Effects of fluorescent protein tdTomato on mouse retina.

Fluorescent proteins (FPs) have been widely used to investigate cellular and molecular interactions and trace biological events in many applications. Some of the FPs have been demonstrated to cause undesirable cellular damage by light-induced ROS production in vivo or in vitro. However, it remains unknown if one of the most popular FPs, tdTomato, has similar effects in neuronal cells. In this study, we discovered that tdTomato expression led to unexpected retinal dysfunction and ultrastructural defects in the transgenic mouse retina. The retinal dysfunction mainly manifested in the reduced photopic electroretinogram (ERG) responses and decreased contrast sensitivity in visual acuity, caused by mitochondrial damages characterized with cellular redistribution, morphological modifications and molecular profiling alterations. Taken together, our findings for the first time demonstrated the retinal dysfunction and ultrastructural defects in the retinas of tdTomato-transgenic mice, calling for a more careful design and interpretation of experiments involved in FPs.

Exploratory study of sea buckthorn enhancing QiangGuYin efficacy by inhibiting CKIP-1 and Notum activating the Wnt/ß-catenin signaling pathway and analysis of active ingredients by molecular docking.

Background: Sea buckthorn (SBT) is a traditional Chinese medicine (TCM), rich in calcium, phosphorus, and vitamins, which can potentially prevent and treat osteoporosis. However, no research has been conducted to confirm these hypotheses. QiangGuYin (QGY) is a TCM compound used to treat osteoporosis. There is a need to investigate whether SBT enhances QGY efficacy. Objectives: The aim of this study was to explore whether SBT enhances QGY efficacy by inhibiting CKIP-1 and Notum expression through the Wnt/ß-catenin pathway. The study also aimed to explore the active components of SBT. Methods: Experimental animals were divided into control, model, QGY, SBT, SBT + Eucommia ulmoides (EU), and SBT + QGY groups. After treatment, bone morphometric parameters, such as estrogen, PINP, and S-CTX levels, and Notum, CKIP-1, and ß-catenin expression were examined. Screening of SBT active components was conducted by molecular docking to obtain small molecules that bind Notum and CKIP-1. Results: The results showed that all the drug groups could elevate the estrogen, PINP, and S-CTX levels, improve femoral bone morphometric parameters, inhibit Notum and CKIP-1 expression, and promote ß-catenin expression. The effect of SBT + EU and SBT + QGY was superior to the others. Molecular docking identified that SBT contains seven small molecules (folic acid, rhein, quercetin, kaempferol, mandenol, isorhamnetin, and ent-epicatechin) with potential effects on CKIP-1 and Notum. Conclusion: SBT improves bone morphometric performance in PMOP rats by inhibiting CKIP-1 and Notum expression, increasing estrogen levels, and activating the Wnt/ß-catenin signaling pathway. Furthermore, SBT enhances the properties of QGY. Folic acid, rhein, quercetin, kaempferol, mandenol, isorhamnetin, and ent-epicatechin are the most likely active ingredients of SBT. These results provide insight into the pharmacological mechanisms of SBT in treating osteoporosis.

Tracking public opinion about online education over COVID-19 in China.

Due to the novel coronavirus disease (COVID-19) outbreak in China, a large number of Chinese students resorted to online learning resources. The increasingly widespread online education enables the investigation of public opinion about this large-scale untraditional mode of learning during this critical period. Sina Weibo Microblogs (the Chinese equivalent of Twitter) related to online education were collected in three distinctive phases: from July 01, 2019 to January 09, 2020 (pre-pandemic); from January 10, 2020 to April 30, 2020 (amid-pandemic); and from May 01, 2020 to Nov 30, 2020 (post-pandemic), respectively. The aim was to obtain broad insight into how online learning was viewed by the public in the Chinese educational landscape. The public opinion during these three periods were analysed and compared. The findings facilitated a better understanding of what the Chinese public perceived about this online learning mode in becoming the dominant channel for teaching and learning during critical periods.

