Immunometabolic remodeling drives adaptation to long-term high-altitude exposure (LTHAE), yet the underlying mechanisms remain elusive. By integrating single-cell transcriptomics and metabolomics from 46 lowlanders following a 90-day LTHAE, we identified a strategy of “innate activation and adaptive suppression.” Neutrophils exhibited enhanced maturation and phagocytosis, whereas adaptive immunity was dampened, characterized by suppressed B cell function and T cell responsiveness. Metabolically, LTHAE induced systemic shifts in steroid and amino acid metabolism associated with immune remodeling. Furthermore, multi-omics integration indicated a conserved upregulation of the glycolysis-TCA-OXPHOS axis across immune lineages, a metabolic adaptation supported by enzymatic assays in hematopoietic tissues of a hypobaric hypoxia mouse model. Collectively, these findings provide integrated insights into immune-metabolic landscape remodeling and suggest a potential mutual regulatory relationship between immune and metabolic state following LTHAE, offering a molecular foundation for high-altitude adaptation research.

Background:Contemporary healthcare requires medical professionals with advanced scientific literacy. Current undergraduate medical curricula may not consistently develop this critical skillset. This study evaluates the effectiveness and challenges of an academic competition-based learning (ACBL) for enhancing scientific literacy in medical undergraduates. Methods:The International Genetically Engineered Machine (iGEM) Competition based program was developed using a two-round modified Delphi study. 30 students participated in an iGEM-based academic competition during 18 months. Scientific literacy domains were assessed through validated questionnaires during a five-year follow-up period. Results:iGEM participants demonstrated significantly greater improvement in literature review, experimental design, technical execution, presentation skills, and research management compared to controls (p < 0.01). Significant gains were observed in scientific knowledge acquisition and scientific reasoning (p < 0.01). Scores for active learning, critical thinking, and collaborative communication were significantly higher in the iGEM group (p < 0.05). Participants identified laboratory resources, space, equipment and funding as primary implementation constraints. Conclusion:ACBL is an innovative and effective strategies to develop students' scientific literacy for professional competitiveness, which highlights the potential of ACBL as a transformative approach in medical education.

It is important to systematically identify tumor suppressor genes (TSGs) to improve our understanding of tumorigenesis and develop strategies for early diagnosis and mitigating disease progression. In the present study, we used an in vivo genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) screen and identified FPS/FES-related (FER) as a TSG. Single-cell RNA sequencing (scRNA-seq) revealed that normal cells with low FER expression exhibited elevated malignant transformation potential and stemness properties. FER knockout promoted the tumorigenic transformation, characterized by high colony-forming efficiency and suspension growth ability, acquired tumorigenicity in vivo, increased metabolic activity, dedifferentiation properties, and immune evasion. Moreover, analysis revealed that low FER expression tumors share molecular phenotypes with FER knockout cells, suggesting the consistent role of FER in tumor initiation and progression. Taken together, our findings not only provide insights into the essential role of FER as a tumor suppressor in tumor initiation and progression but also highlight its potential as a target for future clinical diagnosis. (c) 2024 The Pathological Society of Great Britain and Ireland.

Dax1 (Nr0b1; Dosage-sensitive sex reversal-adrenal hypoplasia congenital on the X-chromosome gene-1) is an important component of the transcription factor network that governs pluripotency in mouse embryonic stem cells (ESCs). Functional evaluation of alternative splice variants of pluripotent transcription factors has shed additional insight on the maintenance of ESC pluripotency and self-renewal. Dax1 splice variants have not been identified and characterized in mouse ESCs. We identified 18 new transcripts of Dax1 with putative protein-coding properties and compared their protein structures with known Dax1 protein (Dax1-472). The expression pattern analysis showed that the novel isoforms were cotranscribed with Dax1-472 in mouse ESCs, but they had transcriptional heterogeneity among single cells and the subcellular localization of the encoded proteins differed. Cell function experiments indicated that Dax1-404 repressed Gata6 transcription and functionally replaced Dax1-472, while Dax1-38 and Dax1-225 partially antagonized Dax1-472 transcriptional repression. This study provided a comprehensive characterization of the Dax1 splice variants in mouse ESCs and suggested complex effects of Dax1 variants in a self-renewal regulatory network.

