Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome.

Baculovirus expression vector system (BEVS) is a powerful and versatile platform for recombinant protein production in insect cells. As the most frequently used baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes 155 open reading frames (ORFs), including a considerable number of non-essential genes for the virus replication in cell culture. Studies have shown that protein production in BEVS can be improved by removing some viral dispensable genes, and these AcMNPV vectors also offer the possibility of accommodating larger exogenous gene fragments. In this study, we, respectively, deleted 14 DNA fragments from AcMNPV genome, each of them containing at least two contiguous genes that were known nonessential for viral replication in cell culture or functionally unknown. The effects of these fragment-deletions on virus replication and exogenous protein production were examined. The results showed that 11 of the 14 fragments, containing 43 genes, were dispensable for the virus replication in cultured cells. By detecting the expression of intracellularly expressed and secreted reporter proteins, we demonstrated that nine of the fragment-deletions benefited protein production in Sf9 cells and/or in High Five cells. After combining the deletion of some dispensable fragments, we obtained two AcMNPV vectors shortened by more than 10 kb but displayed an improved capacity for recombinant protein production. The deletion strategies used in this study has the potential to further improve the BEVS.

Extracellular Hsp90α, which participates in vascular inflammation, is a novel serum predictor of atherosclerosis in type 2 diabetes.

INTRODUCTION: Atherosclerosis is the main pathological change in diabetic angiopathy, and vascular inflammation plays an important role in early atherosclerosis. Extracellular heat shock protein 90 (eHsp90) is secreted into the serum and is involved in various physiological and pathophysiological processes. However, the specific mechanism of eHsp90 in early atherosclerosis remains unclear. This study explored the relationship between Hsp90 and diabetic lower extremity arterial disease and investigated the expression of eHsp90 in vascular endothelial cells under environmental stimulation and the function and mechanism of eHsp90α involved in diabetic atherosclerosis. RESEARCH DESIGN AND METHODS: One hundred and three selected patients were divided into three groups: the diabetes mellitus group (n=27), the diabetic lower extremity arterial disease group (n=46), and the diabetic critical limb ischemia group (n=30). The relationships among serum Hsp90, oxidative stress indexes, and patient outcomes and the correlations among the indexes were analyzed. H&E staining and immunohistochemistry were used to observe the vasculature of amputated feet from patients with diabetic foot. An oxidative stress endothelial injury model was established under high glucose in vitro to explore the role of eHsp90 release in atherosclerosis progression. RESULTS: The level of serum Hsp90 was upregulated with aggravation of diabetic vascular disease. Hsp90α was correlated with malondialdehyde to some extent and was an independent risk factor in the progression of diabetic vascular disease, with predictive ability. The expression area of Hsp90α was consistent with the area of inflammatory infiltration in the vessel lumen. Vascular endothelial cells were found to increase eHsp90α secretion under stress. Then inhibition of eHsp90α can reduce the degree of cellular inflammation and damage. Endothelial cell-conditioned medium and recombinant human Hsp90α increased monocyte migration via the low-denisity lipoprotein receptor-related protein 1 (LRP1) receptor to promote disease progression. CONCLUSIONS: eHsp90α plays a critical role in the early inflammatory injury stage of atherosclerosis. TRIAL REGISTRATION NUMBER: NCT04787770.

Development of a UPLC-MS/MS method for determination of a dual EZH1/2 inhibitor UNC1999 in rat plasma.

Aim: We aimed to establish and validate a simple and sensitive UPLC-MS/MS method for the determination of UNC1999, a dual inhibitor against EZH1 and EZH2 in plasma samples. Materials & methods: UNC1999 in rat plasma was processed with protein precipitation method and then separated on a C18 column and detected under positive ionization mode. The method presented good linearity over the range of 1.0-2000 ng/ml with good accuracy and precision. UNC1999 was absorbed slowly and achieved a maximum concentration of 118.8 ± 12.0 ng/ml 1.5 h after oral administration. Conclusion: The method provides a favorable character in selectivity, linearity, accuracy, precision, recovery, matrix effects and stabilities and was suitable for describing the pharmacokinetic profile of UNC1999.

Renewable energy technological innovation, market forces, and carbon emission efficiency.

Renewable energy technological innovation (RETI) is an important way to reduce carbon emissions and achieve sustainable development. Exploring whether RETI is beneficial to the improvement of carbon emission efficiency and how the market environment affects the role of RETI on carbon emission efficiency is critical to the design of effective policies. Therefore, based on the data from 25 provinces in China from 2002 to 2015, the Tobit fixed-effect model and the panel threshold model are used to investigate the impact and the mechanism of RETI on total factor carbon performance index (TCPI) from a market perspective. The results show that: (1) RETI can effectively improve TCPI, but this effect is affected by market factors; (2) With the reduction of market segmentation or the increase of market potential, the improvement effect of RETI on TCPI is significantly enhanced; (3) The panel threshold model further verifies that the impact of RETI on TCPI has a significant single threshold effect in terms of market segmentation and market potential; (4) There is an inverted "U-shaped" relationship between market segmentation and TCPI, and the increase of market potential is conducive to the improvement of TCPI. This paper provides corresponding policy implications for China to achieve the dual goals of economic transformation and carbon emission reduction.

Development of a UPLC-MS/MS Method for the Quantification of VS-5584 and Its Application in Pharmacokinetic Studies in Rats.