Circular Rims2 Deficiency Causes Retinal Degeneration.

Circular RNAs (circRNAs) refer to a newly recognized family of non-coding RNA with single-stranded RNAs. Despite emerging evidence indicating that circRNAs are abundantly expressed in various tissues, especially in the brain and retina, the role of circRNAs in retinal function and diseases is still largely unknown. Circular Rims2 (circRims2) is highly expressed and conserved in both the human and mouse brains. However, little is known about the expression and function of circRims2 in the retina. In the current study, the high-throughput RNA-seq analysis reveals a high expression of circRims2 in the retina. In addition, it is found that circRims2 is mainly located in plexiform layers that contain synapses between retinal neurons. Knocking down circRims2 with short hairpin RNA through subretinal adeno-associated viral (AAV) delivery in the mice leads to the decrease of the thickness of the outer and inner segment (OS/IS) layers and outer nuclear layer (ONL), and cessation of scotopic and photopic electroretinogram responses. Furthermore, the current study finds that circRims2 deficiency evokes retinal inflammation and activates the tumor necrosis factor (TNF) signaling pathway. Therefore, circRims2 may play an important role in the maintenance of retinal structure and function, and circRims2 deficiency may lead to pathogenic changes in the retina.

Research on automatic labeling of imbalanced texts of customer complaints based on text enhancement and layer-by-layer semantic matching.

Due to its potential impact on business efficiency, automated customer complaint labeling and classification are of great importance for management decision making and business applications. The majority of the current research on automated labeling uses large and well-balanced datasets. However, customer complaint labels are hierarchical in structure, with many labels at the lowest hierarchy level. Relying on lower-level labels leads to small and imbalanced samples, thus rendering the current automatic labeling practices inapplicable to customer complaints. This article proposes an automatic labeling model incorporating the BERT and word2vec methods. The model is validated on electric utility customer complaint data. Within the model, the BERT method serves to obtain shallow text tags. Furthermore, text enhancement is used to mitigate the problem of imbalanced samples that emerge when the number of labels is large. Finally, the word2vec model is utilized for deep text analysis. Experiments demonstrate the proposed model's efficiency in automating customer complaint labeling. Consequently, the proposed model supports enterprises in improving their service quality while simultaneously reducing labor costs.

Abundant Neural circRNA Cdr1as Is Not Indispensable for Retina Maintenance.

Cdr1as is the abundant circular RNA (circRNA) in human and vertebrate retinas. However, the role of Cdr1as in the retina remains unknown. In this study, we aimed to generate a Cdr1as knockout (KO) mouse model and investigate the retinal consequences of Cdr1as loss of function. Through in situ hybridization (ISH), we demonstrated that Cdr1as is mainly expressed in the inner retina. Using CRISPR/Cas9 targeting Cdr1as, we successfully generated KO mice. We carried out ocular examinations in the KO mice until postnatal day 500. Compared with the age-matched wild-type (WT) siblings, the KO mice displayed increased b-wave amplitude of photopic electrophysiological response and reduced vision contrast sensitivity. Through small RNA profiling of the retinas, we determined that miR-7 was downregulated, while its target genes were upregulated. Taken together, our results demonstrated for the first time that Cdr1as ablation led to a mild retinal consequence in mice, indicating that Cdr1as abundance is not indispensable for retinal development and maintenance.

ATP1A3 mutation as a candidate cause of autosomal dominant cone-rod dystrophy.

