Toward Better Food Security Using Concepts from Industry 5.0.

The rapid growth of the world population has increased the food demand as well as the need for assurance of food quality, safety, and sustainability. However, food security can easily be compromised by not only natural hazards but also changes in food preferences, political conflicts, and food frauds. In order to contribute to building a more sustainable food system-digitally visible and processes measurable-within this review, we summarized currently available evidence for various information and communication technologies (ICTs) that can be utilized to support collaborative actions, prevent fraudulent activities, and remotely perform real-time monitoring, which has become essential, especially during the COVID-19 pandemic. The Internet of Everything, 6G, blockchain, artificial intelligence, and digital twin are gaining significant attention in recent years in anticipation of leveraging the creativity of human experts in collaboration with efficient, intelligent, and accurate machines, but with limited consideration in the food supply chain. Therefore, this paper provided a thorough review of the food system by showing how various ICT tools can help sense and quantify the food system and highlighting the key enhancements that Industry 5.0 technologies can bring. The vulnerability of the food system can be effectively mitigated with the utilization of various ICTs depending on not only the nature and severity of crisis but also the specificity of the food supply chain. There are numerous ways of implementing these technologies, and they are continuously evolving.

Pyruvate Kinase Muscle-1 Expression Appears to Drive Lactogenic Behavior in CHO Cell Lines, Triggering Lower Viability and Productivity: A Case Study.

Chinese hamster ovary (CHO) cell lines are used to express a variety of therapeutic proteins. However, lactogenic behavior displayed by some CHO cell lines during manufacturing processes may result in a decline in viability, productivity, and possible alterations in product quality. In cultured cells, lactate is produced during glycolysis through irreversible conversion of phosphoenolpyruvate to pyruvate and then lactate via sequential function of pyruvate kinase and lactate dehydrogenase (LDH) enzymes. In the process of cell line development (CLD), two lactogenic cell lines expressing different antibody molecules are identified. The lactogenic behaviors of these cell lines can be differentially mitigated through optimization of either nutrient feeds or culture pH, depending on the cell line. Analysis of various proteins involved in the glycolysis pathway reveal a direct correlation between the pyruvate kinase muscle-1 (PKM-1) isoform levels and lactogenic behavior. CRISPR mediated knockout of the PKM-1 isoform abolishes lactate accumulation even under lactogenic conditions. Furthermore, a cell line lacking expression of both PKM-1 and PKM-2 enzymes capable of maintaining productivity, viability, and growth without displaying lactogenic behavior is identified. Targeted deletion of PKM in CHO cells may be tolerated due to expression of PKL (liver) and PKR (red blood cell) isoforms of pyruvate kinase. All together, these findings suggest that PKM-1 up-regulation during antibody production could trigger lactogenic behavior and that this enzyme is dispensable for CHO cell survival.

Short- and long-term effects on mAb-producing CHO cell lines after cryopreservation.

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Qp ) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Qp at increased cell age: 17 of the 24 cell lines displayed ≥20% Qp decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Qp decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Qp decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463-477, 2018.

More similar than different: Host cell protein production using three null CHO cell lines.

To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to <20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration-can accumulate a considerable amount of immunogenic HCP (∼1-2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis.

An integrated breast cancer risk assessment and management model based on fuzzy cognitive maps.

BACKGROUND: There is a growing demand for women to be classified into different risk groups of developing breast cancer (BC). The focus of the reported work is on the development of an integrated risk prediction model using a two-level fuzzy cognitive map (FCM) model. The proposed model combines the results of the initial screening mammogram of the given woman with her demographic risk factors to predict the post-screening risk of developing BC. METHODS: The level-1 FCM models the demographic risk profile. A nonlinear Hebbian learning algorithm is used to train this model and thus to help on predicting the BC risk grade based on demographic risk factors identified by domain experts. The risk grades estimated by the proposed model are validated using two standard BC risk assessment models viz. Gail and Tyrer-Cuzick. The level-2 FCM models the features of the screening mammogram concerning normal, benign and malignant cases. The data driven Hebbian learning algorithm (DDNHL) is used to train this model in order to predict the BC risk grade based on these mammographic image features. An overall risk grade is calculated by combining the outcomes of these two FCMs. RESULTS: The main limitation of the Gail model of underestimating the risk level of women with strong family history is overcome by the proposed model. IBIS is a hard computing tool based on the Tyrer-Cuzick model that is comprehensive enough in covering a wide range of demographic risk factors including family history, but it generates results in terms of numeric risk score based on predefined formulae. Thus the outcome is difficult to interpret by naive users. Besides these models are based only on the demographic details and do not take into account the findings of the screening mammogram. The proposed integrated model overcomes the above described limitations of the existing models and predicts the risk level in terms of qualitative grades. The predictions of the proposed NHL-FCM model comply with the Tyrer-Cuzick model for 36 out of 40 patient cases. With respect to tumor grading, the overall classification accuracy of DDNHL-FCM using 70 real mammogram screening images is 94.3%. The testing accuracy of the proposed model using 10-fold cross validation technique outperforms other standard machine learning based inference engines. CONCLUSION: In the perspective of clinical oncologists, this is a comprehensive front-end medical decision support system that assists them in efficiently assessing the expected post-screening BC risk level of the given individual and hence prescribing individualized preventive interventions and more intensive surveillance for high risk women.

Protein kinase A (PknA) of Mycobacterium tuberculosis is independently activated and is critical for growth in vitro and survival of the pathogen in the host.

The essential mycobacterial protein kinases PknA and PknB play crucial roles in modulating cell shape and division. However, the precise in vivo functional aspects of PknA have not been investigated. This study aims to dissect the role of PknA in mediating cell survival in vitro as well as in vivo. We observed aberrant cell shape and severe growth defects when PknA was depleted. Using the mouse infection model, we observe that PknA is essential for survival of the pathogen in the host. Complementation studies affirm the importance of the kinase, juxtamembrane, and transmembrane domains of PknA. Surprisingly, the extracytoplasmic domain is dispensable for cell growth and survival in vitro. We find that phosphorylation of the activation loop at Thr(172) of PknA is critical for bacterial growth. PknB has been previously suggested to be the receptor kinase, which activates multiple kinases, including PknA, by trans-phosphorylating their activation loop residues. Using phospho-specific PknA antibodies and conditional pknB mutant, we find that PknA autophosphorylates its activation loop independent of PknB. Fluorescently tagged PknA and PknB show distinctive distribution patterns within the cell, suggesting that although both kinases are known to modulate cell shape and division, their modes of action are likely to be different. This is supported by our findings that expression of kinase-dead PknA versus kinase-dead PknB in mycobacterial cells leads to different cellular phenotypes. Data indicate that although PknA and PknB are expressed as part of the same operon, they appear to be regulating cellular processes through divergent signaling pathways.

Culture temperature modulates aggregation of recombinant antibody in cho cells.

During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤ 5% aggregates at harvest. Aggregate levels increased 4-12-fold in 33°C cultures when compared to 37°C, with a corresponding 2-4-fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25-75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2-5-fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery.