Dulaglutide (glucagon like peptide-1/IgG-Fc fusion protein, GLP-1-Fc) is a long lasting GLP-1 agonist, which consists of two arms of GLP-1 moieties fused to IgG Fc fragment. Dulaglutide is a safe and ...effective medication for type 2 diabetes. In an attempt to develop a biosimilar version of dulaglutide, we found that up to 75% of GLP-1-Fc displayed
N
-terminal truncations in one or both GLP-1 arms. We proposed that the
N
-terminal heterogeneity was caused by mis-cleavage of signal peptide and solved this problem through signal peptide optimization. Murine immunoglobulin kappa light chain signal peptide (KASP) significantly improves GLP-1-Fc
N
-terminal integrity and homogeneity. 92.8–95.7% of GLP-1-Fc molecules directed by KASP contain intact
N
-terminus. The productivity of GLP-1-Fc could reach 2.2 g/L in shaking flask fed batch culture. KASP is an optimal signal peptide for GLP-1-Fc expression in Chinese hamster ovary (CHO) cells.
Protein aggregation is a major challenge in the development of therapeutic monoclonal antibodies (mAbs). Several stressors can cause protein aggregation, including temperature shifts, mechanical ...forces, freezing-thawing cycles, oxidants, reductants, and extreme pH. When antibodies are exposed to low pH conditions, aggregation increases dramatically. However, low pH treatment is widely used in protein A affinity chromatography and low pH viral inactivation procedures. In the development of an IgG4 subclass antibody, mAb1-IgG4 showed a strong tendency to aggregate when temporarily exposed to low pH conditions. Our findings showed that the aggregation of mAb1-IgG4 under low pH conditions is determined by the stability of the Fc. The CH2 domain is the least stable domain in mAb1-IgG4. The L309E, Q311D, and Q311E mutations in the CH2 domain significantly reduced the aggregation propensity, which could be attributed to a reduction in the hydrophobicity of the CH2 domain. Protein stabilizers, such as sucrose and mannose, could also attenuate low pH-induced mAb1-IgG4 aggregation by shielding hydrophobic areas and increasing protein stability. Our findings provide valuable strategies for managing the aggregation of protein therapeutics with a human IgG4 backbone.
Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to ...generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells.
In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95–100% of productivity after 40 days of continuous culture (~48–56 generations).
Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.
How to cite: Li J, Wei S, Cao C, et al. Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.07.002.
Three-dimensional (3-D) shape monitoring is essential to prevent fatigue damage to cables. It can be challenging to monitor certain unique functional cables over time with conventional optical or ...acoustic techniques (such as underwater cables, umbilical cables, etc.). As a result, the cable is compared to a space curve in this article, and a method for reconstructing curves using a MEMS-IMU array is suggested. This method is less disturbed by the surrounding environment with low cost for long-distance and long-term use. After solving some technical issues, such as reconstruction algorithm and error, we applied the whole system to the submarine landing cable to evaluate the fatigue state of the submarine cable by quantifying cable displacement. The results demonstrate that in harsh weather, the cable's local movement is significant, which could wear the armor.
Rockburst is one of the critical safety concerns for underground engineering. The acoustic emission (AE) activity that occurs during rockburst is one of its notable characteristics. Because rockburst ...is induced by complexity factors, the prediction of rockburst has been investigated widely, yet few methods have been applied in depth. In this study, dynamic process of rockburst was investigated, and the information entropy of the dominant frequency was determined to be an indicator of rockburst. A series of true triaxial tests were conducted, and AE signals associated with the evolution of rock failure were collected. The AE data were treated with the fast Fourier transform to acquire the dominant frequency. Then, the information entropy of the dominant frequency was obtained, and a significant drop of the entropy value right after it reached the peak was observed. Finally, based on the comprehensive analysis of AE energy, dominant-frequency entropy, and other precursory information of rock failure, a multiparameter synergetic method of predicting rockburst is proposed. This research has important theoretical significance for improving the prediction accuracy of rockburst.
China’s first Mars exploration mission (HuoXing-1) has been named as ‘Tianwen-1’ meaning Heaven Inquiry. Tianwen-1 was launched on July 23, 2020. In this paper, the scientific objectives of earlier ...and current Mars exploration missions worldwide are reviewed, and the scientific objectives, payloads and preliminary scientific investigation plan of China’s first Mars exploration mission are introduced, and expected scientific achievements are analyzed.
Sex difference in the incidence rate of idiopathic pulmonary fibrosis (IPF) indicates that estrogen has a certain protective effect on the disease. Nevertheless, there is a dearth of study ...investigating the association between factors pertaining to endogenous estrogen exposure level, such as age at menarche (AAM) in women, and IPF. Our study intended to employ Mendelian randomization (MR) method to elucidate the causal association between AAM and IPF.
