Aberrantly high activation of oncogenic transcription factors has been implicated in initiation and progression of malignant diseases. However, the landscape of dysregulated TFs in HNSCC remains poorly characterized, and especially their biological contributions to remodeling of cancer immune microenvironment are unknown. Here, by globally investigating oncogenic TF-target interactions in clinic, we identified that KLF7 is one of the most potential oncogenic TFs in HNSCC. In vitro and in vivo experiments showed that KLF7 governs not only the autonomous malignant behaviors but also recruitment of macrophage in the cancer microenvironment, consequently promoting progression of HNSCC in a tumor-associated macrophage (TAM) dependent manner. Mechanistically, we found that LOX is a bona fide target directly transcriptionally activated by KLF7 in HNSCC cells. In vivo assay showed that LOX-driven crosslinking of extracellular matrix conducted a stiff extracellular matrix environment for macrophage recruitment and consequent disruption of CD8+ T cell mediated killing effect in cancer microenvironment. More importantly, we elucidated the clinical significance of the correlation of KLF7, LOX and TAM in HNSCCs. Our study reveals an extrinsic mechanism of the oncogenic role of KLF7 and further demonstrates that a KLF7/LOX/TAM signaling could be a potential therapeutic vulnerability for HNSCC patients.

Inflammatory bowel disease (IBD) is prevalent, and no satisfactory therapeutic options are available because the mechanisms underlying its development are poorly understood. In this study, we discovered that increased expression of methyltransferase-like 3 (METTL3) in macrophages was correlated with the development of colitis and that depletion of METTL3 in macrophages protected mice against dextran sodium sulfate (DSS)-induced colitis. Mechanistic characterization indicated that METTL3 depletion increased the YTHDF3-mediated expression of phosphoglycolate phosphatase (PGP), which resulted in glucose metabolism reprogramming and the suppression of CD4+ T helper 1 (Th1) cell differentiation. Further analysis revealed that glucose metabolism contributed to the ability of METTL3 depletion to ameliorate colitis symptoms. In addition, we developed two potent small molecule METTL3 inhibitors, namely, F039-0002 and 7460-0250, that strongly ameliorated DSS-induced colitis. Overall, our study suggests that METTL3 plays crucial roles in the progression of colitis and highlights the potential of targeting METTL3 to attenuate intestinal inflammation for the treatment of colitis.

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive malignancy characterized by the aberrant accumulation of immature and dysfunctional B cells in bone marrow (BM). Although chemotherapy and other therapies have been widely applied, some patients such as relapsed or drug-refractory (R/R) B-ALL patients exhibit limited response. YT521-B homologous domain-containing protein 1 (YTHDC1) is a nuclear reader of N6-methyladenosine (m6A) RNA modification, which has been implicated in different malignancies including leukemia. In the current study, we show that YTHDC1 is highly expressed in B-ALL cells. YTHDC1 knockdown attenuated B-ALL cell proliferation and cell cycle progression in vitro, and prolonged survival of mice in the human B-ALL xenograft model in vivo attributable to compromised leukemogenesis. Mechanistically, YTHDC1 knockdown significantly increased the accumulation of endogenous and chemotherapeutic agents-induced DNA damage in B-ALL cells. Furthermore, we identified that YTHDC1 binds to and stabilizes m6A-modified KMT2C mRNA. KMT2C is a key enzyme catalyzing histone H3K4 methylation required for the expression of DNA damage response (DDR)-related genes, implying that YTHDC1 inhibitors might improve chemotherapy by attenuating DDR via reducing KMT2C. Indeed, with molecular docking and biochemical experiments, we identified EPZ-5676 as a YTHDC1 inhibitor, and combination of EPZ-5676 with Cytarabine (Ara-c) significantly improved the efficacy of chemotherapy in B-ALL mouse models using YTHDC1high primary and lined B-ALL cells. Collectively, YTHDC1 is required for DDR in B-ALL cells by upregulating DDR-related gene expression via stabilizing m6A-modified KMT2C mRNA, thereby leading to increased histone H3K4 methylation, and targeted inhibition of YTHDC1 is a potentially new therapeutic strategy against B-ALL, especially YTHDC1high B-ALL.

