Improving product quality and productivity of an antibody-based biotherapeutic using inverted frustoconical shaking bioreactors

The Chinese hamster ovarian (CHO) cells serve as a common choice in biopharmaceutical production, traditionally cultivated in stirred tank bioreactors (STRs). Nevertheless, the pursuit of improved protein quality and production output for commercial purposes demand exploration into new bioreactor types. In this context, inverted frustoconical shaking bioreactors (IFSB) present unique physical properties distinct from STRs. This study aims to compare the production processes of an antibody-based biotherapeutic in both bioreactor types, to enhance production flexibility. The findings indicate that, when compared to STRs, IFSB demonstrates the capability to produce an antibody-based biotherapeutic with either comparable or enhanced bioprocess performance and product quality. IFSB reduces shear damage to cells, enhances viable cell density (VCD), and improves cell state at a 5-L scale. Consequently, this leads to increased protein expression (3.70 g/L vs 2.56 g/L) and improved protein quality, as evidenced by a reduction in acidic variants from 27.0% to 21.5%. Scaling up the culture utilizing the Froude constant and superficial gas velocity ensures stable operation, effective mixing, and gas transfer. The IFSB maintains a high VCD and cell viability at both 50-L and 500-L scales. Product expression levels range from 3.0 to 3.6 g/L, accompanied by an improved acidic variants attribute of 20.6%–22.7%. The IFSB exhibits superior productivity and product quality, underscoring its potential for incorporation into the manufacturing process for antibody-based biotherapeutics. These results establish the foundation for IFSB to become a viable option in producing antibody-based biotherapeutics for clinical and manufacturing applications

Surface Quality Evolution Model and Consistency Control Method of Large Shaft Multi-Pass Grinding

Large shaft usually achieves high surface quality through multi-pass grinding in practice. Common surface quality indexes include surface roughness and glossiness, which are not only required numerically, but also require high consistency of distribution along the whole shaft. In multi-pass grinding, these two indexes are affected by the process parameters and the surface quality of the previous grinding pass, which leads to the difficulty of modeling. In addition, due to the uneven distribution of actual grinding depth, the surface quality along the whole shaft is usually inconsistent, resulting in the need for multiple spark-out grinding passes to ensure consistency. In this study, the surface quality evolution models for surface roughness and glossiness based on Elman neural network are developed, which build regressions between process parameters, surface quality indexes of the previous grinding pass, and surface quality indexes of the current grinding pass. Moreover, a consistency control method of surface quality is proposed by adjusting the actual grinding depth within the dimensional accuracy tolerance range at the rough grinding stage. Experimental results show that the surface roughness and glossiness prediction errors of the surface quality evolution models are only 5.5% and 5.1%. The consistency control method guarantees the consistency of surface quality, reduces the grinding passes, and increases the grinding efficiency

A20 haploinsufficiency caused by loss‐of‐function TNFAIP3 mutation likely leads to progression of antiphospholipid syndrome to marginal zone lymphomas following coronavirus disease 2019 vaccination: A case study

Abstract Antiphospholipid syndrome (APS) is a rare autoimmune systemic disorder. Previously, no report suggests that APS could progress to extranodal marginal zone lymphomas (EMZL). In this study, we met an unusual APS patient with such progression to EMZL. The patients had been diagnosed with APS two years ago and was in a stable condition after regular treatment until his readmission to our hospital and re‐diagnosed with EMZL recently. Coincidentally, we noticed that the patient had been immunized against inactivated COVID‐19 vaccine just 2 days before his readmission. Furthermore, we performed whole‐exome sequencing and identified a heterozygous, new variant in TNFAIP3 (tumor necrosis factor, α‐induced protein 3) which encoded A20 protein, a key molecule controlling NF‐κB signaling. This variation caused a loss of a base A in TNFAIP3 gene at position 443_444, leading to a frameshift mutation and the production of a truncated A20 Lys148fs*67. A20 Lys148fs*67 failed to suppress TNF‐α‐induced NF‐κB activation and might act through haploinsufficiency. Vaccines work by triggering an immune response to a virus or bacterium within the body. A20 negatively regulates NF‐κB signaling to protect immune system from overactivation. In our case, the newly identified mutation in the TNFAIP3 led to the production of a loss‐of‐function A20 Lys148fs*67, which lost the ability to inhibit inflammation. The patient with such a heterozygous mutation, when facing with the “second hit” of COVID‐19 vaccination challenge, might produce excessive amounts of inflammatory cytokines and formed "cytokine storm" duo to A20 haploinsufficiency, eventually leading to the progression from APS to EMZL

