Dual-role transcription factors stabilize intermediate expression levels.

Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.

Induction of mouse totipotent stem cells by a defined chemical cocktail.

In mice, only the zygotes and blastomeres from 2-cell embryos are authentic totipotent stem cells (TotiSCs) capable of producing all the differentiated cells in both embryonic and extraembryonic tissues and forming an entire organism1. However, it remains unknown whether and how totipotent stem cells can be established in vitro in the absence of germline cells. Here we demonstrate the induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells using a combination of three small molecules: the retinoic acid analogue TTNPB, 1-azakenpaullone and the kinase blocker WS6. The resulting chemically induced totipotent stem cells (ciTotiSCs), resembled mouse totipotent 2-cell embryo cells at the transcriptome, epigenome and metabolome levels. In addition, ciTotiSCs exhibited bidirectional developmental potentials and were able to produce both embryonic and extraembryonic cells in vitro and in teratoma. Furthermore, following injection into 8-cell embryos, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach to totipotent stem cell induction and maintenance provides a defined in vitro system for manipulating and developing understanding of the totipotent state and the development of multicellular organisms from non-germline cells.

Non-viral approaches for somatic cell reprogramming into cardiomyocytes.

Heart disease is the leading cause of human deaths worldwide. Due to lacking cardiomyocytes with replicative capacity and cardiac progenitor cells with differentiation potential in adult hearts, massive loss of cardiomyocytes after ischemic events produces permanent damage, ultimately leading to heart failure. Cellular reprogramming is a promising strategy to regenerate heart by induction of cardiomyocytes from other cell types, such as cardiac fibroblasts. In contrast to conventional virus-based cardiac reprogramming, non-viral approaches greatly reduce the potential risk that includes disruption of genome integrity by integration of foreign DNAs, expression of exogenous genes with oncogenic potential, and appearance of partially reprogrammed cells harmful for the physiological functions of tissues/organs, which impedes their in-vivo applications. Here, we review the recent progress in development of non-viral approaches to directly reprogram somatic cells towards cardiomyocytes and their therapeutic application for heart regeneration.

Application of Fixation of Orbicularis Oculi Muscle and Orbital Septal Tissue Flap at the Upper Edge of Incision in Double Eyelid Plasty.

PURPOSE: In order to make the postoperative effect of open double eyelid more close to the physiological and anatomical structure of double eyelid, we improved the traditional open double eyelid operation according to the anatomical characteristics of the upper eyelid. We fixed part of the orbicularis oculi muscle above the incisal margin with the orbital septum flap to make the double eyelid formed after surgery more natural and beautiful. MATERIALS AND METHODS: A total of 76 patients who received open double blepharoplasty in department of plastic surgery from February 2019 to May 2022 were selected as this study objects, all of whom were female. Their ages ranged from 18 to 32 years, with a mean of (23.6 ± 5.2) years. The surgical method is open double blepharoplasty by fixing part of the orbicularis oculi muscle above the incisal margin with the flap of the orbital septum. RESULTS: In this study, all 76 patients underwent successful surgery, with an average operation time of (1.5 ± 0.2) h. The postoperative double eyelid curvature was smooth and the double eyelid width was basically symmetrical. In terms of the doctors' satisfaction evaluation of the postoperative effect, 64 cases were very satisfied and 12 cases were satisfied. In terms of patients' satisfaction evaluation of the postoperative effect, 60 patients were very satisfied, 15 patients were satisfied and 1 patient was dissatisfied. CONCLUSIONS: Through this study, we found that this surgical method invented by us has short operation time, good operation effect, few postoperative complications and high patient satisfaction, which is worthy of promotion and application in plastic surgery clinical practice. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to www.springer.com/00266 .

Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism.