Inherited and age-related retinal degenerations are the commonest causes of blindness without effective treatments. Retinal progenitor cells (RPCs), which have the multipotency to differentiate into various retinal cell types, are regarded as a promising source of cell transplantation therapy for retinal degenerative diseases. However, the self-limited expansion of RPCs causes difficulty in cell source supply and restrict its clinical treatment. In this work, we found that inhibition of microRNA-449a (miR-449a) in RPCs can promote proliferation and inhibit apoptosis of RPCs, partially through upregulating Notch signaling. Further optimization of transduction miR-449a inhibitor into RPCs by endothelial cell-derived exosomes can promote the survival of RPCs transplanted in vivo and reduce cell apoptosis in retinal degeneration mouse models. In summary, these studies have shown that exosome-miR-449a inhibitor can effectively promote the expansion of RPCs in vitro and enhance transplanted RPCs survival in vivo, which might provide a novel intervention strategy for retinal degenerations in the future.

Supplementary Figures 1-12, Tables 1-2 from Notch Signaling Determines the M1 versus M2 Polarization of Macrophages in Antitumor Immune Responses

Alzheimer’s disease (AD) is a heterogeneous disease with complex clinicopathological characteristics. To date, the role of m6A RNA methylation in monocyte-derived macrophages involved in the progression of AD is unknown. In our study, we found that methyltransferase-like 3 (METTL3) deficiency in monocyte-derived macrophages improved cognitive function in an amyloid beta (Aβ)-induced AD mouse model. The mechanistic study showed that that METTL3 ablation attenuated the m6A modification in DNA methyltransferase 3A (Dnmt3a) mRNAs and consequently impaired YTH N6-methyladenosine RNA binding protein 1 (YTHDF1)-mediated translation of DNMT3A. We identified that DNMT3A bound to the promoter region of alpha-tubulin acetyltransferase 1 (Atat1) and maintained its expression. METTL3 depletion resulted in the down-regulation of ATAT1, reduced acetylation of α-tubulin and subsequently enhanced migration of monocyte-derived macrophages and Aβ clearance, which led to the alleviated symptoms of AD. Collectively, our findings demonstrate that m6A methylation could be a promising target for the treatment of AD in the future.

With the better understanding of human genome and genetic contributions to human complex diseases, more and more clinical genetic tests have been developed for the prevention, diagnosis, and treatment of human diseases. This has raised new challenges to classroom teaching of medical genetics (MG) in medical education of undergraduates, who are relatively naive about the theory and technology of MG. A clear understanding of genotype-phenotype relationships is critical in MG learning. In the current study, we incorporated a practical approach of classroom MG teaching by using alcohol tolerance, a phenotype that is widely experienced by students and closely related to genetic polymorphism. To verify this teaching approach, which we named as student experience-based teaching (SEBT), undergraduates of two classes were recruited, with one accepting SEBT and the other accepting conventional lecture-based teaching (LBT). Final examination of the MG course indicated that while the two classes showed similar results to multiple-choice questions, the SEBT class showed significantly better performance to short-answer questions about gene polymorphism and phenotype, suggesting that SEBT might improve understanding. Thus, using student experience-based phenotypic instances and explaining the phenotype by practical genetic tests could improve understanding of genotype-phenotype relationships in classroom MG teaching.

B cell development in bone marrow (BM) is a multi-staged process involving pro-B, pre-B, immature B, and mature B cells, among which pre-B cells undergo vigorous proliferation, differentiation, apoptosis, and gene rearrangement. While several signaling pathways participate in pre-B cell development have been clarified, detailed intrinsic mechanisms regulating pre-B cell proliferation and survival have not been fully understood. In the current study, we report that miR-582 regulates pre-B cell proliferation and survival. miR-582 is enriched in pre-B cells. Deletion of miR-582 in mice expanded the BM pre-B cell population in a cell-autonomous manner as shown by competitive BM transplantation. We show that forced miR-582 overexpression inhibited pre-B cell proliferation and survival, whereas downregulation of miR-582 by siRNA significantly promoted pre-B cell proliferation and survival in vitro. We identified that Hif1α and Rictor are authentic targets of miR-582 in pre-B cells as shown by reporter assays. Moreover, miR-582 overexpression reduced the expression of Hif1α and its downstream molecule Glut1, as well as Rictor and mTORC2 activity as shown by attenuated AKT and FoxO1 phosphorylation, while miR-582 knockdown showed opposite effects. miR-582 knockdown-induced increases in pre-B proliferation and survival was abrogated by Hif1α and Rictor inhibitors. Together, miR-582 functions as a negative regulator of pre-B cell proliferation and survival by simultaneously targeting Hif1α and mTORC2 signaling that regulates metabolism in early B cell development.

