Arabidopsis cell suspension culture and RNA sequencing reveal regulatory networks underlying plant-programmed cell death.

Programmed cell death (PCD) facilitates selective, genetically controlled elimination of redundant, damaged, or infected cells. In plants, PCD is often an essential component of normal development and can mediate responses to abiotic and biotic stress stimuli. However, studying the transcriptional regulation of PCD is hindered by difficulties in sampling small groups of dying cells that are often buried within the bulk of living plant tissue. We addressed this challenge by using RNA sequencing and Arabidopsis thaliana suspension cells, a model system that allows precise monitoring of PCD rates. The use of three PCD-inducing treatments (salicylic acid, heat, and critical dilution), in combination with three cell death modulators (3-methyladenine, lanthanum chloride, and conditioned medium), enabled isolation of candidate core- and stimuli-specific PCD genes, inference of underlying regulatory networks and identification of putative transcriptional regulators of PCD in plants. This analysis underscored a disturbance of the cell cycle and mitochondrial retrograde signaling, and repression of pro-survival stress responses, as key elements of the PCD-associated transcriptional signature. Further, phenotyping of Arabidopsis T-DNA insertion mutants in selected candidate genes validated the potential of generated resources to identify novel genes involved in plant PCD pathways and/or stress tolerance.

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture.

Activin A belongs to the transforming growth factor (TGF) family member, which exhibits a wide range of biological activities, including the regulation of cellular proliferation and differentiation and the promotion of neuronal survival. The isolation of AA from natural sources can only produce limited quantities of this bioactive protein. In this study, the whole gene of the precursor form of recombinant human activin A (rhAA) contains a signal peptide, and a pro-region and a mature region were cloned into an expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. To obtain the mature (active) form of rhAA, an enterokinase cleavage site was inserted between the pro-region and mature region of rhAA. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with recombinant vectors by the Agrobacterium-mediated method, and the integration of the target gene into the plant genome was confirmed by genomic PCR. The transcript expression of rhAA in transgenic rice calli was confirmed by a Northern blot analysis of mRNA. The production of rhAA was verified by Western blot analysis and ELISA. The accumulation of secreted rhAA in the culture medium was purified by Ni2+-NTA. The mature form of AA was released from the precursor form of rhAA after proteolytically processing with enterokinase. Western blot shows that the mature AA was split into monomer and homodimer with molecular weights of 14 kDa and 28 kDa under reducing and non-reducing conditions, respectively. These results suggest that the mature form of rhAA could be produced and purified using transgenic rice cell suspension culture.

Microcarrier-based clinical-grade manufacturing of therapeutic Wharton's jelly mesenchymal stromal cells.

Due to their immunomodulatory and anti-inflammatory properties, tissue repair capabilities and regenerative potential, Wharton's jelly mesenchymal stem/stromal cells (WJMSCs) have been widely investigated as potential treatment for diverse clinical indications. WJMSCs have been found to be well-tolerated and safe, positioning them as a promising candidate for cellular therapy. To address the commercial need for manufacturing WJMSCs for clinical applications, the production scale should be capable of generating large quantities of cells that retain their expected identity, purity and potency. This study aimed to establish a current Good Manufacturing Practice (cGMP) compliant robust and scalable expansion process representing a critical step towards a cGMP-compliant large-scale production platform for WJMSC-based clinical applications. Using our in-house cGMP-manufactured WJMSCs, which are currently being tested in a Phase Ib clinical trial (NCT03158896) using two-dimensional (2D) planar systems, we optimized various culture parameters including type of microcarrier, seeding density, agitation and culture feed regime in a 3D microcarrier-based culture system in spinner flasks. The results showed that cell adhesion was potentiated under intermittent stirring (3 min of agitation at 25 rpm followed by a period of non-agitation for 30 min), with reduced supplementation (0.05%) during the initial 8 h of cultivation with an initial cell concentration of 0.45 × 105 cells/mL. Microcarrier-based WJMSC expansion in spinner flasks achieved greater cell densities of 1.67 × 106 cells/mL with a maximum of 37-fold expansion, yielding ∼84 × 106 cells after 6 days of culture with a 95% harvest efficiency. Additionally, post 3D expansion, WJMSCs maintained their phenotypic characteristics, differentiation potential, normal karyotype, functional properties and sterility in the culture systems evaluated. This cGMP-compliant expansion process described herein demonstrates a successful transition of an established 2D planar culture process of clinical grade WJMSCs to 3D microcarrier-based suspension process generating higher cell yields, is cost-effective and represents an important step toward fulfilling the commercial demand of clinical grade mesenchymal stromal cells.