Metabolism has been shown to integrate with epigenetics and transcription to modulate cell fate and function. Beyond meeting the bioenergetic and biosynthetic demands of T-cell differentiation, whether metabolism might control T-cell fate by an epigenetic mechanism is unclear. Here, through the discovery and mechanistic characterization of a small molecule, (aminooxy)acetic acid, that reprograms the differentiation of T helper 17 (TH17) cells towards induced regulatory T (iTreg) cells, we show that increased transamination, mainly catalysed by GOT1, leads to increased levels of 2-hydroxyglutarate in differentiating TH17 cells. The accumulation of 2-hydroxyglutarate resulted in hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which is essential for fate determination towards TH17 cells. Inhibition of the conversion of glutamate to α-ketoglutaric acid prevented the production of 2-hydroxyglutarate, reduced methylation of the Foxp3 gene locus, and increased Foxp3 expression. This consequently blocked the differentiation of TH17 cells by antagonizing the function of transcription factor RORγt and promoted polarization into iTreg cells. Selective inhibition of GOT1 with (aminooxy)acetic acid ameliorated experimental autoimmune encephalomyelitis in a therapeutic mouse model by regulating the balance between TH17 and iTreg cells. Targeting a glutamate-dependent metabolic pathway thus represents a new strategy for developing therapeutic agents against TH17-mediated autoimmune diseases.

The role of NPY2R/NFATc1/DYRK1A regulatory axis in sebaceous glands for sebum synthesis.

BACKGROUND: Sebaceous glands (SGs) synthesize and secret sebum to protect and moisturize the dermal system via the complicated endocrine modulation. Dysfunction of SG are usually implicated in a number of dermal and inflammatory diseases. However, the molecular mechanism behind the differentiation, development and proliferation of SGs is far away to fully understand. METHODS: Herein, the rat volar and mammary tissues with abundant SGs from female SD rats with (post-natal day (PND)-35) and without puberty onset (PND-25) were arrested, and conducted RNA sequencing. The protein complex of Neuropeptide Y receptor Y2 (NPY2R)/NPY5R/Nuclear factor of activated T cells 1 (NFATc1) was performed by immunoprecipitation, mass spectrum and gel filtration. Genome-wide occupancy of NFATc1 was measured by chromatin immunoprecipitation sequencing. Target proteins' expression and localization was detected by western blot and immunofluorescence. RESULTS: NPY2R gene was significantly up-regulated in volar and mammary SGs of PND-25. A special protein complex of NPY2R/NPY5R/NFATc1 in PND-25. NFATc1 was dephosphorylated and activated, then localized into nucleus to exert as a transcription factor in volar SGs of PND-35. NFATc1 was especially binding at enhancer regions to facilitate the distal SG and sebum related genes' transcription. Dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) contributed to NFATc1 phosphorylation in PND-25, and inactivated of DYRK1A resulted in NFATc1 dephosphorylation and nuclear localization in PND-35. CONCLUSIONS: Our findings unmask the new role of NPY2R/NFATc1/DYRK1A in pubertal SG, and are of benefit to advanced understanding the molecular mechanism of SGs' function after puberty, and provide some theoretical basis for the treatment of acne vulgaris from the perspective of hormone regulation.

Acetylated Rhamnose Triterpenoid Saponins from Glechoma longituba Analyzed by LC-Q-TOFMS.

The aim of the present work is to isolate a series of triterpene derivatives with rhamnosyl linking acetyl groups from Glechoma longituba according to the structural characteristics of previously described triterpene saponins. The extract ion chromatography spectrum of the crude extract of G. longituba was detected and analyzed by HPLC-HR-ESI-MS to determine possible components, and these metabolites were traced and separated by combining high-resolution mass spectrometry and predicted liquid chromatography retention time. Three 11α, 12α-epoxypentacyclic oleanolic acid triterpene saponins (glechomanosides H-J) and one ursane triterpene aldehyde saponin with a C-28 aldehyde group were isolated from G. longituba. The structure of these compounds was confirmed by NMR and compared with those of previously characterized compounds. The strategy described in this report enables a rapid, reliable, and complete analysis of glycoside compounds containing different numbers of acetyl groups at different positions on the sugar.

The structure and stability analysis of the pea seed legumin glycosylated by oligochitosan.

