Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells.

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: • The recombinant therapeutic protein degradation in CHO cell systems is reviewed. • Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. • Reducing the degradation can improve the quality of recombinant therapeutic proteins.

The rapid identification of chemical constituents in Fufang Xiling Jiedu capsule, a modern Chinese medicine, by ultra-performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry and data mining strategy.

Fufang Xiling Jiedu capsule is an effective Chinese medicine widely used for the treatment of cold and influenza. However, its chemical constituents had not been determined, which entailed a huge obstacle to further pharmacological studies, clinical-safe medication administration, and quality evaluation. To identify the chemical constituents in Fufang Xiling Jiedu capsule, an efficient and systematic approach using ultra-high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry in conjunction with a data mining strategy was adopted in this study. As a result, 145 compounds were qualitatively identified, including 26 phenolic acids, 46 flavonoids, 39 triterpenes, and 34 other compounds, among which 6 were potentially new and 144 were being reported from Fufang Xiling Jiedu capsule for the first time. This research not only provides useful information for quality control of Fufang Xiling Jiedu capsule and its involved single herbs but also serve as basis data for further study of Fufang Xiling Jiedu capsule in vivo. Moreover, it provides a reference for the characterization of the chemical constituents of other Chinese medicine preparations.

Zwitterionic Cellulose Nanofibrils with High Salt Sensitivity and Tolerance.

To improve the salt tolerance/sensitivity of cellulose nanofibrils (CNFs), zwitterionic cellulose nanofibrils (ZCNFs) were prepared from softwood bleached kraft pulp fibers via a sequential process of anionic modification with 2,2,6,6-tetramethylepiperidin-1-oxyl (TEMPO)-mediated oxidation, cationic modification with (2,3-epoxypropyl) trimethylammonium chloride (EPTMAC), and high-pressure homogenization. To produce ZCNFs with different contents of cation group, EPTMAC loadings of 0.15 to 1.15 g/g fiber were explored during cationization. The obtained ZCNFs were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectra (XPS), thermogravimetric analysis (TGA), transmission electron microscopy (TEM), and rheological measurements. The salt tolerance of the ZCNFs was investigated by adding mixed salts into the ZCNF dispersions. The results demonstrated that the ZCNFs with both anionic and cationic charges were produced. Compared with the TEMPO-mediated oxidized cellulose nanofibrils (TOCNFs), the ZCNFs exhibited an excellent "salt-thickening" behavior under the studied salt concentrations (2-24% w/w). Moreover, increasing the content of the cation group increased the salt tolerance/sensitivity of ZCNFs. This work demonstrated that introducing cationic charges to the anionic charged TOCNFs imparts the produced ZCNFs with excellent salt sensitivity and tolerance, which could expand the application of nanocellulose in oil recovery or wastewater treatment.

TGF-ß and BMP signals regulate insect diapause through Smad1-POU-TFAM pathway.

The transforming growth factor-ß (TGF-ß) superfamily signaling pathway contains two general branches, known as TGF-ß and bone morphogenetic protein (BMP), that regulate development in animals. It is well known that TGF-ß superfamily signaling participates in the regulation of dauer (lifespan extension) in Caenorhabditis elegans, but little is known about the molecular mechanisms of lifespan extension in the pathway. Diapause, a programmed developmental arrest in insects, is similar to dauer in C. elegans. In this study, we find that TGF-ß superfamily signaling regulates Helicoverpa armigera diapause via a novel mechanism. Both TGF-ß and BMP signals are weaker in the brains of diapause-destined pupae than in nondiapause-destined pupae, and the levels of p-Smad1, POU, TFAM, and mitochondrial activity are decreased in diapause pupae. Development in nondiapause pupae is delayed by an injection of TGF-ß or BMP receptor inhibitors. Both TGF-ß and BMP signals can activate a common target, Smad1. ChIP and EMSA assays indicate that Smad1 can bind to the POU promoter to regulate its expression. POU can improve the transcription of TFAM, which regulates mitochondrial activity. This is the first report showing that both TGF-ß and BMP signals regulate development or diapause through the Smad1-POU-TFAM-mitochondrial activity in insects.

