The molecular mechanisms that underpin invasive ductal breast cancer (IDC) invasion and metastasis are incompletely understood. The oncogene, mouse double minute 2 (MDM2), has been implicated in the pathogenesis of numerous cancers, where it stimulates the expression of matrix metalloproteinase 9 (MMP9), an important enzyme in the breakdown of the extracellular matrix. However, its role in breast cancer remains poorly understood. This study assessed the clinical significance of MDM2 expression in IDC and used in vitro expression assays to determine the molecular roles of MDM2. Immunohistochemical staining for MMP9 and MDM2 was performed using archived tumor blocks from 321 women who underwent surgical resection for IDC at the First Affiliated Hospital of Nanjing Medical University, China between January 2002 and December 2003. MCF-7 and MDA-MD-231 cell lines were transfected with siRNA targeted against MDM2, or MDM2 was overexpressed using transiently expressed vectors. The invasion, cell migration and proteolytic capabilities of cells that over- or underexpressed MDM2 was then assessed and compared against control cells, in addition to the consequent effects on MMP9 expression using RT-PCR. In vivo, 54.9% and 49.6% of samples were positive for MMP9 and MDM2 expression, respectively, and their expression was significantly correlated (r² = 0.171, P = 0.012). Moreover, MDM2 expression was markedly correlated with disease-free survival (HR 2.56, 95% CI 1.02-6.40, P = 0.038). In vitro, MDM2 overexpression significantly enhanced cell invasion, migration and proteolysis compared with control cells, and the converse effects were observed after MDM2-siRNA treatment. MDM2 overexpression induced MMP9 expression in a dose-dependent manner. Taken together, these results suggest that high levels of MDM2 are associated with a poorer prognosis in IDC. This might result from increased tumor invasiveness due to enhanced MMP9 expression causing increased extracellular matrix breakdown.

It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock-in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis.

Telocytes (TCs) are a newly discovered type of mesenchymal cell that are closely related to the tissue's internal environment. The study aimed to investigate the morphological identification of TCs in the epididymis of adult yak and their role in the local microenvironment. In this study, transmission electron microscopy (TEM), scanning electron microscopy, immunofluorescence, qRT-PCR, and western blotting were used to analyze the cell morphology of TCs. The results showed that there are two types of TCs in the epididymal stroma of yak by TEM; one type is distributed around the capillaries with full cell bodies, longer TPs, and a large number of secretory vesicles; the other is distributed outside the basement membrane with irregularly long, striped, large nuclei and short telopodes (TPs). In addition, these TCs formed complex TC cell networks through TPs with epididymal interstitial capillaries and basal fibroblasts. TCs often appear near the capillaries and basement membrane by special staining. The surface markers of TCs (CD34, vimentin, and CD117) were positively expressed in the epididymal stroma and epithelium by immunohistochemistry, and immunofluorescence co-expression of vimentin + CD34 and CD117 + CD34 was observed on the surface of TCs. The trends in the mRNA and protein expression of TCs surface markers revealed expression was highest in the caput epididymis. In summary, this is first report of TCs in the epididymis of yak, and two phenotypes of TCs were observed. The existence and distribution characteristics of TCs in the epididymis of plateau yaks provide important clues for further study of the adaptation to reproductive function in the plateau.

MTNR1A and MTNR1B, two high-affinity MT membrane receptors found in mammals, mediate the activity of MT on the HPGA to regulate animal reproduction. Nevertheless, the expression patterns and function of the MTNR1A and MTNR1B genes in the HPTA of seasonal estrus sheep and perennial estrus sheep have not been elucidated. We studied the expression of MTNR1A and MTNR1B in the hypothalamic-pituitary-testicular axis (HPTA) of Tibetan sheep at different reproductive stages using histochemistry, enzyme linked immunosorbent assay (ELSIA), scanning electron microscopy, transmission electron microscopy, quantitative Real-time PCR (qRT-PCR), and Western blot (WB), and analyzed the relationship between their expression and reproductive hormone receptors. We also compared relevant characteristics between seasonal Tibetan sheep and non-seasonal Small Tail Han sheep in the same pastoral area. The results showed that MTNR1A and MTNR1B were expressed in all tissues of the Tibetan sheep HPTA, and both were co-expressed in the cytoplasm of epididymis basal and halo cells located at common sites of the epididymis basement membrane, forming an immune barrier. The qRT-PCR analysis showed that not only MTNR1A but also N-acetyltransferase (AANAT), hydroxyindole-oxygen- methyltransferase (HIOMT), androgen receptor (AR), and estrogen receptor α (ERα) mRNA expression was significantly upregulated in the testis and epididymis of Tibetan sheep during the breeding season, whereas no clear upregulation of these genes was observed in the tissues of Small Tail Han sheep. MTNR1A and MTNR1B are important regulators of the HPTA in sheep. MTNR1A mediates seasonal estrus regulation in Tibetan sheep. Both MTNR1A and MTNR1B may play important roles in formation of the blood-epididymal barrier. The results of this study should help advance research on the mechanism of reproductive regulation of the HPTA in male animals and provide reference data for improving the reproductive rate of seasonal breeding animals.