BACKGROUND: The functionality of pea proteins is relatively weak relative to that of soybean proteins, which limits the application of pea proteins in food and nutritional applications. Glycosylation is a promising approach to influence the protein structure and in turn change the functional properties of pea proteins. RESULTS: In this study, the effect of transglutaminase-induced oligochitosan glycosylation on the structural and functional properties of pea seed legumin was studied. Different oligochitosan-modified legumin complexes (OLCs) were prepared by applying different molar ratios of legumin to oligochitosan (1:1 to 1:4) induced by transglutaminase (10 U g-1 protein). Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), glucosamine, and free amino analysis showed that the legumin could be covalently bonded with the oligochitosan and were influenced by the applying dose of the oligochitosan. Infrared spectroscopy, fluorescence, and scanning electron microscopy analysis indicated that the structure of the different OLC samples could be changed to different extents. Moreover, although the emulsifying activity decreased, the emulsification stability, thermal stability, and in vitro digestive stability of the OLCs were remarkably improved relative to that of the untreated legumin. CONCLUSION: Oligochitosan glycosylation could change the structure of the legumin and consequently improve its emulsification stability, thermal stability, and in vitro digestive stability. This study will facilitate the legumin functionalization by the glycosylation approach to fabricate protein-oligochitosan complex for potential food and nutritional applications. © 2020 Society of Chemical Industry.

Synthesis and biological evaluation of novel millepachine derivative containing aminophosphonate ester species as novel anti-tubulin agents.

A new series of millepachine derivative containing aminophosphonate ester moieties were designed and synthesized, and evaluated for their anticancer activities using MTT assay. Among all the compounds, compound 9m exhibited the most potent cytotoxic activity against all tested human cancer cell lines including multidrug resistant phenotype, which inhibited cancer cell growth with IC50 values ranging from 0.85 to 3.09 µM, respectively. In addition, its low cytotoxicity toward human normal liver cells HL-7702 and sensitivity toward to doxorubicin or cisplatin-resistant cells indicated the possibility for cancer therapy. Furthermore, 9m significantly induced cell apoptosis and cell cycle arrest in G2/M phase and dramatically disrupted the microtubule organization. Moreover, a decrease in MMP, an increase in reactive oxygen species (ROS) generation and Bax/Bcl-2 ratio, accompanied by activated caspase-3 and -9, were observed in HepG-2 cells after incubation with 9m, indicating that the mitochondrial pathway was involved in the 9m-mediated apoptosis.

Progress in the reprogramming of somatic cells.

Pluripotent stem cells can differentiate into nearly all types of cells in the body. This unique potential provides significant promise for cell-based therapies to restore tissues or organs destroyed by injuries, degenerative diseases, aging, or cancer. The discovery of induced pluripotent stem cell (iPSC) technology offers a possible strategy to generate patient-specific pluripotent stem cells. However, because of concerns about the specificity, efficiency, kinetics, and safety of iPSC reprogramming, improvements or fundamental changes in this process are required before their effective clinical use. A chemical approach is regarded as a promising strategy to improve and change the iPSC process. Dozens of small molecules have been identified that can functionally replace reprogramming factors and significantly improve iPSC reprogramming. In addition to the prospect of deriving patient-specific tissues and organs from iPSCs, another attractive strategy for regenerative medicine is transdifferentiation-the direct conversion of one somatic cell type to another. Recent studies revealed a new paradigm of transdifferentiation: using transcription factors used in iPSC generation to induce transdifferentiation or called iPSC transcription factor-based transdifferentiation. This type of transdifferentiation not only reveals and uses the developmentally plastic intermediates generated during iPSC reprogramming but also produces a wide range of cells, including expandable tissue-specific precursor cells. Here, we review recent progress of small molecule approaches in the generation of iPSCs. In addition, we summarize the new concept of iPSC transcription factor-based transdifferentiation and discuss its application in generating various lineage-specific cells, especially cardiovascular cells.

Slow Cooling and Controlled Ice Nucleation Enabling the Cryopreservation of Human T Lymphocytes with Low-Concentration Extracellular Trehalose.

