Impact of Dietary Variations on Kuruma Shrimp (Penaeus japonicus) Assessed through Individual-Based Rearing and Insights into Individual Differences.

This study developed an individual-rearing method to compare the effects of live feed (sandworms Perinereis aibuhitensis), formulated pellet diets, and a mixture of live feed and formula feed on the Kuruma shrimp Penaeus japonicus, aiming to minimize the influence of non-dietary factors on the growth of P. japonicus, like cannibalism. Results indicated that live feed, with its higher protein, essential amino acids, and fatty acid content, led to significantly better growth and feeding performance in P. japonicus (p < 0.05) compared to pellet diets. A mixed diet resulted in a lower average daily protein intake yet maintained a growth and feeding performance comparable to live feed. The intestinal microbiota of shrimp, dominated by Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria, showed significant shifts with diet changes. Specifically, formulated feed increased the relative abundance of Vibrio and Photobacterium while decreasing Shimia and Rhodobacterales (p < 0.05), and feeding live food resulted in a more complex and stable bacterial network. Notably, individual variances in growth and feeding were observed among shrimps, with some on formulated diets showing growth comparable to those on live feed. Each shrimp's final weight, specific growth rate, protein efficiency rate, and average daily food intake positively correlated with its initial body weight (p < 0.05), and daily intake varied cyclically with the molting cycle. These findings suggest that individual-rearing is an effective approach for detailed feed evaluation and monitoring in P. japonicus, contributing to improved feed selection, development, and feeding strategies.

Theory of Mind and physical bullying in preschool children: the role of peer rejection and gender differences.

BACKGROUND: Preschool represents the budding and initial stage of bullying behavior, where perpetration of physical bullying predominates as the primary form of bullying. An in-depth understanding of the factors linked to preschool physical bullying behavior is crucial for enabling early prevention and intervention strategies. OBJECTIVE: The purpose of this study was to examine the relationship between Theory of Mind and physical bullying behavior in 4-6 years old children in kindergarten and the mediating role of peer rejection and gender in this relationship. METHODS: Data on perpetration of physical bullying and peer rejection were obtained from 310 preschool children (age range = 52-79 months, M = 66.85, SD = 7.04) by the peer nomination method, and their Theory of Mind was measured by the Theory of Mind Development Scale. RESULTS: The results showed that Theory of Mind negatively predicted perpetration of physical bullying in preschool children and that Theory of Mind was related to perpetration of physical bullying in preschool children only through peer rejection. Boys were also found to have a stronger association between peer rejection and perpetration of physical bullying in preschool children than girls. CONCLUSION: Peer rejection may play a mediating role between Theory of Mind and perpetration of physical bullying in preschool children. In addition, the relationship between peer rejection and perpetration of physical bullying appears to be stronger for boys than for girls. This contributes to our understanding of the relationship between Theory of Mind and perpetration of physical bullying in preschool children and has implications for how bullying prevention and intervention can be tailored to the gender of young children.

Eliminating drug target interference with specific antibody or its F(ab')2 fragment in the bridging immunogenicity assay.

Background: DB-1003 is a humanized anti-IgE monoclonal antibody with higher affinity than omalizumab. In the affinity capture elution (ACE)-based bridging electrochemiluminescent immunoassay (ECLIA) for antibodies to DB-1003, monkey serum IgE caused false-positive results. Materials & methods: The target-specific antibody or its F(ab')2 fragment was used to mitigate drug target interference in an ACE-based bridging ECLIA for the detection of anti-DB-1003 antibodies. Results: The sensitivity of the developed assay was at least 100 ng/ml. When the anti-drug antibody concentration was 250 ng/ml, the assay tolerated at least 20.0 µg/ml of the monkey IgE. Conclusion: Incorporating the target-specific antibody or its F(ab')2 fragment can overcome the interference from monkey serum IgE in ACE-based bridging ECLIA for anti-DB-1003 antibody detection.

Theabrownin of raw and ripened pu-erh tea varies in the alleviation of HFD-induced obesity via the regulation of gut microbiota.