Downregulation of ZMYND11 induced by miR-196a-5p promotes the progression and growth of GBM.

ZMYND11 (zinc finger MYND-type containing 11) has been widely regarded to be involved in a variety of cancers as a potential suppressor. However, the biological role and mechanism of ZMYND11 in glioblastoma multiform (GBM) remain unknown. In this study, we found that ZMYND11 expression was remarkably decreased in GBM tissues from 20 cases and cell line (U87) compared to normal brain tissue from 10 cases (P < 0.001). Furthermore, we explored that ZMYND11 upregulation significantly suppressed U87 cells proliferation and invasion, induced cell cycle arrest and apoptosis in vitro. Subsequently, we identified increased ZMYND11 inhibited the tumor growth using tumor cells xenograft experiment on rude mice. Moreover, we explored that ZMYND11 was a new direct and functional target of miR-196a-5p in U87 via luciferase reporter assay. In addition, we confirmed the negative correlation between miR-196a-5p and ZMYND11 in GBM tissue and U87 cells by changing the expression level of miR-196a-5p with lentivirus and plasmid vector. Furthermore, we demonstrated that decreased ZMYND11 could reverse suppressive effect of downregulated miR-196a-5p on U87 by rescue experiment. Taken together, ZMYND11 was demonstrated to be a potential and extremely promising suppressor of GBM, while miRNA-196a-5p was quite an important target of treatment of GBM.

Acevaltrate promotes apoptosis and inhibits proliferation by suppressing HIF-1α accumulation in cancer cells.

Acevaltrate is a natural product isolated from the roots of Valeriana glechomifolia F.G.Mey. (Valerianaceae) and has been shown to exhibit anti-cancer activity. However, the mechanism by which acevaltrate inhibits tumor growth is not fully understood. We here demonstrated the effect of acevaltrate on hypoxia-inducible factor-1α (HIF-1α) expression. Acevaltrate showed a potent inhibitory activity against HIF-1α induced by hypoxia in various cancer cells. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently. Further analysis revealed that acevaltrate inhibited HIF-1α protein synthesis and promoted degradation of HIF-1α protein, without affecting the expression level of HIF-1α mRNA. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by acevaltrate. In addition, acevaltrate promoted apoptosis and inhibited proliferation, which was potentially mediated by suppression of HIF-1α. We also found that acevaltrate administration inhibited tumor growth in mouse xenograft model. Taken together, these results suggested that acevaltrate was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of acevaltrate against cancers.

CBR1 decreases protein carbonyl levels via the ROS/Akt/CREB pathway to extend lifespan in the cotton bollworm, Helicoverpa armigera.

Reactive oxygen species (ROS) are considered a major cause of ageing and ageing-related diseases through protein carbonylation. Little is known about the molecular mechanisms that confer protection against ROS. Here, we observed that, compared with nondiapause-destined pupae, high protein carbonyl levels are present in the brains of diapause-destined pupae, which is a 'non-ageing' phase in the moth Helicoverpa armigera. Protein carbonyl levels respond to ROS and decrease metabolic activity to induce diapause in order to extend lifespan. However, protein carbonylation in the brains of diapause-destined pupae still occurs at a physiological level compared to young adult brains. We find that ROS activate Akt, and Akt then phosphorylates the transcription factor CREB to facilitate its nuclear import. CREB binds to the promoter of carbonyl reductase 1 (CBR1) and regulates its expression. High CBR1 levels reduce protein carbonyl levels to maintain physiological levels. This is the first report showing that the moth brain can naturally control protein carbonyl levels through a distinct ROS-Akt-CREB-CBR1 pathway to extend lifespan.

A review of novel research technology to explore the mystery of traditional Chinese medicine: Terahertz.

