Trace metal optimization in CHO cell culture through statistical design of experiments.

A majority of the biotherapeutics industry today relies on the manufacturing of monoclonal antibodies from Chinese hamster ovary (CHO) cells, yet challenges remain with maintaining consistent product quality from high-producing cell lines. Previous studies report the impact of individual trace metal supplemental on CHO cells, and thus, the combinatorial effects of these metals could be leveraged to improve bioprocesses further. A three-level factorial experimental design was performed in fed-batch shake flasks to evaluate the impact of time wise addition of individual or combined trace metals (zinc and copper) on CHO cell culture performance. Correlations among each factor (experimental parameters) and response variables (changes in cell culture performance) were examined based on their significance and goodness of fit to a partial least square's regression model. The model indicated that zinc concentration and time of addition counter-influence peak viable cell density and antibody production. Meanwhile, early copper supplementation influenced late-stage ROS activity in a dose-dependent manner likely by alleviating cellular oxidative stress. Regression coefficients indicated that combined metal addition had less significant impact on titer and specific productivity compared to zinc addition alone, although titer increased the most under combined metal addition. Glycan analysis showed that combined metal addition reduced galactosylation to a greater extent than single metals when supplemented during the early growth phase. A validation experiment was performed to confirm the validity of the regression model by testing an optimized setpoint of metal supplement time and concentration to improve protein productivity.

Advances in Biotechnological Production and Metabolic Regulation of Astragalus membranaceus.

Legume medicinal plants Astragalus membranaceus are widely used in the world and have very important economic value, ecological value, medicinal value, and ornamental value. The bioengineering technology of medicinal plants is used in the protection of endangered species, the rapid propagation of important resources, detoxification, and the improvement of degraded germplasm. Using bioengineering technology can effectively increase the content of secondary metabolites in A. membranaceus and improve the probability of solving the problem of medicinal plant resource shortage. In this review, we focused on biotechnological research into A. membranaceus, such as the latest advances in tissue culture, including callus, adventitious roots, hairy roots, suspension cells, etc., the metabolic regulation of chemical compounds in A. membranaceus, and the research progress on the synthetic biology of astragalosides, including the biosynthesis pathway of astragalosides, microbial transformation of astragalosides, and metabolic engineering of astragalosides. The review also looks forward to the new development trend of medicinal plant biotechnology, hoping to provide a broader development prospect for the in-depth study of medicinal plants.

Polyacetylenes from the adventitious roots of Centella asiatica with glucose uptake stimulatory activity.

Centella asiatica (L.) Urban is an herbaceous perennial plant of the Apiaceae family that has diverse medicinal uses. Its active components are saponin, phenolics, and polyacetylenes. Plant cell cultures have been exploited for the efficient production of metabolites with pharmacological activity. In this work, we prepared adventitious root cultures of C. asiatica and analyzed their content and biological activity. Adventitious root extracts were found to increase glucose uptake by differentiated L6 skeletal muscle cells and to be more efficient than the extract of whole plants. Chromatographic fractionation of the extracts from adventitious roots of C. asiatica led to the isolation of two known polyacetylenes, araliadiol (1) and 8-acetoxy-1,9-pentadecadiene-4,6-diyn-3-ol (2), in addition to a new polyacetylene, which we have named centellidiol (3). All the three polyacetylenes stimulated glucose uptake in a dose-dependent manner. The methanol extract of adventitious roots contained 0.53% and 0.82% of compounds 1 and 2, respectively, which are values that were 15 and 21 times higher that are found in mother plants. We therefore suggest that the high content of these polyacetylenes contributes to the high efficacy of C. asiatica adventitious root cultures. Overall, adventitious root cultures of C. asiatica can be part of a secure supply of effective ingredients including polyacetylenes.

Application of Data Modeling, Instrument Engineering and Nanomaterials in Selected Medid the Scientific Recinal Plant Tissue Culture.