PURPOSE: Pu-erh tea can be classified into raw pu-erh tea and ripened pu-erh tea. Theabrownin (TB) is one of the major components of pu-erh tea. The difference of the anti-obesity activity between raw pu-erh tea TB (R-TB) and ripened pu-erh tea TB (F-TB) has not been comprehensively investigated yet. Therefore, this article aimed to systemically study the anti-obesity activity and the underlying mechanism of R-TB and F-TB. METHOD: High-fat diet (HFD)-induced C57BL/6J mice with obesity were gavaged with R-TB or F-TB to assess the effect of R-TB and F-TB on the amelioration of obesity, the expression of lipid metabolism-related genes, and the regulation of gut flora imbalance. RESULTS: Administration of both R-TB and F-TB could suppress body weight gain, improve insulin sensitivity and glucose homeostasis, regulate the lipid level and reduce the chronic inflammation in obese mice. The underlying anti-obesity mechanism of R-TB and F-TB might involve the regulation of lipogenesis and lipolysis, amelioration of the gut microbiota disorder and promotion of microbial metabolism. Interestingly, R-TB was more efficient in the regulation of blood glucose, reduction of inflammation and suppression of partial adipogenesis-related genes and protein, while F-TB was more effective in the inhibition of lipolysis-related genes and protein. In addition, F-TB might be more effective in adjusting the dysbacteria caused by HFD back to normal by promoting the proliferation of the beneficial microbiota, such as Lactobacillus and Lachnospiraceae_NK4A136_group. CONCLUSION: Taken together, both R-TB and F-TB had the potential to be developed as beneficial dietary supplements or functional foods for ameliorating obesity and obesity-related metabolic disorders, but their effects and the ability to regulate the intestinal flora varied.

Development, Validation and Application of a Bridging ELISA for Detection of Antibodies against GQ1001 in Cynomolgus Monkey Serum.

Immunogenicity is a major issue associated with the PK, efficacy, and safety evaluation of therapeutic protein products during pre-clinical and clinical studies. A multi-tiered approach consisting of screening, confirmatory, and titration assays has been widely adopted for anti-drug antibody testing. GQ1001, a recombinant humanized anti-human epidermal growth factor receptor 2 monoclonal antibody covalently linked to a cytotoxin of DM1, possesses a novel format of antibody-drug conjugates. In this study, we reported the development, validation, and application of an acid-dissociation bridging enzyme-linked immunosorbent assay for the detection of antibodies against GQ1001 in cynomolgus monkey serum. The sensitivity of the screening assay was 126.141 ng/mL in undiluted serum. The screening assay and confirmatory assay were neither affected by the naïve monkey serum nor by 2% and 5% (v/v) erythrocyte hemolysates. Moreover, the assay was not subject to interference by 2500 ng/mL of human IgG1 in the samples. Drug interference at low positive control (150 ng/mL) and high positive control (8000 ng/mL) of anti-GQ1001 antibodies was not observed when GQ1001 concentrations were below 3.125 µg/mL and 100 µg/mL, respectively. Furthermore, no hook effect was observed for the positive antibodies in the concentration range of 8 to 64 µg/mL. The validated assay was, thereafter, successfully applied to a single-dose toxicity study of GQ1001. Anti-drug antibody positive rates among dosing animals and testing samples were reported, and no significant impact was found on toxicokinetic outcomes.

PNPLA3 148M/M Is More Susceptible to Palmitic Acid-Induced Endoplasmic Reticulum Stress-Associated Apoptosis in HepG2 Cells.