VS-5584 is a small-molecular compound that showed equivalent activity against mTOR and all class I PI3K isoforms and demonstrated preclinical activity in diverse cancer cell lines and xenograft tumor model, and rational combination of VS-5584 and other target therapies achieved promising outcomes in oncology. In the present study, we established and validated a simple and sensitive UPLC-MS/MS method for the determination of VS-5584 in plasma samples. VS-5584 was separated via an Acquity UPLC BEH C18 column, with a mobile phase composed of acetonitrile and 0.2% formic acid in water (40 : 60). The calibration curve displayed a good linearity in the range of 1.0-1000 ng/mL, with satisfactory accuracy (-13.6% < RE% < 8.8%) and precision (CV%, less than 9.2%). The validated method was then applied to a pharmacokinetic study in rats. After administration of 10 mg/kg, VS-5584 was absorbed quickly and reached a peak concentration of 473.2 ± 72.0 ng/mL after 20 min. The established method allows for the quantification of VS-5584 in rat plasma in detail and can be utilized to successfully describe the pharmacokinetic profile of VS-5584.

Development of a ultra-performance LC-MS/MS method for quantification of GW788388 and its applications in pharmacokinetic study in rats.

Aim: GW788388 is a selective and orally active TGF-ß1 receptor inhibitor that shows potent activity in renal fibrosis. We aimed to establish and validate a simple and sensitive ultra-performance LC-MS/MS method for the determination of GW788388 in plasma samples. Methodology & results: GW788388 in rat plasma was processed with protein precipitation method and then separated on a C18 column. The calibration curve presented a good linearity in the range of 1.0-1200 ng/ml, with satisfactory accuracy (relative error, [-17.5% < relative error <11.7%) and precision (CV <8.9%) for all quality control samples. After oral administration, GW788388 was absorbed quickly and reached a peak concentration of 595.3 ± 60.2 ng/ml after 20 min. Conclusion: The validated method provides a quantification method of GW788388 in rat plasma in detail, and can be utilized to successfully describe the pharmacokinetic profile of GW788388.

Quantitation of capmatinib, a mesenchymal-epithelial transition factor inhibitor by UPLC-MS/MS in rat plasma and its application to a pharmacokinetic study.

Aim: Capmatinib is an orally bioavailable mesenchymal-epithelial transition factor inhibitor with anticancer activity, which has proved preclinical activity in multiple cancer trials. The present study aimed to develop a fast and reliable assay approach to quantify capmatinib in rat plasma. Methodology & results: After protein precipitation with acetonitrile, the chromatographic separation was achieved with an Acquity UPLC BEH C18 column, and subsequently detected with positive electrospray ionization via a triple quadrupole tandem mass spectrometer. The target quantitative ion pairs m/z 412.99 â†’ 381.84 for capmatinib and 387.00 â†’ 355.81 for the internal standard, respectively. The calibration curve for the assay was linear over the range of 1.0-4000 ng/ml. Conclusion: The method shows an excellent performance in linearity, accuracy, precision, stability, and has been successfully applied to a pharmacokinetic study after oral administration of capmatinib at three doses (5, 10 and 20 mg/kg) in rats.

[Summarize drug dosage forms in treatment of age-related macular degeneration disease].

In this review, the authors summarized the drugs in treatment of the age-related macular degeneration (AMD or ARMD), including the pathogenesis of the age-related macular degeneration at home and abroad, dosage forms used in the treatment, and the drugs research and development directions in the future. AMD disease is the third largest blinding diseases all over the world, with an incidence of 6.62%. The dosage form of the traditional medicine is mostly oral formulations, playing a role in body, while the newly dosage form is topical drug delivery formulation. Traditional Chinese medicine (TCM) has certain advantages in the treatment of AMD disease and the development of topical drug delivery preparations with newly preparation technologies would have a very bright prospect in the future.

Cdc42 Promotes Schwann Cell Proliferation and Migration Through Wnt/ß-Catenin and p38 MAPK Signaling Pathway After Sciatic Nerve Injury.

Schwann cells (SCs) are unique glial cells in the peripheral nerve and may secrete multiple neurotrophic factors, adhesion molecules, extracellular matrix molecules to form the microenvironment of peripheral nerve regeneration, guiding and supporting nerve proliferation and migration. Cdc42 plays an important regulatory role in dynamic changes of the cytoskeleton. However, there is a little study referred to regulation and mechanism of Cdc42 on glial cells after peripheral nerve injury. The present study investigated the role of Cdc42 in the proliferation and migration of SCs after sciatic nerve injury. Cdc42 expression was tested, showing that the mRNA and protein expression levels of Cdc42 were significantly up-regulated after sciatic nerve injury. Then, we isolated and purified SCs from injuried sciatic nerve at day 7. The purified SCs were transfected with Cdc42 siRNA and pcDNA3.1-Cdc42, and the cell proliferation, cell cycle and migration were assessed. The results implied that Cdc42 siRNA remarkably inhibited Schwann cell proliferation and migration, and resulted in S phase arrest. While pcDNA3.1-Cdc42 showed a contrary effect. Besides, we also observed that Cdc42 siRNA down-regulated the protein expression of ß-catenin, Cyclin D1, c-myc and p-p38, which were up-regulated by pcDNA3.1-Cdc42. Meanwhile, the inhibitor of Wnt/ß-catenin and p38 MAPK signaling pathway IWP-2 and SB203580 significantly inhibited the effect of pcDNA3.1-Cdc42 on cell proliferation and migration. Overall, our data indicate that Cdc42 regulates Schwann cell proliferation and migration through Wnt/ß-catenin and p38 MAPK signaling pathway after sciatic nerve injury, which provides further insights into the therapy of the sciatic nerve injury.