Cone-rod dystrophy (CORD) is an inherited retinal degenerative disease characterized by progressive loss of cone and rod photoreceptors. Although several genes have been reported to cause autosomal dominant CORD (adCORD), the genetic causes of adCORD have not been fully elucidated. Here, we identified the ATP1A3 gene, encoding the α3 subunit of Na+, K+-ATPase, as a novel gene associated with adCORD. Using whole-exome sequencing (WES), we found a candidate mutation of ATP1A3 that co-segregated with the disease in an analysis of two affected patients and one healthy relative in an adCORD family. According to our RNA-seq data, we demonstrated that the Atp1a3 mRNA level was extremely high in the murine retina. Overexpression of mutant ATP1A3 in vitro led to a reduced oxygen consumption rate (OCR), reflecting the limited mitochondrial reserve capacity. Furthermore, we generated transgenic mice expressing the ATP1A3 cDNA with patient variant and found decreased electroretinogram (ERG) responses. Moreover, the mutant ATP1A3 is highly expressed in photoreceptor inner segment, where mitochondria are enriched. These results suggest that the ATP1A3 mutation is a new genetic cause responsible for adCORD and indicate that ATP1A3 plays an important role in retinal function.

Curcumin inhibited HGF-induced EMT and angiogenesis through regulating c-Met dependent PI3K/Akt/mTOR signaling pathways in lung cancer.

The epithelial-mesenchymal transition (EMT) and angiogenesis have emerged as two pivotal events in cancer progression. Curcumin has been extensively studied in preclinical models and clinical trials of cancer prevention due to its favorable toxicity profile. However, the possible involvement of curcumin in the EMT and angiogenesis in lung cancer remains unclear. This study found that curcumin inhibited hepatocyte growth factor (HGF)-induced migration and EMT-related morphological changes in A549 and PC-9 cells. Moreover, pretreatment with curcumin blocked HGF-induced c-Met phosphorylation and downstream activation of Akt, mTOR, and S6. These effects mimicked that of c-Met inhibitor SU11274 or PI3 kinase inhibitor LY294002 or mTOR inhibitor rapamycin treatment. c-Met gene overexpression analysis further demonstrated that curcumin suppressed lung cancer cell EMT by inhibiting c-Met/Akt/mTOR signaling pathways. In human umbilical vein endothelial cells (HUVECs), we found that curcumin also significantly inhibited PI3K/Akt/mTOR signaling and induced apoptosis and reduced migration and tube formation of HGF-treated HUVEC. Finally, in the experimental mouse model, we showed that curcumin inhibited HGF-stimulated tumor growth and induced an increase in E-cadherin expression and a decrease in vimentin, CD34, and vascular endothelial growth factor (VEGF) expression. Collectively, these findings indicated that curcumin could inhibit HGF-promoted EMT and angiogenesis by targeting c-Met and blocking PI3K/Akt/mTOR pathways.

miR-206 regulates cisplatin resistance and EMT in human lung adenocarcinoma cells partly by targeting MET.

MicroRNAs (miRNAs) play a critical role in drug resistance and epithelial-mesenchymal transition (EMT). The aims of this study were to explore the potential role of miR-206 in governing cisplatin resistance and EMT in lung cancer cells. We found that both lung adenocarcinoma A549 cisplatin-resistant cells (A549/DDP) and H1299 cisplatin-resistant cells (H1299/DDP) acquired mesenchymal features and were along with low expression of miR-206 and high migration and invasion abilities. Ectopic expression of miR-206 mimics inhibited cisplatin resistance, reversed the EMT phenotype, decreased the migration and invasion in these DDP-resistant cells. In contrast, miR-206 inhibitors increased cisplatin resistance, EMT, cell migration and invasion in non-DDP-resistant cells. Furthermore, we found that MET is the direct target of miR-206 in lung cancer cells. Knockdown of MET exhibited an EMT and DDP resistant inhibitory effect on DDP-resistant cells. Conversely, overexpression of MET in non-DDP- resistant cells produced a promoting effect on cell EMT and DDP resistance. In lung adenocarcinoma tissues, we demonstrated that low expression of miR-206 were also correlated with increased cisplatin resistance and MET expression. In addition, we revealed that miR-206 overexpression reduced cisplatin resistance and EMT in DDP-resistant cells, partly due to inactivation of MET/PI3K/AKT/mTOR signaling pathway, and subsequent downregulation of MDR1, ZEB1 and Snail expression. Finally, we found that miR-206 could also sensitize A549/DDP cells to cisplatin in mice model. Taken together, our study implied that activation of miR-206 or inactivation of its target gene pathway could serve as a novel approach to reverse cisplatin resistance in lung adenocarcinomas cells.