The Transcriptome of Brassica napus L. Roots under Waterlogging at the Seedling Stage

Although rapeseed (Brassica napus L.) is known to be affected by waterlogging, the genetic basis of waterlogging tolerance by rapeseed is largely unknown. In this study, the transcriptome under 0 h and 12 h of waterlogging was assayed in the roots of ZS9, a tolerant variety, using digital gene expression (DGE). A total of 4432 differentially expressed genes were identified, indicating that the response to waterlogging in rapeseed is complicated. The assignments of the annotated genes based on GO (Gene Ontology) revealed there were more genes induced under waterlogging in “oxidation reduction”, “secondary metabolism”, “transcription regulation”, and “translation regulation”; suggesting these four pathways are enhanced under waterlogging. Analysis of the 200 most highly expressed genes illustrated that 144 under normal conditions were down-regulated by waterlogging, while up to 191 under waterlogging were those induced in response to stress. The expression of genes involved under waterlogging is mediated by multiple levels of transcriptional, post-transcriptional, translational and post-translational regulation, including phosphorylation and protein degradation; in particular, protein degradation might be involved in the negative regulation in response to this stress. Our results provide new insight into the response to waterlogging and will help to identify important candidate genes

The Rules and Functions of Nucleocytoplasmic Shuttling Proteins

Biological macromolecules are the basis of life activities. There is a separation of spatial dimension between DNA replication and RNA biogenesis, and protein synthesis, which is an interesting phenomenon. The former occurs in the cell nucleus, while the latter in the cytoplasm. The separation requires protein to transport across the nuclear envelope to realize a variety of biological functions. Nucleocytoplasmic transport of protein including import to the nucleus and export to the cytoplasm is a complicated process that requires involvement and interaction of many proteins. In recent years, many studies have found that proteins constantly shuttle between the cytoplasm and the nucleus. These shuttling proteins play a crucial role as transport carriers and signal transduction regulators within cells. In this review, we describe the mechanism of nucleocytoplasmic transport of shuttling proteins and summarize some important diseases related shuttling proteins

PARP1 in Carcinomas and PARP1 Inhibitors as Antineoplastic Drugs

Poly (ADP-ribose) polymerase 1 (PARP1), the best-studied isoform of the nuclear enzyme PARP family, plays a pivotal role in cellular biological processes, such as DNA repair, gene transcription, and so on. PARP1 has been found to be overexpressed in various carcinomas. These all indicate the clinical potential of PARP1 as a therapeutic target of human malignancies. Additionally, multiple preclinical research studies and clinical trials demonstrate that inhibition of PARP1 can repress tumor growth and metastasis. Up until now, PARP1 inhibitors are clinically used not only for monotherapy to suppress various tumors, but also for adjuvant therapy, to maintain or enhance therapeutic effects of mature antineoplastic drugs, as well as protect patients from chemotherapy and surgery-induced injury. To supply a framework for understanding recent research progress of PARP1 in carcinomas, we review the structure, expression, functions, and mechanisms of PARP1, and summarize the clinically mature PARP1-related anticancer agents, to provide some ideas for the development of other promising PARP1 inhibitors in antineoplastic therapy

Design, Synthesis, Mode of Action and Herbicidal Evaluation of Quinazolin-4(3H)-one Derivatives Based on Aryloxyphenoxypropionate Motif

To discover new acetyl-CoA carboxylase (ACCase) inhibiting-based herbicides, twenty-nine novel quinazolin-4(3H)-one derivatives were designed and synthesized based on the aryloxyphenoxypropionate motif. The bioassay results showed that most of the target compounds showed better pre-emergent herbicidal activity against monocotyledonous weeds in a greenhouse. Especially, when applied at 375 g ha−1 under pre-emergence conditions, compound QPP-7 displayed excellent herbicidal activity against monocotyledonous weeds (i.e., E. crusgalli, D. sanguinalis, P. alopecuroides, S. viridis, E. indica, A. fatua, E. dahuricu, S. alterniflora) with inhibition rate >90%, and displayed excellent crop safety to O. sativa, T. aestivum, G. spp, and A. hypogaea. The study of structure-activity relationship (SAR) revealed that the herbicidal activity of target compounds is strongly influenced by the spatial position of R group and the bulk of R1 group on quinazolin-4(3H)-one, and the (R = 6-F, R1 = Me) pattern is confirmed as the optimal orientation. Furthermore, the molecular docking study and the good inhibitory activity of QPP-7 against E. crusgalli ACCase enzyme (IC50 = 54.65 nM) indicated that it may be a ACCase inhibitor. Taken together, the present work demonstrated that compound QPP-7 could serve as a potential lead structure for further developing novel ACCase inhibiting-based herbicide