Background: Patatin-like phospholipase domain-containing 3 (PNPLA3) is a major susceptibility gene for nonalcoholic fatty liver disease (NAFLD), and its rs738409 (I148M) polymorphism is associated with the occurrence and progression of NAFLD. Endoplasmic reticulum (ER) stress-related hepatocyte lipoapoptosis contributes to the progress of NAFLD. PNPLA3 is also known as a member of the calcium-independent phospholipase A2ε family, which can hydrolyze fatty acids to generate lysophosphatidylcholine (LPC) that induces ER stress-related hepatocyte lipoapoptosis. Whether the PNPLA3 risk genotype 148M/M is involved in more severe ER stress-associated lipoapoptosis is unclear. Methods: A PNPLA3148I knock-in HepG2 cell model was constructed based on HepG2 expressing PNPLA3 148M/M using the Cas9/sgRNA system. PNPLA3 148M/M, I/M, and I/I cells were treated with 0.3 mM palmitic acid (PA) for 24 h to induce lipid deposition. Cellular lipid deposition was detected by oil red staining. Apoptosis was observed by TUNEL. LPC was determined by ELISA, and the expression of PNPLA3, the ER stress marker Bip, molecules involved in the ER stress PERK/elF-2a pathway, and its downstream C/EBP homologous protein (CHOP)-mediated apoptotic pathway were detected by western blot. Results: The results showed no difference in PNPLA3 basal expression and basal hepatocyte lipid content between the three genotypes of cells. Lipid deposition and apoptosis were more severe in PNPLA3 148M/M and 148I/M cells than in I/I cells after PA treatment. PA-induced upregulation of protein expression of Bip, ER stress-responsive PERK pathway molecules p-PERK, p-eIF2α, CHOP, and CHOP-associated apoptotic molecules PUMA and Bax were more pronounced in PNPLA3 148M/M cells than in PNPLA3 148I/I cells. The basal LPC levels and the PA-treated increase of LPC levels in the cell culture supernatants did not differ between the three genotypic cells. Conclusion: PNPLA3 148M/M cells were more susceptible to PA-induced lipid deposition and ER stress-related apoptosis than 148I/I cells, and the proapoptotic susceptibility of PNPLA3 148M/M is independent of LPC.

The Multiple Influences of Natural Farming Environment on the Cultured Population Behavior of Kuruma Prawn, Penaeus japonicus.

Recent years have witnessed a tremendous development in shrimp farming around the world, which, however, has raised a variety of issues, possibly due to a lack of knowledge of shrimp behavior in farms. This study focused on the relationship between shrimp behavior and the various factors of natural farming environment through situ surveys, as distinguished from the majority of laboratory studies on shrimp behavior. In the survey, the behaviors of kuruma prawn (Penaeus japonicus) were investigated in the groups of swimming in the water, crawling on the sand, resting on the sand, and hiding in the sand, followed by the quantification of the sex ratio, water quality, density, and light intensity. The results showed the average proportions of resting, hiding, crawling, and swimming activities of 69.87%, 20.85%, 8.24%, and 1.04%, respectively, of P. japonicus. The behavior of hiding, resting, and crawling is significantly affected by the sex ratio of the shrimp (p < 0.05). The proportions of hiding behavior exhibited a negative connection with density and a positive connection with light intensity, while the proportions of resting behavior showed the opposite according to both Pearson correlation analysis and multiple linear regression analysis. The light intensity was the only factor that significantly influenced the swimming behavior, in which the probability of the swimming behavior was reduced from 48% to 5% when light intensity varied from 0 to 10 lx, as determined by the generalized linear model. It could be speculated that P. japonicus prefers a tranquil environment. Female shrimp might exhibit less aggression and more adventure compared to male shrimp. The findings suggested light intensity, followed by density, as the most crucial element influencing the behavior of P. japonicus in the culture environment. These findings will contribute to the comprehension of the behavior of P. japonicus and provide a novel perspective for the formulation of its culture management strategy.

Analysis Model of the Influence of Self-Efficacy on Professional Toughness of Preschool Teachers under the Condition of Ensuring Children's Mental Health and Healthy Family Environment.

Preschool teachers' professional toughness (PT) has received a lot of attention recently, and this research is one of the main areas of study for their ability to manage stress and develop normally. Based on how well a person thinks of his own abilities, SE (self-efficacy) is formed. 300 kindergarten teachers from 10 metropolitan kindergartens were chosen as research samples in order to examine the relationship between the SE of kindergarten teachers and their PT, as well as the mediating effects of professional identity and work passion. A paradigm for measuring teacher performance based on the BPNN (BP neural network) is proposed, and several data processing techniques are developed. The study's findings indicate that teachers' WE (work enthusiasm) fills a limited intermediary role between PT and SE; the intermediary effect value is 0.041 and accounts for 15.7% of the overall effect (0.6559). The conclusion is that in order to help new instructors cope with their negative feelings, it is important to engage in a variety of leisure activities.

Enzyme-linked immunosorbent assays for quantification of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera.