Quantitative intra- and intergeneric taxonomic relationships among Micrococcaceae strains reveal contradictions in the historical assignments of the strains and indicate the need for species reclassification.

This article reports the results of quantitative intra- and intergeneric taxonomic relationships among Micrococcaceae strains and a novel endophytic bacterium (SG) isolated from a suspension culture of Arabidopsis thaliana (L.) Heynh in our laboratory. The known strain Rothia sp. ND6WE1A was used as a reference one for SG. Whole-genome sequencing and phylogenetic analysis were based on the 16S rRNA test. Quantitative analysis for the nucleotide identity (ANI) and calculation of evolutionary distances were based on the identified amino acids (AAI) test indicating the generic assignment of the reference strain within and between the identified monophyletic groups of Micrococcaceae. The amino acid data structure of Rothia sp. ND6WE1A was compared against the UniProt database (250 million records) of close lineage of Micrococcaceae, including other Rothia spp. These data presented unique and evolutionary amino acid alignments, eventually expected in the new SG isolate as well. The metagenomic entries of the respective genome and proteome, characterized at the genus and species levels, could be considered for evolutionary taxonomic reclassification of the isolated and the reference strain (SG + Rothia sp. ND6WE1A). Therefore, our results warrant further investigations on the isolated SG strain.

Integrated Metabolomics and Transcriptomics Analyses of the Biosynthesis of Arbutin and 6'-O-Caffeoylarbutin in Vaccinium dunalianum Cell Suspension Cultures Fed with Hydroquinone.

Arbutin and 6'-O-caffeoylarbutin (CA) from Vaccinium dunalianum Wight are known for their ability to inhibit melanin synthesis. To boost the production of arbutin and CA, precursor feeding with hydroquinone (HQ) was studied in V. dunalianum suspension cells. The effect of HQ on the biosynthesis of arbutin and CA in the suspension cells was investigated using high-performance liquid chromatography (HPLC), and possible molecular mechanisms were analyzed using metabolomics and transcriptomics analyses. HPLC analysis only showed that the addition of HQ significantly enhanced arbutin synthesis in cells, peaking at 15.52 ± 0.28 mg·g-1 after 0.5 mmol·L-1 HQ treatment for 12 h. Subsequently, metabolomics identified 78 differential expression metabolites (DEMs), of which arbutin and CA were significantly up-regulated metabolites. Moreover, transcriptomics found a total of 10,628 differential expression genes (DEGs). The integrated transcriptomics and metabolomics revealed that HQ significantly enhanced the expression of two arbutin synthase (AS) genes (Unigene0063512 and Unigene0063513), boosting arbutin synthesis. Additionally, it is speculated that CA was generated from arbutin and 3,4,5-tricaffeoylquinic acid catalyzed by caffeoyl transferase, with Unigene0044545, Unigene0043539, and Unigene0017356 as potentially associated genes with CA synthesis. These findings indicate that the precursor feeding strategy offers a promising approach for the mass production of arbutin and CA in V. dunalianum suspension cells and provides new insights for CA biosynthesis in V. dunalianum.

Investigation of miniature goat cheese produced from Cynara scolymus L. pistil cell suspension culture extracts elicited with melatonin and salicylic acid.

