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.

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 negatively modulated by nicotine functions as a tumor suppressor in colorectal cancer.

The biological role of vacuolar protein sorting 33B (VPS33B) has not been examined in colorectal cancer (CRC). We report that VPS33B was downregulated in dextran sulfate sodium/azoxymethane (DSS/AOM) -induced CRC mice models and nicotine-treated CRC cells via the PI3K/AKT/c-Jun pathway. Reduced VPS33B is an unfavorable factor promoting poor prognosis in human CRC patients. VPS33B overexpression suppressed CRC proliferation, intrahepatic metastasis and chemoresistance of cisplatin (DDP) in vivo and in vitro through modulating the epidermal growth factor receptor (EGFR)/RAS/ERK/c-Myc/p53/miR-133a-3p feedback loop and the downstream cell cycle or EMT-related factors. Furthermore, NESG1 as a newly identified tumor suppressor interacted with VPS33B via colocalization in the cytoplasm, and it was stimulated by VPS33B through the downregulation of RAS/ERK/c-Jun-mediated transcription. NESG1 also activated VPS33B expression via the RAS/ERK/c-Jun pathway. Suppression of NESG1 increased cell growth, migration and invasion via the reversion of the VPS33B-modulating signal in VPS33B-overexpressed cells. Taken together, VPS33B as a tumor suppressor is easily dysregulated by chemical carcinogens and it interacts with NESG1 to modulate the EGFR/RAS/ERK/c-Myc/p53/miR-133a-3p feedback loop and thus suppress the malignant phenotype of CRC.

miR-296-3p Negatively Regulated by Nicotine Stimulates Cytoplasmic Translocation of c-Myc via MK2 to Suppress Chemotherapy Resistance.

This study aimed to identify mechanisms by which microRNA 296-3p (miR-296-3p) functions as a tumor suppressor to restrain nasopharyngeal carcinoma (NPC) cell growth, metastasis, and chemoresistance. Mechanistic studies revealed that miR-296-3p negatively regulated by nicotine directly targets the oncogenic protein mitogen-activated protein kinase-activated protein kinase-2 (Mapkapk2) (MK2). Suppression of MK2 downregulated Ras/Braf/Erk/Mek/c-Myc and phosphoinositide-3-kinase (PI3K)/Akt/c-Myc signaling and promoted cytoplasmic translocation of c-Myc, which activated miR-296-3p expression by a feedback loop. This ultimately inhibited cell cycle progression, epithelial-to-mesenchymal transition (EMT), and chemoresistance of NPC. In addition, nicotine as a key component of tobacco was observed to suppress miR-296-3p and thus elevate MK2 expression by inducing PI3K/Akt/c-Myc signaling. In clinical samples, reduced miR-296-3p as an unfavorable factor was inversely correlated with MK2 and c-Myc expression. These results reveal a novel mechanism by which miR-296-3p negatively regulated by nicotine directly targets MK2-induced Ras/Braf/Erk/Mek/c-Myc or PI3K/AKT/c-Myc signaling to stimulate its own expression and suppress NPC cell proliferation and metastasis. miR-296-3p may thus serve as a therapeutic target to reverse chemotherapy resistance of NPC.

Determination of glutathione in apoptotic SMMC-7221 cells induced by xylitol selenite using capillary electrophoresis.

OBJECTIVE: To determine the glutathione (GSH) content in a human hepatoma cell line (SMMC-7221) treated with xylitol/selenite, providing a part of an investigation of its anti-cancer mechanisms. RESULTS: The nuclei of SMMC-7221 cells were stained with Hoechst 33258 in an apoptosis assay, and their morphology subsequently changed from circular to crescent shape. The calibration curve (r(2) = 0.992) was established, and GSH content markedly decreased after treated with 0.5 and 1 mg xylitol/selenite l(-1) for 12, 36 and 60 h (12 h: from 95.57 ± 19.57 to 29.09 ± 7.74 and 24.27 ± 11.15; 36 h: from 70.73 ± 11.35 to 19.54 ± 6.39 and 9.35 ± 6.69; 60 h: from 72.63 ± 16.94 to 7.432 ± 3.84 and 0). The depletion rate of GSH was more related to the concentration of xylitol/selenite than the treatment time (from 69.95 ± 1.87 to 100 % vs. 0.22 ± 0.2 to 100 %). CONCLUSIONS: Xylitol/selenite is a promising anti-cancer drug to induce apoptosis in SMMC-7221 cells. It may regulate the apoptosis through the co-action of multiple mechanisms related to GSH depletion.