Antibody-drug conjugates (ADCs) are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies. In this study, we report the generation of a monoclonal antibody against monomethyl auristatin E (MMAE) and the development, validation, and application of sensitive and high-throughput enzyme-linked immunosorbent assays (ELISA) to measure the concentrations of MMAE-conjugated ADCs and total antibodies (tAb, antibodies in ADC plus unconjugated antibodies) in cynomolgus monkey sera. These assays were successfully applied to in vitro plasma stability and pharmacokinetic (PK) studies of SMADC001, an MMAE-conjugated ADC against trophoblast cell surface antigen 2 (TROP-2). The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit, Lys (m-dPEG24)-Cit, and Val-Cit linkers. The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between serum concentrations and the OD450 values, with R 2 at 1.000, and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL, respectively; the intra- and inter-assay accuracy bias% ranged from -12.2% to -5.2%, precision ranged from -12.4% to -1.4%, and the relative standard deviation (RSD) was less than 6.6% and 8.7%, respectively. The total error was less than 20.4%. The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters, which suggested that these can be applied to quantify MMAE-conjugated ADCs, as well as in PK studies. Furthermore, these assays can be easily adopted for development of other similar immunoassays.

CCDC65, a Gene Knockout that leads to Early Death of Mice, acts as a potentially Novel Tumor Suppressor in Lung Adenocarcinoma.

CCDC65 is a member of the coiled-coil domain-containing protein family and was only reported in gastric cancer by our group. We first observed that it is downregulated in lung adenocarcinoma based on the TCGA database. Reduced CCDC65 protein was shown as an unfavorable factor promoting the clinical progression in lung adenocarcinoma. Subsequently, CCDC65-/- mice were found possibly dead of hydrocephalus. Compared with the CCDC65+/+ mice, the downregulation of CCDC65 in CCDC65+/- mice significantly increased the formation ability of lung cancer induced by urethane. In the subsequent investigation, we observed that CCDC65 functions as a tumor suppressor repressing cell proliferation in vitro and in vivo. Molecular mechanism showed that CCDC65 recruited E3 ubiquitin ligase FBXW7 to induce the ubiquitination degradation of c-Myc, an oncogenic transcription factor in tumors, and reduced c-Myc binding to ENO1 promoter, which suppressed the transcription of ENO1. In addition, CCDC65 also recruited FBXW7 to degrade ENO1 protein by ubiquitinated modulation. The downregulated ENO1 further reduced the phosphorylation activation of AKT1, which thus inactivated the cell cycle signal. Our data demonstrated that CCDC65 is a potential tumor suppressor by recruiting FBWX7 to suppress c-Myc/ENO1-induced cell cycle signal in lung adenocarcinoma.

SIRT1 mediates nutritional regulation of SREBP-1c-driven hepatic PNPLA3 transcription via modulation of H3k9 acetylation.

BACKGROUND: Patatin-like phospholipase domain containing 3 (PNPLA3) is the main nonalcoholic fatty liver disease (NAFLD) susceptibility. Its expression is regulated tightly by nutritional and energy status, but the mechanism of epigenetic regulation of PNPLA3 gene by nutritional dietary factors has not been reported. Here, we investigated the effect and mechanism of Sirtuin 1 (SIRT1) regulated H3K9 deacetylation on PNPLA3 transcriptional expression in vivo and in vitro. METHODS: Mouse models of fasting/re-feeding transition and nonalcoholic fatty liver induced by high Sucrose diet were constructed; and HepG2 cells were treated with serum- and glucose-free medium or exposed to high glucose and high insulin, to generate fasting and high-glucose-induced lipid deposition cell states. Enrichment levels of histone H3K9 acetylation and sterol responsive element binding protein-1c (SREBP-1c) at the PNPLA3 promoter were observed by ChIP-qPCR. PNPLA3 gene expression was detected by real-time PCR; SIRT1 protein expression was detected by western blot. And lipid deposition was detected by Oil Red O. RESULTS: H3K9ac levels at SRE regions of PNPLA3 promoter were found to be decreased in mice during fasting and increase during refeeding, and increased in mice with NAFLD induced by high-sucrose diet. The change pattern of PNPLA3 promoter H3K9Ac physiologically (fasting/refeeding) and pathologically was consistent with that of PNPLA3 gene expression, but opposite to that of SIRT1 protein expression. In HepG2 cells, overexpression of SIRT1 inhibited high-glucose induced hyper-acetylation of H3K9 at PNPLA3 promoter, and silent expression of SIRT1 suppressed fasting-induced hypo-acetylation of H3K9. Overexpression of SIRT1 prevented basal and SREBP-1c-driven PNPLA3 gene expression and also prevented the endogenous binding of SREBP-1c to PNPLA3. CONCLUSIONS: We first preliminarily revealed SIRT1 may regulate PNPLA3 gene expression by affecting SREBP-1-driven transcription via acetylation modification of H3K9.