Cryopreservation of human T lymphocytes has become a key strategy for supporting cell-based immunotherapy. However, the effects of ice seeding on the cryopreservation of cells under relatively slow cooling have not been well researched. The cryopreservation strategy with a nontoxic, single-ingredient, and injectable cryoprotective solution remains to be developed. We conducted ice seeding for the cells in a solution of normal saline with 1% (v/v) dimethyl sulfoxide (Me2SO), 0.1 M trehalose, and 4% (w/v) human serum albumin (HSA) under different slow cooling rates. With the positive results, we further applied seeding in the solution of 0.2 M trehalose and 4% (w/v) HSA under the same cooling rates. The optimal concentration of trehalose in the Me2SO-free solutions was then investigated under the optimized cooling rate with seeding, with control groups without seeding, and in a freezing container. In vitro toxicity of the cryoprotective solutions to the cells was also tested. We found that the relative viability of cells (1% [v/v] Me2SO, 0.1 M trehalose and 4% [w/v] HSA) was improved significantly from 88.6% to 94.1% with ice seeding, compared with that without seeding (p < 0.05). The relative viability of cells (0.2 M trehalose and 4% [w/v] HSA) with seeding was significantly higher than that without seeding, 96.3% and 92.0%, respectively (p < 0.05). With no significant difference in relative viability between the solutions of 0.2 M trehalose or 0.3 M trehalose with 4% (w/v) HSA (92.4% and 94.6%, respectively, p > 0.05), the solution of 0.2 M trehalose and 4% (w/v) HSA was selected as the optimized Me2SO-free solution. This strategy could cryopreserve human T lymphocytes without any toxic cryoprotectant and boost the application of cell products in humans by intravenous injection, with the osmolality of the low-concentration cryoprotective solution close to that of human plasma.

The formation of phycocyanin-EGCG complex for improving the color protection stability exposing to light.

Phycocyanin (PC) is a natural pigment-protein complex in food dye applications. In this study, a phycocyanin-epigallocatechin gallate (EGCG) complex (PE) was prepared and the effects of EGCG on the structure and color stability of PC were evaluated. The fluorescence results showed that the binding number n was 62.1 ± 3.41 (EGCG/PC) and the binding constant K was 4.39 (±0.2) × 105 M-1, indicating a weak-binding interaction. Fourier transform-infrared analysis showed that EGCG caused structural changes in PC by partially uncoiling α-helix and increasing ß-sheet content. The EGCG induced a PC association at a reaction molar ratio above 40:1 (EGCG/PC). Moreover, EGCG protected phycocyanobilin against color fading, making PE more stable relative to PC under 8-days storage in light. This study provides a novel scheme to stabilize PC by forming a complex with polyphenols, which will facilitate the PC application as a natural blue pigment in food.

KDM4 inhibitor SD49-7 attenuates leukemia stem cell via KDM4A/MDM2/p21CIP1 axis.

Rationale: Traditional treatments for leukemia fail to address stem cell drug resistance characterized by epigenetic mediators such as histone lysine-specific demethylase 4 (KDM4). The KDM4 family, which acts as epigenetic regulators inducing histone demethylation during the development and progression of leukemia, lacks specific molecular inhibitors. Methods: The KDM4 inhibitor, SD49-7, was synthesized and purified based on acyl hydrazone Schiff base. The interaction between SD49-7 and KDM4s was monitored in vitro by surface plasma resonance (SPR). In vitro and in vivo biological function experiments were performed to analyze apoptosis, colony-formation, proliferation, differentiation, and cell cycle in cell sub-lines and mice. Molecular mechanisms were demonstrated by RNA-seq, ChIP-seq, RT-qPCR and Western blotting. Results: We found significantly high KDM4A expression levels in several human leukemia subtypes. The knockdown of KDM4s inhibited leukemogenesis in the MLL-AF9 leukemia mouse model but did not affect the survival of normal human hematopoietic cells. We identified SD49-7 as a selective KDM4 inhibitor that impaired the progression of leukemia stem cells (LSCs) in vitro. SD49-7 suppressed leukemia development in the mouse model and patient-derived xenograft model of leukemia. Depletion of KDM4s activated the apoptosis signaling pathway by suppressing MDM2 expression via modulating H3K9me3 levels on the MDM2 promoter region. Conclusion: Our study demonstrates a unique KDM4 inhibitor for LSCs to overcome the resistance to traditional treatment and offers KDM4 inhibition as a promising strategy for resistant leukemia therapy.