BACKGROUND: The present study aimed to evaluate the potential of enzymes produced by artichoke (Cynara scolymus L.) flower cell suspension cultures elicited by melatonin (5 µm) and salicylic acid (50 µm) on the production and characteristics of miniature goat cheeses. In this study, five types of fresh miniature goat cheese were produced using whole artichoke flower extract, salicylic acid (50 µm) or melatonin (5 µm) treated artichoke suspension cell culture extracts, a culture extract without elicitor treatment (control) and rennin enzyme. RESULTS: The milk clotting activity values of the enzymes were measured in the range 0.52-0.74. The effect of the enzymes on the titratable acidity, water-soluble nitrogen, ripening index, αs-caseins and ß-caseins of goat cheese was significant (P < 0.05). The highest level of casein degradation was observed in the cheese produced with the enzyme containing melatonin, followed by the cheese produced with the enzyme containing salicylic acid. CONCLUSION: For the enzymes produced by the suspension cell culture method, the addition of melatonin and salicylic acid had a slightly positive effect on the proteolytic activity of the extracts. It was also found that the enzymes obtained from artichokes by the suspension cell culture method did not achieve successful cheese production in terms of chemical, textural and biochemical aspects compared to those obtained from animal enzymes. © 2024 Society of Chemical Industry.

In vitro mutagenesis using habituation and PBR autotrophy based indirect somatic embryogenesis (ISE) system in Kinnow mandarin.

Solid mutant induction using specialized habituation and PBR (Plant bio-regulator) autotrophy-mediated suspension-based ISE system was the prime aim of present investigation. Based on survival of cell clumps after mutagen treatment, the probit analysis was calculated. The result revealed LD50 at 54.31 Gy in gamma, while for EMS (ethyl methanesulfonate), it was 0.1% for 3 h and 0.5% for 1 h. Based on embryogenesis efficiency, a dose rate of 100 Gy and 0.1% EMS for a 3-h exposure were selected for regeneration. As compared to control, significant decrease in the embryogenesis efficiency was recorded at 100 Gy (85.92%) with similar reduction trends in embryo production (79.49%), germination (13.43%), conversion (2.48%), establishment (15.78%) and acclimatization (60.92%). The growth-related parameters such as root and shoot length and number of leaves/regenerant were also significantly reduced to 67.29%, 30.19% and 5.03%, respectively, in the regenerated plants after gamma irradiation as compared to control. In the EMS treatment, at the dose rate of 0.1% for 3-h, the embryogenesis efficiency was reduced to 43.67% with similar diminution trends in embryo production (59.49%), germination (8.95%), conversion (1.94%), establishment (4.37%) and acclimatization (29.9%). The growth-related parameters in the EMS treatment, decreased to 91.00% (root length), 71.34% (shoot length) and 35.03% (no. of leaves). The molecular marker based varied amplifications confirmed the occurrence of mutations in both gamma and EMS induced M1 regenerants. The study highlights the alternative high frequency in vitro mutagenesis protocol for induction of solid mutants in Kinnow mandarin and related citrus species.

Phytosulfokine contributes to suspension culture of Cunninghamia lanceolata through its impact on redox homeostasis.

BACKGROUND: Suspension culture is widely used in the establishment of efficient plant regeneration systems, as well as in the mass production of plant secondary metabolites. However, the establishment of a suspension culture system of Cunninghamia lanceolata is genotype-dependent given that proembryogenic masses (PEMs) are prone to browning during this process in recalcitrant genotypes. Previously, we reported that the plant peptide hormone phytosulfokine (PSK) can tremendously decrease the hydrogen peroxide (H2O2) level and help to initiate somatic embryogenesis (SE) in recalcitrant C. lanceolata genotypes. However, to date, no studies have revealed whether or how PSK may contribute to the establishment of a suspension culture system in these recalcitrant genotypes. RESULTS: Here, we demonstrated that exogenous application of PSK effectively inhibited PEM browning during suspension culture in a recalcitrant genotype of C. lanceolata. Comparative time-series transcriptome profiling showed that redox homeostasis underwent drastic fluctuations when PEMs were cultured in liquid medium, while additional PSK treatment helped to maintain a relatively stable redox homeostasis. Interestingly, PSK seemed to have a dual effect on peroxidases (PRXs), with PSK simultaneously transcriptionally repressing ROS-producing PRXs and activating ROS-scavenging PRXs. Furthermore, determination of H2O2 and MDA content, as well as cell viability, showed that exogenous PSK treatment inhibited PEM browning and safeguarded PEM suspension culture by decreasing the H2O2 level and increasing PEM activity. CONCLUSIONS: Collectively, these findings provide a valuable tool for the future establishment of large-scale C. lanceolata PEM suspension culture without genotype limitations.