Transcriptional enhanced associate domain (TEAD) transcription factors play important roles in embryonic stem cell (ESC) renewal and differentiation. Four TEAD transcription factors (Tead1, Tead2, Tead3 and Tead4) and their various splice variants have been discovered in mice, but the expression pattern of them during pluripotency state transition is unclear. Here, we investigated the expression of TEADs and their splice variants in mouse ESCs at different pluripotent/differentiating states and adult mouse tissues. Our results preliminarily revealed the diversity and heterogeneity of TEAD family, which is helpful for understanding their overlapping and distinctive functions. Furthermore, a novel splice variant of Tead1 was identified and named Tead1 isoform 4.

As a key effector of the Hippo pathway, yes‐associated protein (YAP) is a major regulator of cell proliferation and apoptosis. In this study, 23 hYAP isoforms were identified in HEK293 cells, with 14 isoforms being reported for the first time. These isoforms were classified into hYAP‐a and hYAP‐b isoforms based on the variation in exon 1. The two groups of isoforms showed distinctly different subcellular localizations. hYAP‐a isoforms could activate TEAD‐ or P73‐mediated transcription, affect the proliferation rate, and enhance the cellular chemosensitivity of HEK293 cells. Moreover, different activation abilities and pro‐cytotoxic effects were observed among hYAP‐a isoforms. However, hYAP‐b isoforms were not found to exert any significant biological effects. Our findings add to the knowledge of YAP gene structure and protein‐coding capacity and will help in the elucidation of the function and related molecular mechanisms of the Hippo‐YAP signaling pathway.

The pluripotency of embryonic stem cells (ESCs) is more accurately viewed as a continuous developmental process rather than a fixed state. However, the factors that play general or state-specific roles in regulating self-renewal in different pluripotency states remain poorly defined. In this study, parallel genome-wide CRISPR/Cas9 knockout (KO) screens were applied in ESCs cultured in the serum plus LIF (SL) and in the 2i plus LIF (2iL) conditions. The candidate genes were classified into seven groups based on their positive or negative effects on self-renewal, and whether this effect was general or state-specific for ESCs under SL and 2iL culture conditions. We characterized the expression and function of genes in these seven groups. The loss of function of novel pluripotent candidate genes Usp28, Zfp598, and Zfp296 was further evaluated in mouse ESCs. Consistent with our screen, the knockout of Usp28 promotes the proliferation of SL-ESCs and 2iL-ESCs, whereas Zfp598 is indispensable for the self-renewal of ESCs under both culture conditions. The cell phenotypes of Zfp296 KO ESCs under SL and 2iL culture conditions were different. Our work provided a valuable resource for dissecting the molecular regulation of ESC self-renewal in different pluripotency states.

A comprehensive and precise definition of the pluripotency gene regulatory network (PGRN) is crucial for clarifying the regulatory mechanisms in embryonic stem cells (ESCs). Here, after a CRISPR/Cas9-based functional genomics screen and integrative analysis with other functional genomes, transcriptomes, proteomes and epigenome data, an expanded pluripotency-associated gene set is obtained, and a new PGRN with nine sub-classes is constructed. By integrating the DNA binding, epigenetic modification, chromatin conformation, and RNA expression profiles, the PGRN is resolved to six functionally independent transcriptional modules (CORE, MYC, PAF, PRC, PCGF and TBX). Spatiotemporal transcriptomics reveal activated CORE/MYC/PAF module activity and repressed PRC/PCGF/TBX module activity in both mouse ESCs (mESCs) and pluripotent cells of early embryos. Moreover, this module activity pattern is found to be shared by human ESCs (hESCs) and cancers. Thus, our results provide novel insights into elucidating the molecular basis of ESC pluripotency.

