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.

Epigenomic diversity of cortical projection neurons in the mouse brain.

Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain1, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

Ganoderma triterpenoids attenuate tumour angiogenesis in lung cancer tumour-bearing nude mice.

CONTEXT: Ganoderma lucidum (Leyss. ex Fr.) Karst. (Polyporaceae) triterpenoids (GLTs), the main components and bioactive metabolites of G. lucidum, have antitumour activity. OBJECTIVE: We investigated the effects of GLTs in lung cancer tumour-bearing nude mice and their potential mechanism. MATERIALS AND METHODS: Forty BALB/c nude mice were randomly divided into four groups: saline control, GLT (1 g/kg/day), gefitinib (GEF, 15 mg/kg/day), and GLT (1 g/kg/day) + GEF (15 mg/kg/day) for 14 days. Cell viability was conducted using the Cell Counting Kit-8 assay. The tumour volume, inhibition rate, histopathological, microvessel density (MVD), mRNAs, and proteins were determined. RESULTS: GLTs inhibited the cell viability of A549 cells with an IC50 value of 14.38 ± 0.29 mg/L, while the IC50 value of GEF was 10.26 ± 0.47 µmol/L. The tumour inhibition rate in the GLT + GEF group (51.54%) was significantly decreased relative to the saline control… group (p < 0.05). The MVD in the GLT + GEF group (2.9 ± 0.7) was significantly decreased than that in the saline control group (12.8 ± 1.4, p < 0.05). The angiostatin, endostatin, and Bax protein expression in the GLT, GEF, and GLT + GEF groups were significantly increased compared to those in the saline control group, while the VEGFR2 and Bcl-2 protein expression were decreased. DISCUSSION AND CONCLUSIONS: Our study provided evidence that GLT and GEF combination therapy may be a promising candidate for the treatment of lung cancer and as an experimental basis for clinical treatment.

Ubiquitinated CD36 sustains insulin-stimulated Akt activation by stabilizing insulin receptor substrate 1 in myotubes.

Both the magnitude and duration of insulin signaling are important in executing its cellular functions. Insulin-induced degradation of insulin receptor substrate 1 (IRS1) represents a key negative feedback loop that restricts insulin signaling. Moreover, high concentrations of fatty acids (FAs) and glucose involved in the etiology of obesity-associated insulin resistance also contribute to the regulation of IRS1 degradation. The scavenger receptor CD36 binds many lipid ligands, and its contribution to insulin resistance has been extensively studied, but the exact regulation of insulin sensitivity by CD36 is highly controversial. Herein, we found that CD36 knockdown in C2C12 myotubes accelerated insulin-stimulated Akt activation, but the activated signaling was sustained for a much shorter period of time as compared with WT cells, leading to exacerbated insulin-induced insulin resistance. This was likely due to enhanced insulin-induced IRS1 degradation after CD36 knockdown. Overexpression of WT CD36, but not a ubiquitination-defective CD36 mutant, delayed IRS1 degradation. We also found that CD36 functioned through ubiquitination-dependent binding to IRS1 and inhibiting its interaction with cullin 7, a key component of the multisubunit cullin-RING E3 ubiquitin ligase complex. Moreover, dissociation of the Src family kinase Fyn from CD36 by free FAs or Fyn knockdown/inhibition accelerated insulin-induced IRS1 degradation, likely due to disrupted IRS1 interaction with CD36 and thus enhanced binding to cullin 7. In summary, we identified a CD36-dependent FA-sensing pathway that plays an important role in negative feedback regulation of insulin activation and may open up strategies for preventing or managing type 2 diabetes mellitus.

Integrative Multi-omics Analysis to Characterize Herpes Virus Infection Increases the Risk of Alzheimer's Disease.

Evidence suggests that herpes virus infection is associated with an increased risk of Alzheimer's disease (AD), and innate and adaptive immunity plays an important role in the association. Although there have been many studies, the mechanism of the association is still unclear. This study aims to reveal the underlying molecular and immune regulatory network through multi-omics data and provide support for the study of the mechanism of infection and AD in the future. Here, we found that the herpes virus infection significantly increased the risk of AD. Genes associated with the occurrence and development of AD and genetically regulated by herpes virus infection are mainly enrichment in immune-related pathways. The 22 key regulatory genes identified by machine learning are mainly immune genes. They are also significantly related to the infiltration changes of 3 immune cell in AD. Furthermore, many of these genes have previously been reported to be linked, or potentially linked, to the pathological mechanisms of both herpes virus infection and AD. In conclusion, this study contributes to the study of the mechanisms related to herpes virus infection and AD, and indicates that the regulation of innate and adaptive immunity may be an effective strategy for preventing and treating herpes virus infection and AD. Additionally, the identified key regulatory genes, whether previously studied or newly discovered, may serve as valuable targets for prevention and treatment strategies.

Hesperidin administration suppresses the proliferation of lung cancer cells by promoting apoptosis via targeting the miR­132/ZEB2 signalling pathway.

This aim of the present study was to identify the relationship between hesperidin and microRNA (miR)­132, and to study the role of hesperidin and miR­132 in the pathogenesis of non­small cell lung cancer (NSCLC). Computational analysis and luciferase assays were performed to identify the target of miR­132. Subsequently, reverse transcription­quantitative PCR and western blot assays were used to detect the effect of miR­132 and hesperidin on the expression of haematological and neurological expressed 1 (HN1) and zinc finger E­box binding homeobox 2 (ZEB2). Finally, MTT assays and flow cytometry analysis were used to investigate the effect of hesperidin on cell proliferation and apoptosis. ZEB2 was identified as a target gene of miR­132, and transfection with miR­132 mimic reduced the luciferase activity of the wild­type ZEB2 3'­untranslated region (3'­UTR) but not that of the mutant ZEB2 3'­UTR. By contrast, neither transfection with miR­132 mimic nor hesperidin treatment affected HN1 expression. Furthermore, hesperidin evidently inhibited cell proliferation and promoted apoptosis in a dose­dependent manner. Furthermore, the tumour volume in rats transplanted with NSCLC cells and treated with hesperidin was notably smaller compared with that in rats transplanted with NSCLC cells alone, while treatment with hesperidin significantly reduced the colony formation efficiency of NSCLC cells by increasing miR­132 expression and decreasing ZEB2 expression. To the best of our knowledge, the present study demonstrated for the first time that the administration of hesperidin decreased the expression of ZEB2 by upregulating the expression of miR­132, which in turn promoted apoptosis and inhibited the proliferation of NSCLC cells.