Succinylated ferritin as a novel nanocage-like vehicle of polyphenol: Structure, stability, and absorption analysis.

Ferritin, a protein with an 8-nm cage structure, can encapsulate and deliver bioactive molecules. In this study, succinylation was adopted to modify plant ferritin to fabricate succinylated red been ferritin (SRBF) at pH 8.0. The SRBF was retained as a cage-like shape (12 nm diameter), while its secondary structure was altered, rendering higher negative charge accompanies by decreased surface hydrophobicity. The SRBF also demonstrated favorable property of reversible assembly regulated by pH-transitions (pH 2.0/7.0), thus enabled successful encapsulation of epigallocatechin gallate (EGCG) for fabrication of EGCG-loaded SRBF complexes with a diameter of ~12 nm. Succinylation enhanced the thermal stabilities of ferritin and the embedded EGCG. Moreover, SRBF markedly improved the transport efficiency of EGCG in Caco-2 monolayers relative to EGCG and that encapsulated in unmodified ferritin. These findings have extended the succinylation reaction for the cage-like protein modification, and facilitated the usage of ferritin variant in delivery of bioactive molecules.

Fluorescent sensors for Ca(2+) and Pb(2+) based on binaphthyl derivatives.

Two novel binaphthyl compounds have been synthesized for the selective fluorescent recognition of Ca(2+) or Pb(2+). By introducing different terminal groups to the receptor unit, the fluorescence signals of the receptors are significantly changed: 1 is fluorescence enhancement for Ca(2+), 2 is fluorescence quenching for Pb(2+). The binding properties for metal ions were examined by the absorption and fluorescence spectra. The fluorescence intensity enhancement was ascribed to the complex formation between Ca(2+) and 1 which blocked the photo-induced electron transfer process.

Sox2 and Klf4 as the Functional Core in Pluripotency Induction without Exogenous Oct4.

Ectopic expression of Oct4, Sox2, Klf4, and c-Myc can reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs). For years, Oct4 has been considered the key reprogramming factor core of the four factors. Here, we challenge this view by reporting a core function of Sox2 and Klf4 in reprogramming. We found that polycistronic expression of Sox2 and Klf4 was sufficient to induce pluripotency in the absence of exogenous Oct4, and the stoichiometry of Sox2 and Klf4 was essential. Sox2 and Klf4 cooperatively bound across the genome, leading to epigenetic remodeling of their targets, including pluripotency genes and gradual activation of the pluripotency network. Interestingly, cells of different germ layer origins, fibroblasts (mesoderm) and neural progenitor cells (ectoderm), showed convergent reprogramming trajectories and similar efficiency. This work demonstrates a core function of Sox2 and Klf4 in pluripotency induction and shows that this mechanism is independent of germ layer origin.

Human pancreatic beta-like cells converted from fibroblasts.

Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes.

Conversion of human fibroblasts into functional cardiomyocytes by small molecules.

Reprogramming somatic fibroblasts into alternative lineages would provide a promising source of cells for regenerative therapy. However, transdifferentiating human cells into specific homogeneous, functional cell types is challenging. Here we show that cardiomyocyte-like cells can be generated by treating human fibroblasts with a combination of nine compounds that we term 9C. The chemically induced cardiomyocyte-like cells uniformly contracted and resembled human cardiomyocytes in their transcriptome, epigenetic, and electrophysiological properties. 9C treatment of human fibroblasts resulted in a more open-chromatin conformation at key heart developmental genes, enabling their promoters and enhancers to bind effectors of major cardiogenic signals. When transplanted into infarcted mouse hearts, 9C-treated fibroblasts were efficiently converted to chemically induced cardiomyocyte-like cells. This pharmacological approach to lineage-specific reprogramming may have many important therapeutic implications after further optimization to generate mature cardiac cells.