Abstract Background Tumorigenesis is a complex and multistep process characterized by the progressive acquisition of various hallmarks, including unlimited proliferation, resistance to apoptosis, and increased invasiveness and metastasis. However, the molecular mechanisms underlying tumorigenesis remain poorly understood. Methods An in vivo genome-wide CRISPR–Cas9 screen was employed to identify tumor suppressor genes (TSG). The expression correlation analysis for candidate TSGs was performed in normal and cancer cells using TCGA database. To evaluate the role of FER in tumorigenesis, we firstly used publicly single-cell RNA sequencing data to investigate the association of FER expression and normal cell malignant transformation. Next, we established FER-knockout and -knockdown models in BEAS-2B and MCF10A cell lines. Colony formation assay, cell proliferation assay, EdU assay and apoptosis assay were conducted to determine the role of FER in tumorigenesis. Then RNA-seq was performed to explore the mechanism underlying the role of FER in inhibiting tumorigenesis. Additionally, Pan-Cancer analysis was used to analysis the role of FER in tumor progression. Results In our CRISPR–Cas9 screen, we identified 20 candidate genes, among which FER exhibited the strongest negative correlation with tumorigenesis. Normal cells with low FER expression exhibited elevated malignant transformation potential and stemness properties. FER knockout promoted the tumorigenesis of differentiated epithelial cells by reprogramming them into a cancer stem cell (CSC)-like state, characterized by high colony-forming efficiency and suspension growth ability, increased metabolic activity, dedifferentiation properties, and immune evasion. Furthermore, tumors with low FER expression exhibited poor prognosis and a noticeable CSC-like state. Conclusion Taken together, our findings not only provide insights into the essential role of FER as a stemness barrier in malignant cells during tumor initiation and progression but also highlight its potential as a target for future clinical diagnosis.

The mechanisms underlying self-renewal of embryonic stem cells (ESCs) hold great value in the clinical translation of stem cell biology and regenerative medicine research. To study the mechanisms in ESC self-renewal, screening and identification of key genes maintaining ESC self-renewal were performed by a genome-wide CRISPR-Cas9 knockout virus library. The mouse ESC R1 were infected with CRISPR-Cas9 knockout virus library and cultured for 14 days. The variation of single guide RNA (sgRNA) ratio was analyzed by high-throughput sequencing, followed by bioinformatics analysis to profile the altered genes. Our results showed 1375 genes with increased sgRNA ratio were found to be mainly involved in signal transduction, cell differentiation, and cell apoptosis; 2929 genes with decreased sgRNA ratio were mainly involved in cell cycle regulation, RNA splicing, and biological metabolic processes. We further confirmed our screen specificity by identifying Puf60, U2af2, Wdr75, and Usp16 as novel positive regulators in mESC self-renewal. Meanwhile, further analysis showed the relevance between Puf60 expression and tumor. In conclusion, our study screened key genes maintaining ESC self-renewal and successfully identified Puf60, U2af2, Wdr75, and Usp16 as novel positive regulators in mESC self-renewal, which provided theoretical basis and research clues for a better understanding of ESC self-renewal regulation.

Objective To identify new human lung cancer suppressors in a genome-wide screening library during neoplastic transformation of normal human lung epithelial cells using CRISPR/Cas9-mediated genome editing, and preliminarily validate the Results in non-small cell lung cancer (NSCLC) sample databases. Methods Human normal lung epithelial BEAS-2B cell line with stable expression of Cas9 protein was constructed, and then infected with the Brunello lentiviral library encompassing 77 441 single-guide RNAs (sgRNAs) targeting 19 114 genes. Then the obtained cells were transplanted subcutaneously into nude mice. Genomic DNA of tumor cells (Tumor) and pre-transplantation cells (Input) were extracted. The extracted DNA and plasmid library were amplified for deep sequencing. By analyzing the proportion of sgRNA reads in Tumor cells compared to the Input cells and the Log Fold Change (LFC) of sgRNA reads, tumor suppressor genes that can induce oncogenic transformation of BEAS-2B cells after knockout were screened, and preliminary validation were performed using public data platforms. Results Library infections in BEAS-2B cells stably expressing Cas9 protein and subcutaneous tumorigenesis in nude mice were successfully established. Only cells infected with the library formed tumors at the inoculation sites. Sequencing Results showed that only a small part of sgRNAs were detected in tumor cells. Here, 38 genes with sgRNA reads accounting for more than 1% of the total reads or with 2 or more significantly enriched sgRNA were selected as effective candidate human lung tumor suppressor genes, including known tumor suppressor genes such as NF2 and PTEN etc, and some candidate tumor suppressor genes (AP2M1 and PSENEN) had not been previously reported in lung cancer. Enrichment analysis revealed that these candidate tumor suppressor genes were enriched in key biological pathways involved in cancer, such as Notch and Hippo signaling pathways. It is confirmed in the clinical NSCLC sample databases that 38 candidate tumor suppressor genes were mutated in NSCLC samples. Among the 17 genes with mutation rates greater than 2%, 14 genes were co-mutated with KRAS or TP53, and 3 ones were associated with the prognosis of NSCLC patients. There were 30 candidate genes with their expression in the samples of NSCLC different from those in para-tumor normal tissues, and the expression of 17 candidate genes were correlated with the prognosis of NSCLC patients. Conclusion By using an in vivo genome-wide CRISPR/Cas9 screening library, 38 human lung cancer candidate suppressor genes are identified and preliminarily verified in the clinical NSCLC sample databases.