At present, most precious compounds are still obtained by plant cultivation such as ginsenosides, glycyrrhizic acid, and paclitaxel, which cannot be easily obtained by artificial synthesis. Plant tissue culture technology is the most commonly used biotechnology tool, which can be used for a variety of studies such as the production of natural compounds, functional gene research, plant micropropagation, plant breeding, and crop improvement. Tissue culture material is a basic and important part of this issue. The formation of different plant tissues and natural products is affected by growth conditions and endogenous substances. The accumulation of secondary metabolites are affected by plant tissue type, culture method, and environmental stress. Multi-domain technologies are developing rapidly, and they have made outstanding contributions to the application of plant tissue culture. The modes of action have their own characteristics, covering the whole process of plant tissue from the induction, culture, and production of natural secondary metabolites. This paper reviews the induction mechanism of different plant tissues and the application of multi-domain technologies such as artificial intelligence, biosensors, bioreactors, multi-omics monitoring, and nanomaterials in plant tissue culture and the production of secondary metabolites. This will help to improve the tissue culture technology of medicinal plants and increase the availability and the yield of natural metabolites.

Production of Secondary Metabolites from Cell Cultures of Sageretia thea (Osbeck) M.C. Johnst. Using Balloon-Type Bubble Bioreactors.

Sageretia thea is used in the preparation of herbal medicine in China and Korea; this plant is rich in various bioactive compounds, including phenolics and flavonoids. The objective of the current study was to enhance the production of phenolic compounds in plant cell suspension cultures of Sageretia thea. Optimum callus was induced from cotyledon explants on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 0.5 mg L-1), naphthalene acetic acid (NAA, 0.5 mg L-1), kinetin (KN; 0.1 mg L-1) and sucrose (30 g L-1). Browning of callus was successfully avoided by using 200 mg L-1 ascorbic acid in the callus cultures. The elicitor effect of methyl jasmonate (MeJA), salicylic acid (SA), and sodium nitroprusside (SNP) was studied in cell suspension cultures, and the addition of 200 µM MeJA was found suitable for elicitation of phenolic accumulation in the cultured cells. Phenolic and flavonoid content and antioxidant activity were determined using 2,2 Diphenyl 1 picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethybenzothiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP) assays and results showed that cell cultures possessed highest phenolic and flavonoid content as well as highest DPPH, ABTS, and FRAP activities. Cell suspension cultures were established using 5 L capacity balloon-type bubble bioreactors using 2 L of MS medium 30 g L-1 sucrose and 0.5 mg L-1 2,4-D, 0.5 mg L-1 NAA, and 0.1 mg L-1 KN. The optimum yield of 230.81 g of fresh biomass and 16.48 g of dry biomass was evident after four weeks of cultures. High-pressure liquid chromatography (HPLC) analysis showed the cell biomass produced in bioreactors possessed higher concentrations of catechin hydrate, chlorogenic acid, naringenin, and other phenolic compounds.

Response of Cnidium officinale Makino Plants to Heat Stress and Selection of Superior Clones Using Morphological and Molecular Analysis.

Cnidium officinale is a medicinal plant cultivated for its rhizomes, which are used in Chinese, Japanese, and Korean traditional medicine. This medicinal crop is highly susceptible to heat stress and cannot be cultivated in regions of higher temperatures. In the present study, ten clones from Korea (clones 1, 2, 5, 6, 8, 11, 14, 15, 22, and 26) were evaluated for their heat tolerance in vitro at 25, 30, 32.5, and 35 °C, and growth characteristics including plant height, the number of leaves and roots were evaluated. The initial experiment was conducted to find the threshold level for significant damage to the plant, while the second experiment was to screen the germplasm to select heat-tolerant clones. Most of the clones were sensitive to heat stress (clones 1, 2, 8, 11, 14, 15, 22, and 26), and few clones (clones 5 and 6) could perform well at an elevated temperature of 32.5 °C. Molecular analysis of the expression of heat-responsive genes, including heat shock protein (CoHSP), catalase (CoCAT), and cystine protease (CoCP), was performed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) carried out with heat-tolerant and heat-sensitive clones. Two of the heat-tolerant clones (clones 5 and 6) showed significant expression of CoHSP and CoCAT genes at elevated temperature treatment. These clones can be used for further evaluation and cultivation.

Establishment of an Efficient In Vitro Propagation of Cnidium officinale Makino and Selection of Superior Clones through Flow Cytometric Assessment of DNA Content.