自闭症谱系障碍(ASD)是一种具有明显遗传倾向的神经发育障碍疾病,其主要症状是社会交往障碍,语言交流障碍和重复刻板行为.近些年来,ASD的患病率表现为逐年上升,以儿童和青少年居多,给家庭和社会带来了沉重的教育与经济负担,全社会对于ASD的发病机制以及临床遗传检测日益关注.近些年来由于高通量测序技术的大规模应用及诱导多能干细胞技术的发展,在分子水平发现了许多新的ASD易感基因以及非编码突变,基于这些突变建立了新的ASD遗传检测手段;同时也借助ASD病人来源的诱导多能干细胞进行了发病机制的深入研究.本文将从ASD相关的基因组变异、遗传检测及ASD干细胞模型研究三方面的进展进行综述,希望进一步提高全社会对ASD的发病机制及遗传特征的认识,从而更有效地预防和治疗ASD.

Epigenetic regulations on the maintenance of neural stem cells (NSCs) are complicated and far from been fully understood. Our previous findings have shown that after blocking Notch signaling in NSCs in vivo, the stemness of NSCs decreases, accompanied by the downregulated expression of miR-582-5p. In the current study, we further investigated the function and mechanism of miR-582-5p in the maintenance of NSCs in vitro and in vivo. After transfecting a mimic of miR-582-5p, the formation of neurospheres and proliferation of NSCs and intermediate progenitor cells (NS/PCs) were enhanced, and the expression of stemness markers such as Sox2, Nestin, and Pax6 also increased. The results were reversed after transfection of an inhibitor of miR-582-5p. We further generated miR-582 knock-out (KO) mice to investigate its function in vivo, and we found that the number of NSCs in the subventricular zone (SVZ) region decreased and the number of neuroblasts increased in miR-582 deficient mice, indicating reduced stemness and enhanced neurogenesis of NSCs. Moreover, RNA-sequencing and molecular biological analysis revealed that miR-582-5p regulates the stemness and proliferation of NSCs by inhibiting secretory protein FAM19A1. In summary, our research uncovered a new epigenetic mechanism that regulates the maintenance of NSCs, therefore providing novel targets to amplify NSCs in vitro and to promote neurogenesis in vivo during brain pathology and aging.

以学生为主体的互动式教学模式越来越被高等教育界认可,我们把自主提问驱动的基于问题学习法(PBL)专题讨论课引入基础医学教学,让学生为兴趣所驱动,在教师的辅导下,展开自主学习与课堂讨论.学生的课堂表现、过程性评价成绩及问卷反馈显示,自主提问驱动的PBL专题讨论显著提升了教学效果,在未来的实践中需要进一步建设指导教师团队和配套学习资源.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a malignancy characterized by the aberrant accumulation of immature B-cell precursors in bone marrow and other lymphoid organs. Although several intrinsic regulatory signals participating in BCP-ALL have been clarified, detailed intrinsic and extrinsic mechanisms that regulate BCP-ALL progression have not been fully understood. In the current study, we report that miR-582 is downregulated in BCP-ALL cells compared with normal B cells. Forced overexpression of miR-582 attenuated BCP-ALL cell proliferation and survival. We found that miR-582 overexpression disturbed the mitochondrial metabolism of BCP-ALL cells, leading to less ATP but more ROS production. Mechanistically, we identified PPTC7 as a direct target of miR-582. MiR-582 overexpression inhibited the activity of CoQ10, which is downstream of PPTC7 and played an important positive regulatory role in mitochondrial electron transportation. Finally, we found that overexpression of miR-582 upregulated the expression of immune checkpoint molecule CD276 and reduced NK cell-mediated cytotoxicity against BCP-ALL cells. CD276 blockade significantly increased NK cell-mediated cytotoxicity against miR-582-overexpressing BCP-ALL cells. Together, our research demonstrates that miR-582 acts as a negative regulator of BCP-ALL cells by reducing proliferation and survival, but protects BCP-ALL cells from NK cell-mediated cytotoxicity, suggesting that miR-582 may be a new therapeutic biomarker for BCP-ALL with CD276 blocker.

When seeded in small numbers in medium containing 10−6 M aminopterin and fetal calf serum, V79 Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10−6 M aminopterin and dialyzed serum, provided that the medium was supplemented with 10−5 M hypoxanthine and sufficient 5·10−6 M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V79 cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT+ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT+ cells to prevent the growth of the latter in HAT medium. One of the HGPRT− lines showed a high rate of spontaneous reversion (118/105 cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to 4105 cells, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.