Hippo signaling pathway is involved in many biological processes including the fate decision of embryonic stem cells (ESCs). Yes-associated protein (Yap) function as a key effector of Hippo pathway, but its role in ESCs is still controversial. So far, only two isoforms of Yap have been identified and they have both overlapping and distinct functions. Here, we identify six novel isoforms of mouse Yap, bringing the total number of isoforms to eight. According to the differences in the first exon, they are divided into two subtypes (a and b). Isoform-a and isoform-b exhibit different subcellular localizations. Moreover, isoform-a can fully reverse the impaired self-renewal phenotype induced by Yap knockout (KO). Upon overexpression, isoform-a moderately promotes mESCs self-renewal and markedly delays differentiation. On the contrary, no significant pro-self-renewal phenotype is observed when isoform-b overexpressed in wildtype (WT) mESCs or re-expressed in Yap KO cell lines. These finding not only help to clarify the role of Yap in mESCs, but also lay the foundation for advancing functional researches of Yap in other processes.

Dax1(also known as Nr0b1) is regarded as an important component of the transcription factor network in mouse embryonic stem cells (ESCs). However, the role and the molecular mechanism of Dax1 in the maintenance of different pluripotency states are poorly understood. Here, we constructed a stable Dax1 knockout (KO) cell line using the CRISPR/Cas9 system to analyze the precise function of Dax1. We reported that 2i/LIF-ESCs had significantly lower Dax1 expression than LIF/serum-ESCs. Dax1KO ES cell lines could be established in 2i/LIF and their pluripotency was confirmed. In contrast, Dax1-null ESCs could not be continuously passaged in LIF/serum due to severe differentiation and apoptosis. In LIF/serum, the activities of the Core module and Myc module were significantly reduced, while the PRC2 module was activated after Dax1KO. The expression of most proapoptotic genes and lineage-commitment genes were drastically increased, while the downregulated expression of antiapoptotic genes and many pluripotency genes was observed. Our research on the pluripotent state-dependent role of Dax1 provides clues to understand the molecular regulation mechanism at different stages of early embryonic development.

Yap is the key effector of Hippo signaling; however, its role in embryonic stem cells (ESCs) remains controversial. Here, we identify two Yap splicing isoforms (Yap472 and Yap488), which show equal expression levels but heterogeneous distribution in ESCs. Knockout (KO) of both isoforms reduces ESC self-renewal, accelerates pluripotency exit but arrests terminal differentiation, while overexpression of each isoform leads to the reverse phenotype. The effect of both Yap isoforms on self-renewal is Teads-dependent and mediated by c-Myc. Nonetheless, different isoforms are found to affect overlapping yet distinct genes, and confer different developmental potential to Yap-KO cells, with Yap472 exerting a more pronounced biological effect and being more essential for neuroectoderm differentiation. Constitutive activation of Yaps, particularly Yap472, dramatically upregulates p53 and Cdx2, inducing trophectoderm trans-differentiation even under self-renewal conditions. These findings reveal the combined roles of different Yap splicing isoforms and mechanisms in regulating self-renewal efficiency and differentiation potential of ESCs.