Design, Synthesis, Mode of Action and Herbicidal Evaluation of Quinazolin-4(3<i>H</i>)-one Derivatives Based on Aryloxyphenoxypropionate Motif

To discover new acetyl-CoA carboxylase (ACCase) inhibiting-based herbicides, twenty-nine novel quinazolin-4(3H)-one derivatives were designed and synthesized based on the aryloxyphenoxypropionate motif. The bioassay results showed that most of the target compounds showed better pre-emergent herbicidal activity against monocotyledonous weeds in a greenhouse. Especially, when applied at 375 g ha−1 under pre-emergence conditions, compound QPP-7 displayed excellent herbicidal activity against monocotyledonous weeds (i.e., E. crusgalli, D. sanguinalis, P. alopecuroides, S. viridis, E. indica, A. fatua, E. dahuricu, S. alterniflora) with inhibition rate >90%, and displayed excellent crop safety to O. sativa, T. aestivum, G. spp, and A. hypogaea. The study of structure-activity relationship (SAR) revealed that the herbicidal activity of target compounds is strongly influenced by the spatial position of R group and the bulk of R1 group on quinazolin-4(3H)-one, and the (R = 6-F, R1 = Me) pattern is confirmed as the optimal orientation. Furthermore, the molecular docking study and the good inhibitory activity of QPP-7 against E. crusgalli ACCase enzyme (IC50 = 54.65 nM) indicated that it may be a ACCase inhibitor. Taken together, the present work demonstrated that compound QPP-7 could serve as a potential lead structure for further developing novel ACCase inhibiting-based herbicide

Transcriptional Responses of Sclerotinia sclerotiorum to the Infection by SsHADV-1

The infection by a single-stranded DNA virus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), causes hypovirulence, a reduced growth rate, and other colony morphological changes in its host Sclerotinia sclerotiorum strain DT-8. However, the mechanisms of the decline are still unclear. Using digital RNA sequencing, a transcriptome analysis was conducted to elucidate the phenotype-related genes with expression changes in response to SsHADV-1 infection. A total of 3110 S. sclerotiorum differentially expressed genes (DEGs) were detected during SsHADV-1 infection, 1741 of which were up-regulated, and 1369 were down-regulated. The identified DEGs were involved in several important pathways. DNA replication, DNA damage response, carbohydrate and lipid metabolism, ribosomal assembly, and translation were the affected categories in S. sclerotiorum upon SsHADV-1 infection. Moreover, the infection of SsHADV-1 also suppressed the expression of antiviral RNA silencing and virulence factor genes. These results provide further detailed insights into the effects of SsHADV-1 infection on the whole genome transcription in S. sclerotiorum

Proteomic Analysis of Rapeseed Root Response to Waterlogging Stress

The overall health of a plant is constantly affected by the changing and hostile environment. Due to climate change and the farming pattern of rice (Oryza sativa) and rapeseed (Brassica napus L.), stress from waterlogging poses a serious threat to productivity assurance and the yield of rapeseed in China&rsquo;s Yangtze River basin. In order to improve our understanding of the complex mechanisms behind waterlogging stress and identify waterlogging-responsive proteins, we firstly conducted iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomic analysis of rapeseed roots under waterlogging treatments, for both a tolerant cultivar ZS9 and sensitive cultivar GH01. A total of 7736 proteins were identified by iTRAQ, of which several hundred showed different expression levels, including 233, 365, and 326 after waterlogging stress for 4H, 8H, and 12H in ZS9, respectively, and 143, 175, and 374 after waterlogging stress for 4H, 8H, and 12H in GH01, respectively. For proteins repeatedly identified at different time points, gene ontology (GO) cluster analysis suggested that the responsive proteins of the two cultivars were both enriched in the biological process of DNA-dependent transcription and the oxidation&ndash;reduction process, and response to various stress and hormone stimulus, while different distribution frequencies in the two cultivars was investigated. Moreover, overlap proteins with similar or opposite tendencies of fold change between ZS9 and GH01 were observed and clustered based on the different expression ratios, suggesting the two genotype cultivars exhibited diversiform molecular mechanisms or regulation pathways in their waterlogging stress response. The following qRT-PCR (quantitative real-time polymerase chain reaction) results verified the candidate proteins at transcription levels, which were prepared for further research. In conclusion, proteins detected in this study might perform different functions in waterlogging responses and would provide information conducive to better understanding adaptive mechanisms under environmental stresses