During the 2022 Annual National Terahertz Biophysics Conference, the hypothesis was proposed that bio frequency electromagnetic fields sensitive points, akin to acupuncture points, exist in the human body. This development has prompted numerous researchers to apply terahertz technology to the field of traditional Chinese medicine (TCM). In recent years, terahertz technology has achieved notable progress in the field of TCM, particularly concerning the meridian-collateral system. This review systematically presents the advancements in terahertz technology and its implications on TCM theory from a biophysical perspective. Additionally, it summarizes the utilization of terahertz waves in elucidating aspects of TCM, particularly focusing on the scientific connotation of Qi, the theoretical foundation of the meridian-collateral system, and moxibustion in diagnosing and treating diseases. We aimed to explore the innovative applications and distinct advantages of terahertz technology in TCM and its feasibility as a pioneering technological tool for the modernization of TCM.

COXIV and SIRT2-mediated G6PD deacetylation modulate ROS homeostasis to extend pupal lifespan.

Previous studies have shown that high physiological levels of reactive oxygen species (ROS) in the brain promote pupal diapause, which extends the pupal lifespan. However, the molecular mechanisms of ROS generation are unclear. In this paper, we found that mitochondrial ROS (mtROS) levels in the brains of Helicoverpa armigera diapause-destined pupae (DP) were higher and that the expression of cytochrome oxidase subunit IV (COXIV) was lower than in NP. In addition, downregulating COXIV caused mitochondrial dysfunction which elevated mtROS levels. Protein kinase A (PKA) was downregulated in DP, which led to the downregulated expression of the mitochondrial transcription factor TFAM. Low TFAM activity failed to promote COXIV expression and resulted in the high ROS levels that induced diapause. In addition, low sirtuin 2 expression suppressed glucose-6-phosphate dehydrogenase (G6PD) deacetylation at K382, which led to reduced G6PD activity and low NADPH levels, thereby maintaining high levels of ROS. Two proteins, COXIV and G6PD, thus play key roles in the elevated accumulation of ROS that induce diapause and extend the pupal lifespan.

P-S6K is associated with insect diapause via the ROS/AKT/ S6K/CREB/HIF-1 pathway in the cotton bollworm, Helicoverpa armigera.

Diapause is a complex physiological response that allows insects to survive unfavorable environmental conditions, and many signaling pathways participate in regulating this process. However, little is known about TOR signaling in the regulation of diapause. In this study, we found that the TOR pathway-related proteins TOR and Raptor are expressed at low levels in the brains of diapause-destined pupae of Helicoverpa armigera, consistent with a previous report that TOR signaling is associated with development. Interestingly, another TOR signaling-related protein, p-S6K, was increased in the brains of diapause-destined pupae. Our results showed that p-S6K in the brains of diapause-destined pupae can respond to the upstream signals reactive oxygen species (ROS) and AKT and that S6K activates the level of CREB, which binds to the HIF-1α promoter and increases its expression. Previous study has shown that HIF-1α levels elevated by ROS in the brains of diapause-destined pupae cause low mitochondrial activity for insect diapause. Thus, p-S6K in response to ROS/AKT regulates HIF-1α via activating transcription factor CREB for diapause initiation.

Alternative splicing and expression analysis of HSF1 in diapause pupal brains in the cotton bollworm, Helicoverpa armigera.

BACKGROUND: Diapause is the arrest of the development of insects and can be used for the development of effective agricultural pest management strategies. Heat shock protein 70 (Hsp70) is reported to be up-regulated during diapause to maintain survival in some insect species. However, its regulatory mechanism is unknown. RESULTS: Expression of hsp70 in Helicoverpa armigera was found to be up-regulated in diapause pupal brains. To elucidate the molecular regulatory mechanisms of hsp70, we focused our attention on its transcription factor, heat shock factor 1 (HSF1). Four alternative splicing variants of HSF1 from pupal brains of H. armigera were identified, and subcellular localization analysis indicated that these variants were exclusively expressed in the nucleus. Real-time PCR analysis showed that all of these variants were up-regulated in diapause pupal brains, and their expression patterns were consistent with that of hsp70. Finally, promoter activity assay and Western blotting detection demonstrated that hsp70 was activated and up-regulated by these variants. CONCLUSION: Expression of hsp70 in H. armigera during diapause is regulated by multiple alternatively spliced isoforms of HSF1. The results of this study may provide important information for understanding the regulatory mechanisms of hsps during insect diapause. © 2018 Society of Chemical Industry.