Cnidium officinale is a valuable medicinal plant cultivated in Asia for its rhizomes. This study reports the in vitro regeneration of Cnidium officinale plants and the induction of rhizomes from microshoots. The rhizomatous buds of Cnidium officinale induced multiple shoots on Murashige and Skoog (MS) medium supplemented with 0.5 mg L-1 BA, which led to the regeneration of plants within four weeks of culture. After four weeks of culture, the plants were assessed for fresh weight, the number of leaves, the number of roots, and the length of roots to compare the performance of the different clones. The clones with good growth characteristics were selected with the aid of a flow cytometric analysis of 2C nuclear DNA content. The plants bearing high DNA values showed better growth characteristics. Various factors, namely, sucrose concentration (30, 50, 70, and 90 g L-1), ABA (0, 0.5, 1.0, and 2.0 mg L-1), the synergistic effects of BA (1.0 mg L-1) + NAA (0.5 mg L-1) and BA (1.0 mg L-1) + NAA (0.5 mg L-1) + ABA (1.0 mg L-1) with or without activated charcoal (1 g L-1), and light and dark incubation were tested on rhizome formation from microshoots. The results of the above experiments suggest that MS medium supplemented with 50 g L-1 sucrose, 1.0 mg L-1 ABA, and 1 g L-1 AC is good for the induction of rhizomes from the shoots of Cnidium officinale. Plantlets with rhizomes were successfully transferred to pots, and they showed 100% survival.

Individualized Diagnosis and Prescription in Traditional Medicine: Decision-Making Process Analysis and Machine Learning-Based Analysis Tool Development.

While pattern identification (PI) is an essential process in traditional medicine (TM), it is difficult to objectify since it relies heavily on implicit knowledge. Therefore, this study aimed to propose a machine learning (ML)-based analysis tool to evaluate the clinical decision-making process of PI in terms of explicit and implicit knowledge, and to observe the actual process by which this knowledge affects the choice of diagnosis and treatment in individual TM doctors. Clinical data for the development of the analysis tool were collected using a questionnaire administered to allergic rhinitis (AR) patients and the diagnosis and prescription results of TM doctors based on the completed AR questionnaires. Explicit knowledge and implicit knowledge were defined based on the doctors' explicit scoring and feature evaluations of ML models, respectively. There were many differences between the explicit and implicit importance scores in this study. Implicit importance is more closely related to explicit importance in prescription than in diagnosis. The analysis results for eight doctors showed that our tool could successfully identify explicit and implicit knowledge in the PI process. This is the first study to evaluate the actual process by which explicit and implicit knowledge affect the choice of individual TM doctors and to identify assessment tools for the definition of the decision-making process in diagnosing PI and prescribing herbal treatments by TM clinicians. The assessment tool suggested in this study could be broadly used for the standardization of precision medicine, including TM therapeutics.

Development of Hairy Root Cultures for Biomass and Triterpenoid Production in Centella asiatica.

Centella asiatica (Apiaceae) is a tropical/subtropical medicinal plant, which contains a variety of triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. In this study, we tested the efficiency of hairy root (HR) induction in C. asiatica from leaf and petiole explants. Leaves and petioles collected from C. asiatica plants were suspended in agro-stock for 30 min and co-cultured with Agrobacterium rhizogenes for 3 days to induce HR formation. The transformation efficiency of leaf and petiole explants was approximately 27% and 12%, respectively. A total of 36 HR lines were identified by PCR-based amplification of rol genes, and eight of these lines were selected for further analysis. Among all eight HR lines, the petiole-derived lines HP4 and HP2 displayed the highest growth index (37.8) and the highest triterpenoids concentration (46.57 mg∙g-1), respectively. Although triterpenoid concentration was >2-fold higher in leaves than in petioles of C. asiatica plants, the accumulation of triterpenoids in petiole-derived HR cultures was 1.4-fold higher than that in leaf-derived HR cultures. Additionally, in both leaf- and petiole-derived HR cultures, terpenoid production was higher in HRs than in adventitious roots. These results demonstrate that the triterpenoid content in the explant does not affect the triterpenoid content in the resultant HRs. The HR culture of C. asiatica could be scaled up to enable the mass production of triterpenoids in bioreactors for the pharmaceutical and cosmetic industries.