MiR-206 inhibits HGF-induced epithelial-mesenchymal transition and angiogenesis in non-small cell lung cancer via c-Met /PI3k/Akt/mTOR pathway.

MiR-206 is low expression in lung cancers and associated with cancer metastasis. However, the roles of miR-206 in epithelial-mesenchymal transition (EMT) and angiogenesis in lung cancer remain unknown. In this study, we find that hepatocyte growth factor (HGF) induces EMT, invasion and migration in A549 and 95D lung cancer cells, and these processes could be markedly inhibited by miR-206 overexpression. Moreover, we demonstrate that miR-206 directly targets c-Met and inhibits its downstream PI3k/Akt/mTOR signaling pathway. In contrast, miR-206 inhibitors promote the expression of c-Met and activate the PI3k/Akt/mTOR signaling, and this effect could be attenuated by the PI3K inhibitor. Moreover, c-Met overexpression assay further confirms the significant inhibitory effect of miR-206 on HGF-induced EMT, cell migration and invasion. Notably, we also find that miR-206 effectively inhibits HGF-induced tube formation and migration of human umbilical vein endothelial cells (HUVECs), and the mechanism is also related to inhibition of PI3k/Akt/mTOR signaling. Finally, we reveal the inhibitory effect of miR-206 on EMT and angiogenesis in xenograft tumor mice model. Taken together, miR-206 inhibits HGF-induced EMT and angiogenesis in lung cancer by suppressing c-Met/PI3k/Akt/mTOR signaling. Therefore, miR-206 might be a potential target for the therapeutic strategy against EMT and angiogenesis of lung cancer.

[Non-small cell lung cancer 95D cells co-cultured with 3D-bioprinted scaffold to construct a lung cancer model in vitro].

OBJECTIVE: To fabricate an innovative scaffold for lung cancer cell culture and establish a three-dimensional lung cancer model in vitro, and to reveal the differences in biological functions of lung cancer cells under the two-dimensional and three-dimensional culture conditions. METHODS: We chose agarose and alginate as the scaffold materials, and 3D printing technique was applied to construct cell culture scaffold. 95D cells were co-cultured with this scaffold. The differences of cell morphology, proliferation ability, protein expression, etc. in the cells cultured under 2D and 3D cultural conditions were evaluated by light microscopy using HE staining, MTT assay, scanning electron microscopy, and Western blot analysis. RESULTS: Cells cultured in 2D wells displayed a spindle and polygonal morphology, whereas those grown in the 3D culture aggregated into spheroids, which invaded, migrated and disseminated into the surrounding scaffold. MTT assay showed that the proliferation rates of the 3D-cultured cells for 2-6 days were significantly lower than, but those cultured for 8-9 days were significantly higher than that of the 2D-cultured cells, indicating that proliferative activity of the cells grown in 2D cultures for 8-9 days was inhibited. In contrast, cells grown on 3D scaffolds still maintained a higher proliferation. The Western blot assay showed that the expression of Cdc42, p53, mTOR were significantly down-regulated in 3D scaffold-cultured group (0.529±0.103, 0.820±0.038 vs. 1.967±0.066), compared with that of the 2D-cultured group (3.063±0.139, 1.738±0.122 vs. 2.472±0.151) (P<0.05 for all), while the expression of MMP-2 was up-regulated in the 3D-cultured cells (1.110±0.029), significantly higher than that of the 2D-cultured cells (0.017±0.001) (P<0.05). CONCLUSIONS: The cell morphology, proliferation and associated protein expression of lung cancer cells in 3D-culture systems are distinctively different as compared to those of the 2D-cultural cells. 3D-bioprinted agarose-alginate scaffold can better mimic the growth microenvironment of lung cancer in vivo and may provide a promising model for lung cancer research in vitro.