Deacetylation of metabolic enzymes by Sirt2 modulates pyruvate homeostasis to extend insect lifespan.

Diapause in insects is akin to dauer in Caenorhabditis elegans and hibernation in vertebrates. Diapause causes a profound extension of lifespan by low metabolic activity. However, the detailed regulatory mechanisms for low metabolic activity remain unknown. Here, we showed that low pyruvate levels are present in the brains of diapause-destined pupae of the cotton bollworm Helicoverpa armigera, and three enzymes pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), and phosphoglycerate mutase (PGAM) are closely correlated with pyruvate homeostasis. Notably, Sirt2 can deacetylate the three enzymes to increase their activity in vitro. Thus, low Sirt2 expression in the brains of diapause individuals decreases PK and PEPCK protein levels as well as PGAM activity, resulting in low pyruvate levels and low tricarboxylic acid cycle activity and eventually inducing diapause initiation by low metabolic activity. These findings suggest that pyruvate is a checkpoint for development or lifespan extension, and Sirt2 is a negative regulator to extend lifespan in insects.

TGF-ß signaling regulates p-Akt levels via PP2A during diapause entry in the cotton bollworm, Helicoverpa armigera.

Akt, which is a key kinase in the insulin signaling pathway, plays important roles in glucose metabolism, cell proliferation, transcription and cell migration. Our previous studies indicated that low insulin levels and high p-Akt levels are present in diapause-destined individuals. Here, we show that PI3K, which is upstream of Akt, is low in diapause-destined pupal brains but high in p-Akt levels, implying that p-Akt is modified by factors other than the insulin signaling pathway. Protein phosphatase 2A (PP2A), which is a key regulator in the TGF-ß signaling pathway, can directly bind to and dephosphorylate Akt. Low PP2A expression and activity in diapause-destined individuals suggest that a weak Akt dephosphorylation contributes to p-Akt accumulation. In addition, transforming growth factor-ß receptor I (TßRI), which is upstream of PP2A, increases the activity of PP2A and decreases the p-Akt levels. These results show that TGF-ß signaling decreases p-Akt levels by increasing the activity of PP2A. This is the first report showing that TGF-ß signaling negatively regulates the insulin pathway in insect development or diapause.

Incidence and risk factors of retinopathy of prematurity in two neonatal intensive care units in North and South China.

BACKGROUND: To investigate the incidence and risk factors of retinopathy of prematurity (ROP) in two Neonatal Intensive Care Units in North and South of China, respectively. METHODS: We studied data concerning 472 infants with gestational age (GA) ≤ 34 weeks or birth weight (BW) ≤ 2000 g who were admitted to the Zhujiang Hospital of Southern Medical University and the Fourth Hospital of Shijiazhuang between January 1, 2011 and December 31, 2011. Clinical information about perinatal neonates was collected and was confirmed by reviewing medical charts. The incidence and severity of ROP were assessed in the screened population. Main outcome measures are the incidence and severity of ROP. The relationship of clinical risk factors and the development of ROP were analyzed. RESULTS: The overall incidence of ROP was 12.7%, and the overall incidence of type 1 ROP was 2.3%; 9.4% of infants in Zhujiang Hospital had ROP compared to 15.0% infants in the Fourth Hospital of Shijiazhuang developed ROP, and the difference is statistically significant. ROP was significantly associated with GA (odds ratio [OR]: 0.77 [0.62-0.95], P = 0.015), BW (OR: 0.998 [0.996-0.999], P = 0.008), maternal supplemental oxygen administration before and during delivery (OR: 4.27 [1.21-15.10], P = 0.024) and preeclampsia (OR: 6.07 [1.73-21.36] P = 0.005). The risk factors for ROP are different in two hospitals. In Zhujiang Hospital, BW is the independent risk factors for ROP while GA, BW and preeclampsia in the Fourth Hospital in Shijiazhuang Conclusions: Retinopathy of prematurity incidence is different based on area. Incidence of ROP is still high in China. More efforts need to prevent ROP.