Suspension culture on microcarriers and as aggregates enables expansion and differentiation of pluripotent stem cells (PSCs).

BACKGROUND AIMS: Human pluripotent stem cells (PSCs) hold a great promise for promoting regenerative medical therapies due to their ability to generate multiple mature cell types and for their high expansion potential. However, cell therapies require large numbers of cells to achieve desired therapeutic effects, and traditional two-dimensional static culture methods cannot meet the required production demand for cellular therapies. One solution to this problem is scaling up expansion of PSCs in bioreactors using culture strategies such as growing cells on microcarriers or as aggregates in suspension culture. METHODS: In this study, we directly compared PSC expansion and quality parameters in microcarrier- and aggregate-cultures grown in single-use vertical-wheel bioreactors. RESULTS: We showed comparable expansion of cells on microcarriers and as aggregates by day 6 with a cell density reaching 2.2 × 106 cells/mL and 1.8 × 106 cells/mL and a fold-expansion of 22- and 18-fold, respectively. PSCs cultured on microcarriers and as aggregates were comparable with parallel two-dimensional cultures and with each other in terms of pluripotency marker expression and retention of other pluripotency characteristics as well as differentiation potential into three germ layers, neural precursor cells and cardiomyocytes. CONCLUSIONS: Our study did not demonstrate a clear advantage between the two three-dimensional methods for the quality parameters assessed. This analysis adds support to the use of bioreactor systems for large scale expansion of PSCs, demonstrating that the cells retain key characteristics of PSCs and differentiation potential in suspension culture.

Biosynthesized Ag nanoparticles on urea-based periodic mesoporous organosilica enhance galegine content in Galega.

The biological approach for synthesizing nanoparticles (NPs) using plant extracts is an efficient alternative to conventional physicochemical methods. Galegine, isolated from Galega (Galega officinalis L.), has anti-diabetic properties. In the present study, silver nanoparticles (AgNPs) loaded onto urea-based periodic mesoporous organosilica (AgNPs/Ur-PMO) were bio-synthesized using G. officinalis leaf extract. The synthesized NPs were characterized and confirmed via analysis methods. Different concentrations of biosynthesized AgNPs/Ur-PMO nanoparticles (0, 1, 5, 10, and 20 mg L-1) were used as elicitors in cell suspension culture (CSC) of G. officinalis. The callus cells from hypocotyl explants were treated at their logarithmic growth phase (8th d) and were collected at time intervals of 24, 72, 120, and 168 h. The viability and growth of cells were reduced (by 17% and 35%, respectively) at higher concentrations and longer treatments of AgNPs/Ur-PMO; however, the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) were increased (1.23 and 3.01 fold, respectively in comparison with the control average). The highest total phenolic (2.43 mg g-1 dry weight) and flavonoid (2.22 mg g-1 dry weight) contents were obtained 168 h after treatment with 10 mg L-1 AgNPs/Ur-PMO. An increasing tendency in the antioxidant enzyme activities was also observed in all the elicitor concentrations. Treatment with AgNPs/Ur-PMO (in particular 5 mg L-1 for 120 h) significantly enhanced the galegine content (up to 17.42 mg g-1) about 1.80 fold compared with the control. The results suggest that AgNPs/Ur-PMO can be used as an effective elicitor for enhancing galegine production in the CSC of G. officinalis. KEY POINTS: • The green biosynthesis of AgNPs/Ur-PMO was done using G. officinalis leaf extract • Its toxicity as an elicitor increased with increasing concentration and treatment time • AgNPs/Ur-PMO significantly increased the antioxidant capacity and galegine content.

Biotechnological approaches for the production of gymnemic acid from Gymnema sylvestre R. Br.