A homogeneous time-resolved fluorometric energy transfer assay for the binding assessment of FcRn with IgG antibodies.

We aimed to develop a homogeneous time-resolved fluorometric energy transfer assay for assessment of human neonatal Fc receptor binding activity with IgG-type antibodies. The assay was configured with FcRn-coupled with Eu cryptate via biotin and streptavidin interaction as donor and IgG1 labeled with d2 as acceptor. Only a single incubation step was involved and no wash step was required. The assay demonstrated good accuracy, precision, linearity and specificity. Our further investigation with a rat pharmacokinetics study revealed that the terminal t1/2 for Trastuzumab and its related three ADCs agreed with the EC50 data. The assay can be applied to various IgGs with modifications to identify antibodies with appropriate binding ability to human FcRn.

CCDC65 as a new potential tumor suppressor induced by metformin inhibits activation of AKT1 via ubiquitination of ENO1 in gastric cancer.

The coiled-coil domain containing protein members have been well documented for their roles in many diseases including cancers. However, the function of the coiled-coil domain containing 65 (CCDC65) remains unknown in tumorigenesis including gastric cancer. Methods: CCDC65 expression and its correlation with clinical features and prognosis of gastric cancer were analyzed in tissue. The biological role and molecular basis of CCDC65 were performed via in vitro and in vivo assays and a various of experimental methods including co-immunoprecipitation (Co-IP), GST-pull down and ubiquitination analysis et al. Finally, whether metformin affects the pathogenesis of gastric cancer by regulating CCDC65 and its-mediated signaling was investigated. Results: Here, we found that downregulated CCDC65 level was showed as an unfavourable factor in gastric cancer patients. Subsequently, CCDC65 or its domain (a.a. 130-484) was identified as a significant suppressor in GC growth and metastasis in vitro and in vivo. Molecular basis showed that CCDC65 bound to ENO1, an oncogenic factor has been widely reported to promote the tumor pathogenesis, by its domain (a.a. 130-484) and further promoted ubiquitylation and degradation of ENO1 by recruiting E3 ubiquitin ligase FBXW7. The downregulated ENO1 decreased the binding with AKT1 and further inactivated AKT1, which led to the loss of cell proliferation and EMT signal. Finally, we observed that metformin, a new anti-cancer drug, can significantly induce CCDC65 to suppress ENO1-AKT1 complex-mediated cell proliferation and EMT signals and finally suppresses the malignant phenotypes of gastric cancer cells. Conclusion: These results firstly highlight a critical role of CCDC65 in suppressing ENO1-AKT1 pathway to reduce the progression of gastric cancer and reveals a new molecular mechanism for metformin in suppressing gastric cancer. Our present study provides a new insight into the mechanism and therapy for gastric cancer.

Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11-RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

Profiling pharmacokinetics of double-negative T cells and cytokines via a single intravenous administration in NSG mice.

This study was intended to delineate the profile of double-negative T cells (DNTs) in NOD.Cg-Prkdcscid Il2rgtm1wj /SzJ mice and cytokines released from DNTs in vivo and in vitro. Total 4 × 107 cells of RC1012 injection per mice were intravenously infused. IFN-γ, TNF-α, IL-1ß, IL-2, IL-4, IL-6, IL-10 were measured in vivo and in vitro. A quantitative polymerase chain reaction (PCR) was employed to determine the gene copies of Notch2-NLA per DNT cell from collected organs. Cytokines were significantly increased in vitro (4 h) and in vivo (3 h). DNT cells were distributed into the lung, liver, heart, and kidney earlier, and redistributed to lymphocyte homing spleen and bone marrow, which seemed to frame a two-compartment pharmacokinetics (PK) model but more data are needed to confirm this, and the clearance of DNT cells fell into first-order kinetics.

VPS33B suppresses lung adenocarcinoma metastasis and chemoresistance to cisplatin.