MicroRNAs (miRNAs) are the effectors of a conserved gene-silencing system with broad roles in post-transcriptional regulation. Due to functional overlaps, assigning specific functions to individual miRNAs has been challenging. DICER1 cleaves pre-miRNA hairpins into mature miRNAs, and previously Dicer1 knockout mouse embryonic stem cells have been generated to study miRNA function in early mouse development. Here we report an essential requirement of DICER1 for the self-renewal of human embryonic stem cells (hESCs). Utilizing a conditional knockout approach, we found that DICER1 deletion led to increased death receptor-mediated apoptosis and failure of hESC self-renewal. We further devised a targeted miRNA screening strategy and uncovered essential pro-survival roles of members of the mir-302-367 and mir-371-373 clusters that bear the seed sequence AAGUGC. This platform is uniquely suitable for dissecting the roles of individual miRNAs in hESC self-renewal and differentiation, which may help us better understand the early development of human embryos.

The purpose of this study was to observe the three-dimensional growth and development of the maxillary arch in 10-year-olds with normal occlusion during the late mixed dentition stage.

The melatonin 1A receptor (MTNR1A) is a membrane receptor distributed across the mammalian gonadal axis-associated membrane. Melatonin (MT) can specifically bind with MTNR1A on the cell membrane and regulates mammalian reproductive activities. However, the role of MTNR1A in regulating the reproductive physiological activities of sheep in the Tibetan Plateau remains unclear. In this study, the MT content in Tibetan sheep blood during the estrous cycle was detected by ELISA. The distribution of MTNR1A in the hypothalamus-pituitary-gonadal axis (HPGA) was analyzed by immunohistochemistry and immunofluorescence. Western blot and qRT-PCR were used to detect dynamic changes of MTNR1A mRNA and protein expression, and the protein distributions in the HPGA. The results showed that the average secretion level of MT in Tibetan sheep blood was highest occurred during diestrus and the lowest during proestrus. Additionally, the secretion of MT at night was significantly higher than during the day. The immunopositive products of MTNR1A were primarily distributed around the glial cells in the dorsal hypothalamic nucleus region, chromophobe cells, and eosinophilic cytoplasm in the pituitary gland, follicular granular layer, follicular adventitia, tubal mucosa, cilia, endometrium, interstices, and glands in the uterus. The expression trends of MTNR1A mRNA and proteins in the HPGA during the estrous cycle were the same. The relative expression levels of MTNR1A mRNA and proteins in the hypothalamus and ovaries were the highest during proestrus and the lowest during metestrus; the highest during diestrus in the pituitary and oviducts; the highest during metestrus in the uterus. Collectively, the differences in the secretion of MT in Tibetan sheep blood and the expression of MTNR1A in HPGA suggest that they may be affected by steroid hormone secretion during the estrous cycle of Tibetan sheep, which has a potential impact on the regulation of animal estrous cycle.

Performance degradation will be caused by a variety of interfering factors for pattern recognition-based myoelectric control methods in the long term. This paper proposes an adaptive learning method with low computational cost to mitigate the effect in unsupervised adaptive learning scenarios. We presents a particle adaptive classifier (PAC), by constructing a particle adaptive learning strategy and universal incremental least square support vector classifier (LS-SVC). We compared PAC performance with incremental support vector classifier (ISVC) and non-adapting SVC (NSVC) in a long-term pattern recognition task in both unsupervised and supervised adaptive learning scenarios. Retraining time cost and recognition accuracy were compared by validating the classification performance on both simulated and realistic long-term EMG data. The classification results of realistic long-term EMG data showed that the PAC significantly decreased the performance degradation in unsupervised adaptive learning scenarios compared with NSVC (9.03% ± 2.23%, p < 0.05) and ISVC (13.38% ± 2.62%, p = 0.001), and reduced the retraining time cost compared with ISVC (2 ms per updating cycle vs. 50 ms per updating cycle).