Diabetes is a chronic disease that affects several organs and can be treated using phytochemicals found in medicinal plants. Gymnema sylvestre (Asclepiadaceae) is one such medicinal plant rich in anti-diabetic properties. The plant is commonly known as madhunashini in Sanskrit because of its ability to cure diabetes (sugar). Gymnemic acid (GA) is a phytochemical (a triterpenoid saponin) responsible for the herb's main pharmacological activity. This secondary metabolite has a lot of potential as a phytochemical with pharmacological properties including nephroprotection, hypoglycemia, antioxidant, antimicrobial, and anti-inflammatory. Gymnema has acquired a lot of popularity in recent years due to its low side effects and high efficacy in healing diabetes, which has led to overexploitation by pharmaceutical enterprises for its biomass in the wild for the purification of gymnemic acid. Modern biotechnological techniques involving the establishment of cell and organ cultures from G. sylvestre will assist us in fulfilling the need for gymnemic acid production. The present review provides insights on the establishment of cell and organ cultures for the production of a potent antidiabetic molecule gymnemic acid. Further, the review also delves into the intricacies of the different strategies for improved production of gymnemic acid using various elicitors. There is huge potential for sustainable production of gymnemic acid which could be met by establishment of bioreactor scale production. Understanding and engineering the biosynthetic pathway could also lead to improved GA production. KEY POINTS: • Gymnemic acid is one of the potential anti-diabetic molecules from madhunashini • Cell and organ culture offers potential approach for gymnemic acid production • Elicitation strategies have improved the gymnemic acid production.

Fine-tuning of epithelial taste bud organoid to promote functional recapitulation of taste reactivity.

Taste stem/progenitor cells from posterior mouse tongues have been used to generate taste bud organoids. However, the inaccessible location of taste receptor cells is observed in conventional organoids. In this study, we established a suspension-culture method to fine-tune taste bud organoids by apicobasal polarity alteration to form the accessible localization of taste receptor cells. Compared to conventional Matrigel-embedded organoids, suspension-cultured organoids showed comparable differentiation and renewal rates to those of taste buds in vivo and exhibited functional taste receptor cells and cycling progenitor cells. Accessible taste receptor cells enabled the direct application of calcium imaging to evaluate the taste response. Moreover, suspension-cultured organoids can be genetically altered. Suspension-cultured taste bud organoids harmoniously integrated with the recipient lingual epithelium, maintaining the taste receptor cells and gustatory innervation capacity. We propose that suspension-cultured organoids may provide an efficient model for taste research, including taste bud development, regeneration, and transplantation.

Adipose-derived stem cells (ASCs) culture in spinner flask: improving the parameters of culture in a microcarrier-based system.

Prior to clinical use, extensive in vitro proliferation of human adipose-derived stem cells (ASCs) is required. Among the current options, spinner-type stirred flasks, which use microcarriers to increase the yield of adherent cells, are recommended. Here, we propose a methodology for ASCs proliferation through cell suspension culture using Cultispher-S® microcarriers (MC) under agitation in a spinner flask, with the aim of establishing a system that reconciles the efficiency of cell yield with high viability of the culture during two distinct phases: seeding and proliferation. The results showed that cell adhesion was potentiated under intermittent stirring at 70 rpm in the presence of 10% FBS for an initial cell concentration of 2.4 × 104 cells/mL in the initial 24 h of cultivation. In the proliferation phase, kinetic analysis showed that cell growth was higher under continuous agitation at 50 rpm with a culture medium renewal regime of 50% every 72 h, which was sufficient to maintain the culture at optimal levels of nutrients and metabolites for up to nine days of cultivation, representing an 11.1-fold increase and a maximum cell productivity of 422 cells/mL/h (1.0 × 105 viable cells/mL). ASCs maintained the immunophenotypic characteristics and mesodermal differentiation potential of both cell lines from different donors. The established protocol represents a more efficient and cost-effective method to obtain a high proliferation rate of ASCs in a microcarrier-based system, which is necessary for large-scale use in cell therapy, highlighting that the manipulation of critical parameters optimizes the ASCs production process.

Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells.