Our study utilized AAM as a measure of endogenous estrogen exposure and investigated its causal effect on the risk of IPF through MR. We employed the inverse variance weighted (IVW) method to assess the causal relationship between AAM and IPF risk, with supplementary analyses conducted using the weighted median estimator (WME) and MR-Egger method. Several sensitivity analyses were performed to assess the dependability of MR estimates.
A total of 9 selected single nucleotide polymorphisms (SNPs) significantly associated with AAM were selected as instrumental variables. The IVW method showed that genetically later AAM was associated with an increased risk of IPF (odds ratio OR = 1.0014, 95%confidence interval CI = 1.0005-1.0023, p = 0.001). The median weighting method and the MR-Egger method obtained similar estimates, and no heterogeneity or pleiotropy was found, indicating that the results were robust.
Our MR study suggested a causal relationship between a later onset of menarche and a heightened susceptibility to IPF.
Background The purpose of this study was to investigate the diagnostic value of IL-17 detection in bronchoalveolar lavage fluid (BALF) and plasma samples from nonneutropenic patients with invasive ...pulmonary aspergillosis. Methods We retrospectively collected data on non-neutropenic patients who were suspected to have IPA admitted to the Third Affiliated Hospital of Soochow University between March 2020 to January 2023. IL-17 and GM were measured using enzyme-linked immunosorbent assays. Results A total of 281 patients were enrolled in this study, of which 62 had proven or probable IPA and the remaining 219 patients were controls. The plasma and BALF IL-17 levels were significantly higher in the IPA group compared with the control group. The plasma GM, plasma IL17, BALF GM, and BALF IL17 assays had sensitivities of 56.5%, 72.6%, 68.7%, and 81.2%, respectively, and specificities of 87.7%, 69.4%, 91.9%, and 72.6%, respectively. The sensitivity of IL17 in plasma and BALF was higher than that of GM. Plasma GM in combination with IL-17 increases the sensitivity but does not decrease the diagnostic specificity of GM testing. The diagnostic sensitivity and specificity of BALF GM combined with IL-17 for IPA in non-neutropenic patients were greater than 80% and there was a significant increase in sensitivity compared with BALF GM. Conclusions Plasma and BALF IL-17 levels were significantly higher in non-neutropenic patients with IPA. The sensitivity of plasma and BLAF IL-17 for diagnosing IPA in non-neutropenic patients was superior to that of GM. Combined detection of lavage fluid GM and IL17 significantly improves the diagnosis of IPA in non-neutropenic patients. The combined detection of GM and IL-17 in plasma also contributes to the diagnosis of IPA in patients who cannot tolerate invasive procedures.
Using a 820 nm-thick high-quality Ge0.97Sn0.03 alloy film grown on Si(001) by molecular beam epitaxy, GeSn p-i-n photodectectors have been fabricated. The detectors have relatively high ...responsivities, such as 0.52 A/W, 0.23 A/W, and 0.12 A/W at 1310 nm, 1540 nm, and 1640 nm, respectively, under a 1 V reverse bias. With a broad detection spectrum (800-1800 nm) covering the whole telecommunication windows and compatibility with conventional complementary metal-oxide-semiconductors (CMOS) technology, the GeSn devices are attractive for applications in both optical communications and optical interconnects.
Acute lung injury (ALI) is a life-threatening disease caused by the severe and acute response of the lungs to a variety of direct and indirect insults. Patients with ALI are currently treated mainly ...with respiratory support due to inadequate understanding of ALI progression. Alveolar epithelial cells produced thymic stromal lymphopoietin (TSLP) has been proved to worsen ALI by triggering airway inflammation. However, the regulation mechanism of TSLP expression remains unclear. In this study, we identified the crucial role played by circNCLN in lipopolysaccharide (LPS)-induced ALI. We demonstrated for the first time that miR-291a-3p could directly bind to the 3′UTR of TSLP and suppress TSLP expression in alveolar epithelial cells. Mechanistically, our data identified that circNCLN acts as a molecular sponge to antagonize miR-291a-3p and thereby maintaining the expression of TSLP in alveolar epithelial cells. Importantly, targeting circNCLN by its antisense oligonucleotide (ASO) markedly alleviated LPS-induced ALI. Therefore, our results suggested that circNCLN/miR-291a-3p/TSLP axis may be an important signaling in LPS-induced ALI and circNCLN inhibition may serve as a potential treatment of ALI.
•MiR-291a-3p is down-regulated in LPS-induced ALI.•circNCLN is the circRNA of miR-291a-3p and it exists in alveolar epithelial cells.•circNCLN binds and inhibits miR-291a-3p to maintain TSLP expression in alveolar epithelial cells.•Targeting circNCLN by ASO alleviates LPS-induced ALI.
PDF