Alternative splicing (AS) is a key process to expand the diversity of mRNA and protein from the genome and it is crucial for fate determination of embryonic stem cells (ESCs) by encoding isoforms with different functions to regulate the balance between pluripotency maintenance and differentiation. Since the role of the Hippo pathway in ESCs is controversial, there may be novel isoforms of Taz, a key effector of the Hippo pathway, previously unknown to us. Here, we identified three variants of Taz in mESCs. Apart from the canonical Taz1185, there were also two novel variants, Taz402 and Taz1086. We found their structure and subcellular localization to be different, while they could all interact with TEAD2 with similar binding affinities and activate transcription. Under the LIFlow condition, overexpression of them all induced apoptosis and differentiation of mESCs, among which the phenotype of Taz1086 was the most dramatic. Taken together, we discovered novel variants of Taz and compared their structure and functional differences in mESC pluripotency maintenance. These findings will help us to understand the Taz gene and clarify its role in mESC.

Growing evidence supports the notion that lipid metabolism is critical for embryonic stem cell (ESC) maintenance. Recently, α/β-hydrolase domain-containing (ABHD) proteins have emerged as novel pivotal regulators in lipid synthesis or degradation while their functions in ESCs have not been investigated. In this study, we revealed the role of ABHD11 in ESC function using classical loss and gain of function experiments. Knockout of Abhd11 hampered ESC expansion and differentiation, triggering the autophagic flux and apoptosis. In contrast, Abhd11 overexpression exerted anti-apoptotic effects in ESCs. Moreover, Abhd11 knockout disturbed GSK3β/β-Catenin and ERK signaling transduction. Finally, Abhd11 knockout led to the misexpression of key metabolic enzymes related to lipid synthesis, glycolysis, and amino acid metabolism, and ABHD11 contributed to the homeostasis of lipid metabolism. These findings provide new insights into the broad role of ABHD proteins and highlight the significance of regulators of lipid metabolism in the control of stem cell function.

Embryonic stem cells (ESCs) provide an ideal model for investigating developmental processes and are great sources for developing regenerative medicine. Harnessing apoptosis facilitates accurate recapitulation of signalling events during embryogenesis and allows efficient expansion of the ESCs during differentiation. Bcl2, a key regulator of intrinsic anti-apoptotic pathway, encodes two splicing isoforms. However, the identification and functional comparison of Bcl2 splicing isoforms in mouse ESCs (mESCs) remains to be elucidated. Here, we provide the evidence that both Bcl2 splicing variants are expressed in mESCs. Despite the structural difference, they have similar subcellular localisation. Both Bcl2α and Bcl2β enhance differentiation efficiency of the ESCs and effectively improve the survival and growth of ESCs under serum-free conditions. However, the functional effect of Bcl2α was more potent than that of Bcl2β. Moreover, only Bcl2α could maintain the long-term expansion and pluripotency of ESCs cultured in serum-free medium. Taken together, our results demonstrate previously unknown functional differences in Bcl2 alternative splicing isoforms in ESCs, and lay the foundation for future efforts to engineer ESCs for regenerative medicine.

Embryonic stem (ES) cells are unique in their ability to self-renew indefinitely while maintaining pluripotency. Krüppel-like factor (Klf) 4 is an important member of the Klf family that is known to play a key role in pluripotency and somatic cell reprogramming. However, the identification and functional comparison of Klf4 splicing isoforms in mouse ESCs (mESCs) remains to be elucidated. Here, we identified three novel alternative splicing variants of Klf4 in mESCs-mKlf4-108, mKlf4-375 and mKlf4-1482-that are distinct from the previously known mKlf4-1449. mKlf4-1449 and mKlf4-1482 may stimulate the growth of ESCs, while mKlf4-108 can only promote the growth of ESCs in LIFlow/serum conditions. In addition, both mKlf4-1449 and mKlf4-1482 can inhibit the differentiation of mESCs. However, the ability of mKlf4-1482 to promote self-renewal and inhibit differentiation is not as strong as that of mKlf4-1449. In contrast, both mKlf4-108 and mKlf4-375 may have the ability to induce endodermal differentiation. Taken together, we have identified for the first time the existence of alternative splicing variants of mKlf4 and have revealed their different roles, which provide new insights into the contribution of Klf4 to the self-renewal and pluripotency of mouse ESCs.