Kidney organoids have potential uses in disease modelling, drug screening and regenerative medicine. However, novel cost-effective techniques are needed to enable scaled-up production of kidney cell types in vitro We describe here a modified suspension culture method for the generation of kidney micro-organoids from human pluripotent stem cells. Optimisation of differentiation conditions allowed the formation of micro-organoids, each containing six to ten nephrons that were surrounded by endothelial and stromal populations. Single cell transcriptional profiling confirmed the presence and transcriptional equivalence of all anticipated renal cell types consistent with a previous organoid culture method. This suspension culture micro-organoid methodology resulted in a three- to fourfold increase in final cell yield compared with static culture, thereby representing an economical approach to the production of kidney cells for various biological applications.

Transition from serum-supplemented monolayer to serum-free suspension lentiviral vector production for generation of chimeric antigen receptor T cells.

BACKGROUND AIMS: Lentiviral vectors (LVs) have been used extensively in gene therapy protocols because of their high biosafety profile and capacity to stably express a gene of interest. Production of these vectors for the generation of chimeric antigen receptor (CAR) T cells in academic and research centers is achieved using serum-supplemented static monolayer cultures. Although efficient for pre-clinical studies, this method has a number of limitations. The main hurdles are related to its incompatibility with robust and controlled large-scale production. For this reason, cell suspension culture in bioreactors is desirable. Here the authors report the transition of LV particle production from serum-supplemented monolayer to serum-free suspension culture with the objective of generating CAR T cells. METHODS: A self-inactivating LV anti-CD19 CAR was produced by transient transfection using polyethylenimine (PEI) in human embryonic kidney 293 T cells previously adapted to serum-free suspension culture. RESULTS: LV production of 8 × 106 transducing units (TUs)/mL was obtained in serum-supplemented monolayer culture. LV production in the serum-free suspension conditions was significantly decreased compared with monolayer production. Therefore, optimization of the transfection protocol was performed using design of experiments. The results indicated that the best condition involved the use of 1 µg of DNA/106 cells, 1 × 106 cells/mL and PEI:DNA ratio of 2.5:1. This condition used less DNA and PEI compared with the standard, thereby reducing production costs. This protocol was further improved with the addition of 5 mM of sodium butyrate and resulted in an increase in production, with an average of 1.5 × 105 TUs/mL. LV particle functionality was also assessed, and the results indicated that in both conditions the LV was capable of inducing CAR expression and anti-tumor response in T cells, which in turn were able to identify and kill CD19+ cells in vitro. CONCLUSIONS: This study demonstrates that the transition of LV production from small-scale monolayer culture to scalable and controllable bioreactors can be quite challenging and requires extensive work to obtain satisfactory production.

Adaptation process of engineered cell line FCHO/IL-24 stably secreted rhIL-24 in serum-free suspension culture.

Interleukin-24 (IL-24) displays tumor cell-specific proliferation inhibition in vitro and in vivo. Recombinant human IL-24 (rhIL-24) has significantly higher activity, yet significantly lower expression level in mammalian cells than in bacteria. To further realize therapeutic potential of IL-24, we enhanced rhIL-24 expression in mammalian cell systems by adapting engineered Flp-InTMCHO/IL-24 (FCHO/IL-24) cells (adherent cultured in Ham's F12 medium with 10% serum) to serum-free suspension culture. First, MTT assay showed that among four different media (F12, DMEM/F12, 1640 and DMEM), DMEM/F12 medium was the most suitable media for lower-serum adherent culture. Then, cells were adherently cultured in DMEM/F12 with serum concentration reduced from 10% to 0.5% in a gradient manner. Compared to cells in 10% serum, cells in 0.5% serum displayed significantly lower relative cell viability by 40%, increased G0/G1 phase arrest (8.5 ± 2.4%, p < 0.05), decreased supernatant rhIL-24 concentration by 73%, and altered metabolite profiles, such as glucose, lactate and ammonia concentration. Next, the cells were directly adapted to 0.5% serum suspension culture in 125 mL shake flask at 119 rpm with the optimal cell seeding density of 5 × 105 cells/mL (3.3 times higher than that of adherent culture), under which the concentration of rhIL-24 in culture medium was stable at 3.5 ng/mL. Finally, cells adapted to 0.5% serum proliferated better in serum-free medium Eden™-B300S with higher rhIL-24 expression level compared to CDM4CHO. The successful adaptation of engineered cells FCHO/IL-24 laid foundation for adapting cells from adherent culture to suspension serum-free culture to mass produce rhIL-24 protein for therapeutic purposes.