Curcubinoyl flavonoids from wild ginseng adventitious root cultures.

Wild ginseng (Panax ginseng) adventitious root cultures were prepared by elicitation using methyl jasmonate and investigated further to find new secondary metabolites. Chromatographic fractionation of wild ginseng adventitious root cultures led to the isolation of eleven compounds. The chemical structures of isolated compounds were identified as four known flavanone derivatives (1-4), one new curcubinoyl derivative, jasmogin A (5) and six new curcubinoyl-flavanone conjugates, jasmoflagins A-F (6-11) by extensive spectroscopic analysis. Newly isolated curcubinoyl derivatives showed inhibitory activity against lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. Therefore, our present study suggested that elicitor stimulated plant cell cultures might contribute to the production of new metabolites.

Inhibitory Effects of Schisandra Lignans on Cytochrome P450s and Uridine 5'-Diphospho-Glucuronosyl Transferases in Human Liver Microsomes.

Schisandra chinensis has been widely used as a traditional herbal medicine to treat chronic coughs, fatigue, night sweats, and insomnia. Numerous bioactive components including lignans have been identified in this plant. Lignans with a dibenzocyclooctadiene moiety have been known to possess anti-cancer, anti-inflammatory, and hepatoprotective activity. Fragmentary studies have reported the ability of some lignans to modulate some cytochrome P450 (P450) enzymes. Herein, we investigated the drug interaction potential of six dibenzocyclooctadiene lignans (schisandrin, gomisin A, B, C, and N, and wuweizisu C) on nine P450 enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A) and six uridine 5'-diphosphoglucuronosyl transferase (UGT) enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) using human liver microsomes. We found that lignans with one or two methylenedioxyphenyl groups inhibited CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2E1 activities in a time- and concentration-dependent like their CYP3A inhibition. In comparison, these lignans do not induce time-dependent inhibition of CYP1A2, CYP2A6, and CYP2D6. The time-dependent inhibition of gomisin A against CYP2C8, CYP2C19, and CYP3A4 was also elucidated using glutathione as a trapping reagent of reactive carbene metabolites given that gomisin A strongly inhibits these P450 enzymes in a time-dependent manner. A glutathione conjugate of gomisin A was generated in reactions with human recombinant CYP2C8, CYP2C19, and CYP3A4. This suggests that the time-dependent inhibition of gomisin A against CYP2C8, CYP2C9, and CYP3A4 is due to the production of carbene reactive metabolite. Six of the lignans we tested inhibited the activities of six UGT to a limited extent (IC50 > 15 µM). This information may aid the prediction of possible drug interactions between Schisandra lignans and any co-administered drugs which are mainly metabolized by P450s.

Effect of Heat-killed Lactobacillus casei DKGF7 on a Rat Model of Irritable Bowel Syndrome.

Non-viable bacteria, referred to as "paraprobiotics," have attracted attention as potentially safer alternatives to probiotics. The aim of this study was to investigate the efficacy of heat-killed Lactobacillus casei DKGF7 on the symptomatic improvement of irritable bowel syndrome (IBS) in a rat disease model and to elucidate the underlying mechanisms that contribute to the beneficial effects of heat-killed probiotics. Seven male Wistar rats were induced with IBS by restraint stress and administered heat-killed L. casei DKGF7 for four weeks and then compared with seven rats in the control group. Stool consistency measured four weeks after initial treatment was the primary outcome measure. To investigate the mechanism of action of the heat-killed bacteria on IBS, we measured serum corticosterone levels, inflammatory cytokines in colon tissue, and expression of tight junction proteins (TJPs) in the epithelium. The treatment group showed significantly better stool consistency scores than the control group at week 4, as well as at every measured time point (all p values < 0.05). The treatment group showed lower serum corticosterone levels, lower colonic inflammatory cytokine levels, and higher expression of TJPs compared with the control group. Paraprobiotics such as heat-killed L. casei DKGF7 can improve stool consistency in a rat IBS model, which may indicate a potential therapeutic strategy for IBS treatment.