Metabolic reprogramming is one of the most critical hallmarks in cancer cells. In the past decades, mounting evidence has demonstrated that, besides the Warburg Effect, lipid metabolism dysregulation is also one of the essential characteristics of cancer cell metabolism. Lipids are water-insoluble molecules with diverse categories of phosphoglycerides, triacylglycerides, sphingolipids, sterols, etc. As the major utilization for energy storage, fatty acids are the primary building blocks for synthesizing triacylglycerides. And phosphoglycerides, sphingolipids, and sterols are the main components constructing biological membranes. More importantly, lipids play essential roles in signal transduction by functioning as second messengers or hormones. Much evidence has shown specific alterations of lipid metabolism in cancer cells. Consequently, the structural configuration of biological membranes, the energy homeostasis under nutrient stress, and the abundance of lipids in the intracellular signal transduction are affected by these alterations. Furthermore, lipid droplets accumulate in cancer cells and function adaptively to different types of harmful stress. This chapter reviews the regulation, functions, and therapeutic benefits of targeting lipid metabolism in cancer cells. Overall, this chapter highlights the significance of exploring more potential therapeutic strategies for malignant diseases by unscrambling lipid metabolism regulation in cancer cells.

Formation of glioma stem cells (GSCs) is considered as one of the main reasons of temozolomide (TMZ) resistance in glioma patients. Recent studies have shown that tumor microenvironment-derived signals could promote GSCs formation. But the critical molecule and underlying mechanism for GSCs formation after TMZ treatment is not entirely identified. Our study showed that TMZ treatment promoted GSCs formation by glioma cells; TMZ treatment of biopsy-derived glioblastoma multiforme cells upregulated HMGB1; HMGB1 altered gene expression profile of glioma cells with respect to mRNA, lncRNA and miRNA. Furthermore, our results showed that TMZ-induced HMGB1 increased the formation of GSCs and when HMGB1 was downregulated, TMZ-mediated GSCs formation was attenuated. Finally, we showed that the effect of HMGB1 on glioma cells was mediated by TLR2, which activated Wnt/β-catenin signaling to promote GSCs. Mechanistically, we found that HMGB1 upregulated NEAT1, which was responsible for Wnt/β-catenin activation. In conclusion, TMZ treatment upregulates HMGB1, which promotes the formation of GSCs via the TLR2/NEAT1/Wnt pathway. Blocking HMGB1-mediated GSCs formation could serve as a potential therapeutic target for preventing TMZ resistance in GBM patients.

The quiescence, activation, and subsequent neurogenesis of neural stem cells (NSCs) play essential roles in the physiological homeostasis and pathological repair of the central nervous system. Previous studies indicate that transmembrane protein Ttyh1 is required for the stemness of NSCs, whereas the exact functions in vivo and precise mechanisms are still waiting to be elucidated. By constructing Ttyh1-promoter driven reporter mice, we determined the specific expression of Ttyh1 in quiescent NSCs and niche astrocytes. Further evaluations on Ttyh1 knockout mice revealed that Ttyh1 ablation leads to activated neurogenesis and enhanced spatial learning and memory in adult mice (6–8 weeks). Correspondingly, Ttyh1 deficiency results in accelerated exhaustion of NSC pool and impaired neurogenesis in aged mice (12 months). By RNA-sequencing, bioinformatics and molecular biological analysis, we found that Ttyh1 is involved in the regulation of calcium signaling in NSCs, and transcription factor NFATc3 is a critical effector in quiescence versus cell cycle entry regulated by Ttyh1. Our research uncovered new endogenous mechanisms that regulate quiescence versus activation of NSCs, therefore provide novel targets for the intervention to activate quiescent NSCs to participate in injury repair during pathology and aging.