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.

Pluripotent stem cells are defined by both the ability to unlimitedly self-renew and differentiate to any somatic cell lineage, but understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. We performed four parallel genome-scale CRISPR-Cas9 screens to investigate the interplay between these two aspects of pluripotency. Our comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including many mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control stem cell identity. We further discovered a core set of factors that control both stem cell fitness and pluripotency identity, including an interconnected network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus self-renewal, and offer a valuable model for categorizing gene function in broad biological contexts.

Human disease phenotypes associated with haploinsufficient gene requirements are often not recapitulated well in animal models. Here, we have investigated the association between human GATA6 haploinsufficiency and a wide range of clinical phenotypes that include neonatal and adult-onset diabetes using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome editing coupled with human pluripotent stem cell (hPSC) directed differentiation. We found that loss of one GATA6 allele specifically affects the differentiation of human pancreatic progenitors from the early PDX1+ stage to the more mature PDX1+NKX6.1+ stage, leading to impaired formation of glucose-responsive β-like cells. In addition to this GATA6 haploinsufficiency, we also identified dosage-sensitive requirements for GATA6 and GATA4 in the formation of both definitive endoderm and pancreatic progenitor cells. Our work expands the application of hPSCs from studying the impact of individual gene loci to investigation of multigenic human traits, and it establishes an approach for identifying genetic modifiers of human disease.

Angelicin is an active compound isolated from the Chinese herb Angelica archangelica, which has been reported to exert antitumor effects by inhibiting malignant behaviors in several types of tumor, including proliferation, colony formation, migration and invasion. However, the effects of angelicin on human cervical cancer cells is yet to be elucidated. The present study evaluated the antitumor effects of angelicin on cervical cancer cells. The results demonstrated that cervical cancer cells were more sensitive to angelicin than cervical epithelial cells. At its IC30, angelicin inhibited the proliferation of HeLa and SiHa cells by blocking the cell cycle at the G1/G0 phase and inhibiting other malignant behaviors, including colony formation, tumor formation in soft agar, migration and invasion. At the IC50, angelicin induced cell death potentially by promoting apoptosis. By identifying the hallmarks of autophagy, it was observed that angelicin treatment caused the accumulation of microtubule associated protein 1 light chain 3-β (LC3B) in the cytoplasm of HeLa and SiHa cells. Western blotting results demonstrated that cleaved LC3B-II and autophagy related proteins (Atg)3, Atg7 and Atg12-5 were upregulated following angelicin treatment. It was also determined that the phosphorylation of mTOR was induced by angelicin treatment. Furthermore, the inhibition of angelicin-induced mTOR phosphorylation did not disrupt its inhibitory effect on autophagy, indicating that angelicin inhibited autophagy in an mTOR-independent manner. Taken together, the present results suggested that angelicin regulated malignant behaviors in cervical cancer cells by inhibiting autophagy in an mTOR-independent manner. Findings suggested that autophagy might be a potential therapeutic target for cervical cancer.

Understanding diverse responses of individual cells to the same perturbation is central to many biological and biomedical problems. Current methods, however, do not precisely quantify the strength of perturbation responses and, more importantly, reveal new biological insights from heterogeneity in responses. Here we introduce the perturbation-response score (PS), based on constrained quadratic optimization, to quantify diverse perturbation responses at a single-cell level. Applied to single-cell transcriptomes of large-scale genetic perturbation datasets (e.g., Perturb-seq), PS outperforms existing methods for quantifying partial gene perturbation responses. In addition, PS presents two major advances. First, PS enables large-scale, single-cell-resolution dosage analysis of perturbation, without the need to titrate perturbation strength. By analyzing the dose-response patterns of over 2,000 essential genes in Perturb-seq, we identify two distinct patterns, depending on whether a moderate reduction in their expression induces strong downstream expression alterations. Second, PS identifies intrinsic and extrinsic biological determinants of perturbation responses. We demonstrate the application of PS in contexts such as T cell stimulation, latent HIV-1 expression, and pancreatic cell differentiation. Notably, PS unveiled a previously unrecognized, cell-type-specific role of coiled-coil domain containing 6 (CCDC6) in guiding liver and pancreatic lineage decisions, where CCDC6 knockouts drive the endoderm cell differentiation towards liver lineage, rather than pancreatic lineage. The PS approach provides an innovative method for dose-to-function analysis and will enable new biological discoveries from single-cell perturbation datasets.