Isolation and Characterization of Compounds from Glycyrrhiza uralensis as Therapeutic Agents for the Muscle Disorders.

Skeletal muscle is the most abundant tissue and constitutes about 40% of total body mass. Herein, we report that crude water extract (CWE) of G. uralensis enhanced myoblast proliferation and differentiation. Pretreatment of mice with the CWE of G. uralensis prior to cardiotoxin-induced muscle injury was found to enhance muscle regeneration by inducing myogenic gene expression and downregulating myostatin expression. Furthermore, this extract reduced nitrotyrosine protein levels and atrophy-related gene expression. Of the five different fractions of the CWE of G. uralensis obtained, the ethyl acetate (EtOAc) fraction more significantly enhanced myoblast proliferation and differentiation than the other fractions. Ten bioactive compounds were isolated from the EtOAc fraction and characterized by GC-MS and NMR. Of these compounds (4-hydroxybenzoic acid, liquiritigenin, (R)-(-)-vestitol, isoliquiritigenin, medicarpin, tetrahydroxymethoxychalcone, licochalcone B, liquiritin, liquiritinapioside, and ononin), liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B were found to enhance myoblast proliferation and differentiation, and myofiber diameters in injured muscles were wider with the liquiritigenin than the non-treated one. Computational analysis showed these compounds are non-toxic and possess good drug-likeness properties. These findings suggest that G. uralensis-extracted components might be useful therapeutic agents for the management of muscle-associated diseases.

Progerinin, an optimized progerin-lamin A binding inhibitor, ameliorates premature senescence phenotypes of Hutchinson-Gilford progeria syndrome.

Previous work has revealed that progerin-lamin A binding inhibitor (JH4) can ameliorate pathological features of Hutchinson-Gilford progeria syndrome (HGPS) such as nuclear deformation, growth suppression in patient's cells, and very short life span in an in vivo mouse model. Despite its favorable effects, JH4 is rapidly eliminated in in vivo pharmacokinetic (PK) analysis. Thus, we improved its property through chemical modification and obtained an optimized drug candidate, Progerinin (SLC-D011). This chemical can extend the life span of LmnaG609G/G609G mouse for about 10 weeks and increase its body weight. Progerinin can also extend the life span of LmnaG609G/+ mouse for about 14 weeks via oral administration, whereas treatment with lonafarnib (farnesyl-transferase inhibitor) can only extend the life span of LmnaG609G/+ mouse for about two weeks. In addition, progerinin can induce histological and physiological improvement in LmnaG609G/+ mouse. These results indicate that progerinin is a strong drug candidate for HGPS.

A Randomized, Noninferiority Trial Comparing ICS + LABA with ICS + LABA + LAMA in Asthma-COPD Overlap (ACO) Treatment: The ACO Treatment with Optimal Medications (ATOMIC) Study.

BACKGROUND: Current guidelines for the treatment of asthma and chronic obstructive pulmonary disease overlap (ACO) recommend initial treatment using inhaled corticosteroids (ICSs) plus 1 or more bronchodilators. OBJECTIVE: To clarify which therapeutic effect is better between the ICS + long-acting ß2 agonist (LABA) and ICS + LABA + long-acting muscarinic antagonist (LAMA) treatment in patients with ACO. METHODS: We conducted a multicenter, 48-week, randomized, noninferiority trial. Patients with ACO were enrolled if they were treated with a moderate to high dose of ICS + LABA. In total, 303 patients were involved in the present trial, with 149 receiving ICS + LABA + LAMA. The primary end point was the time to first exacerbation. Secondary outcomes included changes in FEV1, forced vital capacity, FEV1/forced vital capacity ratio, asthma control, blood eosinophil count, and fractional exhaled nitric oxide. RESULTS: In the ICS + LABA treatment group, 29 of 154 patients (18.83%) experienced exacerbation, whereas 28 of 149 patients (18.79%) experienced exacerbation in the ICS + LABA + LAMA treatment group. The results of this noninferiority study were ultimately inconclusive (hazard ratio, 1.1; 95% CI, 0.66-1.84). However, the patients treated with the addition of LAMA showed significant improvements in FEV1 and forced vital capacity (P < .001). Asthma control did not improve in either group. CONCLUSIONS: Although this study was unable to conclude that ICS + LABA treatment is not inferior to ICS + LABA + LAMA in terms of exacerbation, it is obvious that the ICS + LABA + LAMA treatment group had improved lung function in ACO.