Breast cancer stem cells: Multiple capacities in tumor metastasis.

Breast cancer is the leading cause of cancer death among women worldwide. Accumulating evidence indicates that the local recurrent and/or distant metastatic tumors, the major causes of lethality in the clinic, are related to the aggressive phenotype of a small fraction of cancer cells loosely termed as cancer stem cells (CSCs), tumor initiating cells (TICs), or cancer metastasis-initiating cells (CMICs). Breast cancer stem cells (BCSCs) are shown to exhibit unique growth abilities including self-renewal, differentiation potential, and resistance to most anti-cancer agents including chemo- and/or radiotherapy, all of which are believed to contribute to the development and overall aggressiveness of the recurrent or metastatic lesions. It is in the urgent need not only to further define the nature of heterogeneity in each tumor but also to characterize the precise mechanisms governing tumor-host cross-talk which is assumed to be initiated by BCSCs. In this review, we will focus on recently identified key factors, including the BCSCs among circulating tumor cells, interaction of BCSCs with the host, epithelial mesenchymal transition (EMT), tumor microenvironment, the intrinsic resistance due to HER2 expression, potential biomarkers of BCSCs and cancer cell immune signaling. We believe that new evidence coming from both bench and clinical research will help to develop more effective approaches to control or significantly reduce the aggressiveness of metastatic tumors.

Evaluation on the efficacy and safety of domestic bivalirudin during percutaneous coronary intervention.

BACKGROUND: Bivalirudin was widely used as an anticoagulant during coronary interventional procedure in western countries. However, it was not available in China before this clinical trial was designed. This randomized, single-blind and multicenter clinical trial aimed to evaluate the efficacy and the safety of domestic bivalirudin during percutaneous coronary intervention (PCI). METHODS: A randomized, single-blind, multicenter trial was designed. Elective PCI candidates in five centers were randomized into a bivalirudin group and a heparin group, which were treated with domestic bivalirudin and non-fractional heparin during the PCI procedure. The efficacy was evaluated by comparing the activated coagulation time (ACT), the procedural success rate (residual stenosis < 20% in target lesions without any coronary artery related adverse events within 24 hours after PCI), and the survival rate without major adverse cardiac events at 30 days after PCI between the two groups. Safety was evaluated by the major/minor bleeding rate. RESULTS: A total of 218 elective PCI patients were randomized into a bivalirudin group (n = 110) and heparin group (n = 108). Except for two patients needing additional dosing in the heparin group, the ACT values of all other patients in both groups were longer than 225 seconds at 5 minutes after the first intravenous bolus. Procedural success rates were respectively 100.0% and 98.2% in the bivalirudin group and heparin group (P > 0.05). Survival rates without major adverse cardiac events at 30 days after PCI were 100.0% in the bivalirudin group and 98.2% in the heparin group (P > 0.05). Mild bleeding rates were 0.9% and 6.9% (P < 0.05) at 24 hours, and 1.9% and 8.8% (P < 0.05) at 30 days after PCI in the bivalirudin group and heparin group respectively. There was one severe gastrointestinal bleeding case in the heparin group. CONCLUSIONS: Domestic bivalirudin is an effective and safe anticoagulant during elective PCI procedures. The efficacy is not inferior to heparin, but the safety is superior to heparin.