Background Glioma stem cells (GSCs) are glioma cells with stemness and are responsible for a variety of malignant behaviors of glioma. Evidence has shown that signals from tumor microenvironment (TME) enhance stemness of glioma cells. However, identification of the signaling molecules and underlying mechanisms has not been completely elucidated. Methods Human samples and glioma cell lines were cultured in vitro to determine the effects of adenovirus (ADV) infection by sphere formation, RT-qPCR, western blotting, FACS and immunofluorescence. For in vivo analysis, mouse intracranial tumor model was applied. Bioinformatics analysis, gene knockdown by siRNA, RT-qPCR and western blotting were applied for further mechanistic studies. Results Infection of patient-derived glioma cells with ADV increases the formation of tumor spheres. ADV infection upregulated stem cell markers and in turn promoted the capacities of self-renewal and multi-lineage differentiation of the infected tumor spheres. These ADV infected tumor spheres had stronger potential to form xenograft tumors in immune-compromised mice. GSCs formation could be promoted by ADV infection via TLR9, because TLR9 was upregulated after ADV infection, and knockdown of TLR9 reduced ADV-induced GSCs. Consistently, MYD88, as well as total STAT3 and phosphorylated (p-)STAT3, were also upregulated in ADV-induced GSCs. Knockdown of MYD88 or pharmaceutical inhibition of STAT3 attenuated stemness of ADV-induced GSCs. Moreover, we found that ADV infection upregulated lncRNA NEAT1. Knockdown of NEAT1 impaired stemness of ADV-induced GSCs. Lastly, HMGB1, a damage associated molecular pattern (DAMP) that triggers TLR signaling, also upregulated stemness markers in glioma cells. Conclusion ADV, which has been developed as vectors for gene therapy and oncolytic virus, promotes the formation of GSCs via TLR9/NEAT1/STAT3 signaling.

Malformation of blood vessels represents a hallmark of cancers, but the role and regulation of vascular mural cells (vMCs), including vascular smooth muscle cells (vSMCs) and pericytes, in tumors has not been fully understood. SM22α has been identified as a marker of vSMCs. This study aims at elucidating the function and regulation of SM22α+ mural cells (SM22-MCs) in tumor stroma. Gene-modified mice with a SM22α-CreERT2 transgene were employed to deplete SM22-MCs or activate/block Notch signaling in these cells. vSMCs from mouse dorsal aorta (vSMCs-DA) were cultured in vitro. RNA-seq was used to compare gene expression profiles. qRT-PCR and western blotting were used to determine gene expression level. Immunofluorescence was used to observe morphological alterations in tumors. SM22-MCs are essential for stabilizing tumor vasculature. Notch signaling was downregulated in tumor-derived SM22-MCs and vSMCs-DA treated with cancer cell-derived conditioned medium. Notch activation in SM22-MCs normalized tumor vasculature and repressed tumor growth. On the other hand, Notch disruption aggravated abnormal tumor vasculature and promoted growth and metastasis. Gene expression profiling of vSMCs-DA showed that Notch activation enhances their contractile phenotype and suppresses their secretory phenotype, further attenuating the invasion and proliferation of tumor cells. In contrast, Notch blockade in vSMCs-DA mitigated their contractile phenotype while strengthened the secretory phenotype. SM22-MCs facilitate vessel stability in tumors, and they gain a secretory phenotype and promote tumor malignancy in the absence of Notch signaling.

Teacher-led classical instruction lacks interactions feedbacks and thus leads to a low learning efficiency. Serving as advanced educational concept and method, outcome-based education (OBE) and BOPPPS mode gradually become the leading concept in the remodeling of basic medical education. In order to improve the quality of microbial genetics teaching, this article aims to summarize the methods and experience of applying BOPPPS mode to microbial genetics directed by OBE, which hopefully stimulates medical students' interests of initiative learning and active exploration, cultivates students' ability of raising and resolving questions, develops the awareness of cooperative learning, and finally achieves the goal of improved classroom learning.

Tweety-homolog 1 (Ttyh1) is expressed in neural tissue and has been implicated in the generation of several brain diseases. However, its functional significance in pain processing is not understood. By disrupting the gene encoding Ttyh1, we found a loss of Ttyh1 in nociceptors and their central terminals in Ttyh1-deficient mice, along with a reduction in nociceptor excitability and synaptic transmission at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) in the basal state. More importantly, the peripheral inflammation-evoked nociceptor hyperexcitability and spinal synaptic potentiation recorded in spinal-PAG projection neurons were compromised in Ttyh1-deficient mice. Analysis of the paired-pulse ratio and miniature excitatory postsynaptic currents indicated a role of presynaptic Ttyh1 from spinal nociceptor terminals in the regulation of neurotransmitter release. Interfering with Ttyh1 specifically in nociceptors produces a comparable pain relief. Thus, in this study we demonstrated that Ttyh1 is a critical determinant of acute nociception and pain sensitization caused by peripheral inflammation.