Primary cilia mediate early life programming of adiposity through lysosomal regulation in the developing mouse hypothalamus.

Hypothalamic neurons including proopiomelanocortin (POMC)-producing neurons regulate body weights. The non-motile primary cilium is a critical sensory organelle on the cell surface. An association between ciliary defects and obesity has been suggested, but the underlying mechanisms are not fully understood. Here we show that inhibition of ciliogenesis in POMC-expressing developing hypothalamic neurons, by depleting ciliogenic genes IFT88 and KIF3A, leads to adulthood obesity in mice. In contrast, adult-onset ciliary dysgenesis in POMC neurons causes no significant change in adiposity. In developing POMC neurons, abnormal cilia formation disrupts axonal projections through impaired lysosomal protein degradation. Notably, maternal nutrition and postnatal leptin surge have a profound impact on ciliogenesis in the hypothalamus of neonatal mice; through these effects they critically modulate the organization of hypothalamic feeding circuits. Our findings reveal a mechanism of early life programming of adult adiposity, which is mediated by primary cilia in developing hypothalamic neurons.

Effect of a Synbiotic Containing Lactobacillus paracasei and Opuntia humifusa on a Murine Model of Irritable Bowel Syndrome.

The administration of a combination of probiotics and prebiotics is expected to be a promising strategy for improving irritable bowel syndrome (IBS) symptoms. This study aimed to investigate the efficacy of a synbiotic containing Lactobacillus paracasei and Opuntia humifusa extract for symptomatic improvement of IBS in a murine model and to evaluate the mechanism underlying the beneficial effects of this synbiotic. A total of 20 male Wistar rats aged 8 weeks with IBS induced by restraint stress were assigned into four groups and administered L. paracasei as a probiotic and O. humifusa extract as a prebiotic for 4 weeks. The primary outcome was stool consistency at week 4. To evaluate the mechanism underlying the beneficial effects of the synbiotic, fecal microbial analysis was conducted, and the serum corticosterone levels, tumor necrosis factor-α (TNF-α) levels in the colon tissue, and expression of tight junction proteins were investigated. All three treatment groups showed significantly lower scores for stool consistency than the control group at week 4 (all p < 0.001). When compared with the control group, the synbiotic groups showed a significantly greater abundance of L. paracasei in fecal microbial analysis, lower serum corticosterone levels, lower TNF-α levels in the colon tissue, and higher expression of tight junction proteins. This novel synbiotic containing L. paracasei and O. humifusa extract can improve the stool consistency in a murine model of IBS. It may be a promising treatment option for IBS, and human studies are warranted.

Effect of echinalkamide identified from Echinacea purpurea (L.) Moench on the inhibition of osteoclastogenesis and bone resorption.

Plant cell cultures have been exploited to provide stable production and new secondary metabolites for better pharmacological activity. Fractionation of adventitious root cultures of Echinacea purpurea resulted in the isolation of eleven constituents, including three new compounds. The structures of the three new compounds were determined to be an alkylamide (1), a polyacetylene (2) and a lignan (3) on the basis of combined spectroscopic analysis. To discover new types of antiresorptive agents, we screened for new compounds that regulate osteoclast differentiation, and survival. Among three new compounds, echinalkamide (compound 1) had considerably inhibitory effects on RANKL-induced osteoclast differentiation, and on proliferation of osteoclasts and efficiently attenuated osteoclastic bone resorption without toxicity. In addition, echinalamide treatment inhibited the osteoclast-specific gene expression level. Echinalkamide achieved this inhibitory effect by disturbing phosphorylation of MAPK and activation of osteoclast transcription factors c-Fos and NFATc1. Conclusionally, our study investigated that echinalkamide remarkably inhibited osteoclast differentiation and osteoclast specific gene expression through repression of the MAPK-c-Fos-NFATC1 cascade.