cells in vitro.

Midazolam anesthesia protects neuronal cells from oxidative stress-induced death via activation of the JNK-ERK pathway.

Midazolam is an anesthetic agent commonly used during clinical and surgical procedures, which has been shown to exert ROS­suppressing and apoptosis­modulating pharmacological activities in various cellular systems. However, the effects of midazolam on oxidative stress in neuronal cells require elucidation. The present study investigated the effects of midazolam on buthionine sulfoximine (BSO)­ and hydrogen peroxide (H2O2)­induced oxidative stress in primary cortical neuronal cells. In addition, the effects of midazolam on middle cerebral artery occlusion (MCAO) in mice and on ethanol­induced neuroapoptosis in the brains of neonatal mice were determined. Subsequently, cell viability was detected using the MTT assay; intracellular reactive oxygen species (ROS) generation was determined using the 2',7'­dichlorodihydrofluorescein diacetate method with confocal microscopy; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was conducted to detect apoptotic cells; immunohistochemistry was performed to detect activated caspase­3; neuronal deficit and infarct volume analyses were conducted; and quantitative polymerase chain reaction and western blotting were performed to detect the expression levels of genes and proteins associated with apoptosis and cell survival pathways. The results demonstrated that BSO (10 mM) and H2O2 (1 mM) suppressed proliferation of cortical neuronal cells by inducing apoptosis. These effects were suppressed following treatment with midazolam in a dose­dependent manner. In addition, BSO and H2O2 induced ROS generation in neuronal cells; however, this was effectively suppressed by midazolam (100 µM). Beneficial synergistic effects were detected when midazolam was used in combination with the known antioxidant trolox. BSO and H2O2 also suppressed the protein expression levels of c­Jun N­terminal kinases (JNK), phosphorylated (p)JNK, extracellular signal­regulated kinases (ERK)1/2, pERK1/2, AKT and nuclear factor­κB; however, expression was recovered following treatment with midazolam. Midazolam also activated protein kinase C­Îµ, which was suppressed by BSO, in cortical neuronal cells. In MCAO mice, midazolam post­conditioning significantly suppressed infarct size and reduced the number of TUNEL­positive cells. In addition, the expression levels of caspase­3 and poly (ADP­ribose) polymerase were suppressed in a dose­dependent manner. In neonatal mice, midazolam reduced ethanol­induced activated caspase­3 staining and apoptotic TUNEL staining. The results of the present study demonstrated that midazolam may protect against neuronal degeneration and neuroapoptosis induced by physiological and oxidative stress.

Electrophoretic deposition of chitosan/gelatin coatings with controlled porous surface topography to enhance initial osteoblast adhesive responses.

Electrophoretically deposited (EPD) coatings have often been employed recently for the addition of different new chemical compositions to classic chitosan coatings to improve the biocompatibility and therapeutic potential of coated implants. However, little attention has been paid to enhance the cell response to EPD coatings via integrating the effects of chemical components and surface topography. Here, we fabricated EPD chitosan/gelatin (CS/G) coatings with controlled porous surface topography by controlling bubble generation in the EPD process via changing the gelatin content in solution from 0, 0.01, 0.1, and 1 to 10 mg ml-1. The pure chitosan coating surface was characterized by homogeneous large pores of 500 µm. After 0.01 mg ml-1 gelatin was added, 180 µm small pores appeared on the walls of large pores. As the gelatin content increased to 0.1 mg ml-1, a number of small pores increased noticeably. When the gelatin content reached 1 mg ml-1, large pores disappeared, and the coating displayed homogeneous small pores. 10 mg ml-1 gelatin concentration led to coatings consisting of small pores with not integral and continuous structures. The initial osteoblastic responses, including cell adherence progress, spreading area, proliferation rate, and focal adhesion-related gene expression, gradually improved from 0 to 0.01, 0.1, and 1 mg ml-1 gelatin content, but decreased from 1 to 10 mg ml-1. All these results indicated that the initial cell responses to coatings reached a peak when it was 1 mg ml-1 gelatin and they had homogeneous small pores, which might contribute to the synergistic effects of the porous surface structure and components. This work would be beneficial for expanding the potential application of EPD coatings.