The presence of VPS33B in tumors has rarely been reported. Downregulated VPS33B protein expression is an unfavorable factor that promotes the pathogenesis of lung adenocarcinoma (LUAD). Overexpressed VPS33B was shown to reduce the migration, invasion, metastasis, and chemoresistance of LUAD cells to cisplatin (DDP) in vivo and in vitro. Mechanistic analyses have indicated that VPS33B first suppresses epidermal growth factor receptor (EGFR) Ras/ERK signaling, which further reduces the expression of the oncogenic factor c-Myc. Downregulated c-Myc expression reduces the rate at which it binds the p53 promoter and weakens its transcription inhibition; therefore, decreased c-Myc stimulates p53 expression, leading to decreased epithelial-to-mesenchymal transition (EMT) signal. NESG1 has been shown to be an unfavorable indicator of non-small-cell lung cancer (NSCLC). Here, NESG1 was identified as an interactive protein of VPS33B. In addition, NESG1 was found to exhibit mutual stimulation with VPS33B via reduced RAS/ERK/c-Jun-mediated transcription repression. Knockdown of NESG1 activated EGFR/Ras/ERK/c-Myc signaling and further downregulated p53 expression, which thus activated EMT signaling and promoted LUAD migration and invasion. Finally, we observed that nicotine suppressed VPS33B expression by inducing PI3K/AKT/c-Jun-mediated transcription suppression. Our study demonstrates that VPS33B as a tumor suppressor is significantly involved in the pathogenesis of LUAD.

miR-1254 induced by NESG1 inactivates HDGF/DDX5-stimulated nuclear translocation of ß-catenin and suppresses NPC metastasis.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Chinese and other Southeast Asians. We aimed to explore the precise mechanism for NESG1 in NPC for understanding the pathogenesis of NPC. Transwell, Boyden assays, and wounding healing were respectively performed for cell metastasis. The microRNA (miRNA) microarray and luciferase reporter assays were designed to clarify NESG1-modulated miRNAs and miR-1254-targeted protein. Western blotting assays examined the pathways regulated by miR-1254, the (Hepatoma-Derived Growth Factor) HDGF/DDX5 complex, and NESG1. The chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and co-immunoprecipitation (coIP) assays were used to explore the DNA-protein complex and protein-protein complex. NESG1 suppressed NPC migration and invasion via Wnt/ß-catenin signaling. Further, miR-1254 was confirmed as a positive downstream modulator of NESG1 reducing metastatic abilities of NPC cells in vivo and in vitro. Transduction of HDGF significantly restored cell migration and invasion ability in miR-1254-overexpressing NPC cells. In clinical samples, miR-1254 expression was negatively correlated with HDGF and positively correlated with NESG1 expression. miR-1254 acts as an independent prognostic factor for NPC, which was induced by NESG1 to suppress NPC metastasis via inactivating Wnt/ß-catenin pathway and its downstream EMT signals.

Construction of a Prognostic Immune Signature for Squamous-Cell Lung Cancer to Predict Survival.

Background: Limited treatment strategies are available for squamous-cell lung cancer (SQLC) patients. Few studies have addressed whether immune-related genes (IRGs) or the tumor immune microenvironment can predict the prognosis for SQLC patients. Our study aimed to construct a signature predict prognosis for SQLC patients based on IRGs. Methods: We constructed and validated a signature from SQLC patients in The Cancer Genome Atlas (TCGA) using bioinformatics analysis. The underlying mechanisms of the signature were also explored with immune cells and mutation profiles. Results: A total of 464 eligible SQLC patients from TCGA dataset were enrolled and were randomly divided into the training cohort (n = 232) and the testing cohort (n = 232). Eight differentially expressed IRGs were identified and applied to construct the immune signature in the training cohort. The signature showed a significant difference in overall survival (OS) between low-risk and high-risk cohorts (P < 0.001), with an area under the curve of 0.76. The predictive capability was verified with the testing and total cohorts. Multivariate analysis revealed that the 8-IRG signature served as an independent prognostic factor for OS in SQLC patients. Naive B cells, resting memory CD4 T cells, follicular helper T cells, and M2 macrophages were found to significantly associate with OS. There was no statistical difference in terms of tumor mutational burden between the high-risk and low-risk cohorts. Conclusion: Our study constructed and validated an 8-IRG signature prognostic model that predicts clinical outcomes for SQLC patients. However, this signature model needs further validation with a larger number of patients.