Direct interaction between miR-203 and ZEB2 suppresses epithelial-mesenchymal transition signaling and reduces lung adenocarcinoma chemoresistance.

miR-203 is a tumor suppressor which participates in the pathogenesis of many tumors including lung adenocarcinoma. However, the role of miR-203 in suppressing chemotherapy resistance to cisplatin (cis-diamminedichloroplatinum; DDP) as well as its molecular mechanism is still to be determined in lung adenocarcinoma. In this study, we found that miR-203 decreased lung cancer cell migration and invasion, and that increased miR-203 expression sensitized lung adenocarcinoma cells to DDP in vitro Furthermore, ZEB2 was found to be a direct target of miR-203, which induces epithelial-mesenchymal transition (EMT) signal. Knock-down of ZEB2 significantly increased DDP chemosensitivity in lung adenocarcinoma. More interestingly, we also demonstrated that ZEB2 could directly bind to E-box of the miR-203 promoter and suppress its expression in lung adenocarcinoma. Our data reveal that miR-203 serves as a negative feedback by directly suppressing the upstream ZEB2 gene, which inhibits EMT signaling and reduces chemoresistance of DDP. Together, these results highlight a feedback loop between miR-203 and ZEB2, which participates in the pathogenesis of lung adenocarcinoma.

Enhanced liver functions of HepG2 cells in the alginate/xyloglucan scaffold.

A scaffold provides a framework and initial support for the cells to attach, proliferate and differentiate, and form an extracellular matrix (ECM) in tissue engineering. Here, xyloglucan (XG) was used as a new synthetic ECM for HepG2 cell attachment in alginate capsules. The effects of XG on HepG2 cells on adherent behavior, albumin secretion, ammonia elimination, cell proliferation and gene expression of Connexin 32 and epithelial-cadherin were investigated. Xyloglucan could also promote the HepG2 cell-matrix interactions and the cell clusters formation of HepG2 cells in three dimensional scaffold, thus enhance the liver-specific functions in the three-dimensional space.

[Measurement of Rho-kinase and CD4+CD25+ regulatory T cells in the peripheral blood in asthmatic patients].

OBJECTIVE: To determine the levels of Rho-kinase and CD4+CD25+ regulatory T cells in patients with asthma, and the relationship between Rho-kinase and CD4+CD25+ regulatory T cells. METHODS: We included 16 patients with moderate to severe asthma in the research group and 14 healthy people as the control group. The levels of Rho-kinase in the 2 groups were measured by ELISA. The level of CD4+CD25+ regulatory T cells in the 2 groups was measured by flow cytometry. The pulmonary function was measured by spirometer. RESULTS: The level of Rho-kinase in the research group was higher than that in the healthy controls (P<0.05). The level of CD4+CD25+ regulatory T cells in the healthy controls was higher than that of the research group (P<0.05). There was no correlation between the level of Rho-kinase in the peripheral blood of the 2 groups and forced expiratiory volume at the first second/ forced vital capacity (FEV1%) (r=-0.491, P>0.05). The level of CD4+CD25+ regulatory T cells in the peripheral blood of the 2 groups showed a positive correlation with FEV1% (r=0.380, P=0.038). There was no correlation between the level of Rho-kinase and the level of CD4+CD25+ regulatory T cells in the peripheral blood of the 2 groups (r=-0.438, P>0.05). CONCLUSION: Rho-kinase and CD4+CD25+ regulatory T cells may play a key role in the pathogenesis of asthma.

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.

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.

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.

Dynamic sieving capillary electrophoresis analysis of xylitol selenite-induced apoptosis in SMMC-7221 cells.

DNA ladder fragments, regarded as a biochemical hallmark of apoptosis, have been separated quickly and successfully by capillary electrophoresis. Inter-nucleosomal DNA fragmentations induced by xylitol selenite were determined for the first time, while hydroxypropylmethylcellulose (HPMC) was served as the sieving matrix in dynamic sieving capillary electrophoresis. The calibration curve (r(2) = 0.991) was established and multiples of two different nucleosomes (140 and 180 bp) were formed in the presence of xylitol selenite. Selenium compounds inhibited carcinogenesis in animal models, SMMC-7221 cells and several other cells by increasing apoptosis. The described method was useful in elucidating the anticancer activities of xylitol selenite and other selenium compounds, which was more effective to detect small fragments than slab gel electrophoresis.

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.

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.

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.

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.

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.

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.