YAP signaling induces PIEZO1 to promote oral squamous cell carcinoma cell proliferation.

Most cancer cells are exposed to altered extracellular environments, such as an increase in extracellular matrix (ECM) stiffness and soluble signals consisting of growth factors and cytokines. It is therefore conceivable that changes in tumor extracellular environments affect tumor cell behavior. The Hippo pathway reportedly responds to the extracellular environment and regulates the nuclear localization of the transcription co-activator, yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ). Inactivation of the Hippo pathway with nuclear translocation of YAP/TAZ stimulates cell proliferation. Its pathway also regulates gene expression, but the precise molecule(s) meditating the cell-proliferating effect of YAP signaling on oral squamous cell carcinoma (OSCC) is unclear. First, we examined the effects of YAP signaling on OSCC tumorigenesis. Loss-of-function experiments using siRNA or an inhibitor, and immunohistochemical analyses of tissue specimens obtained from OSCC patients demonstrated that YAP signaling was involved in OSCC cell proliferation. Second, we identified Piezo-type mechanosensitive ion channel component 1 (PIEZO1), a Ca2+ channel, as a transcriptional target of YAP signaling and showed that elevated PIEZO1 was required for PIEZO1 agonist-dependent Ca2+ entry and cell proliferation in OSCC cells. Experiments using three-dimensional and suspension culture revealed that PIEZO1 was involved in OSCC cellular growth. Finally, YAP overexpression in the nucleus and/or cytoplasm was immunohistochemically detected in tumor lesions with frequent expression of both PIEZO1 and Ki-67, but not in non-tumor regions of OSCC specimens. These results suggest that the YAP/PIEZO1 axis promotes OSCC cell growth. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Enhanced production of Bacopa saponins by repeated batch strategy in bioreactor.

Cultivation of cell suspension culture of Bacopa monnieri targeting the production of bacosides was explored in a 5-l stirred tank reactor using statistically optimized conditions. The bioreactor cultivation conditions were modified and this led to profuse biomass growth (2.81 ± 0.20 g/l) and total bacosides (1.26 ± 0.23 mg/g in cells and 0.60 ± 0.11 mg/l in fermenter broth) production in 9 days. The values of static volumetric mass transfer coefficient (kLa), dimensionless mixing time (Nm) were measured in the bioreactor. The culture grew efficiently and produced enhanced amount of bacoside A (5.59 ± 0.41 mg/g total bacosides in cells and 3.12 ± 0.13 mg/l in the fermenter broth) using one cycle of repeated batch strategy adopted in the bioreactor for 15 days. The intracellular concentration of bacoside A3 (1.18 ± 0.11 mg/g), bacopaside II (2.09 ± 0.35 mg/g), bacopaside X (0.79 ± 0.17 mg/g) and bacopasaponin C (2.24 ± 0.23 mg/g) were significantly higher in repeated batch as compared to batch bioreactor cultivation. The yield of total bacosides in the fermenter broth was 5-times higher in repeated batch as compared to batch cultivation. This strategy can be helpful for the enhanced production of other valuable triterpenoid saponins.

Enhancement of recombinant human IL-24 (rhIL-24) protein production from site-specific integrated engineered CHO cells by sodium butyrate treatment.

Interleukin-24 (IL-24) has specific inhibitory effects on the proliferation of various tumor cells with almost no toxicity to normal cells. The antitumor activity of recombinant human IL-24 protein produced in mammalian cells is much higher than that of bacteria, but its expression level is extremely low. Sodium butyrate (NaBu) was utilized as a media additive to increase protein expression in Chinese hamster ovary cells. The site-specific integrated engineered cells FCHO/IL-24 were treated with NaBu under different culture conditions (10% and 0.5% serum adherent culture, 0.5% serum suspension culture). First, 3 days of 1 mmol/L NaBu treatment significantly increased rhIL-24 expression level in FCHO/IL-24 cells by 119.94 ± 1.5% (**p < 0.01), 57.49 ± 2.4% (**p < 0.01), and 20.17 ± 3.03% (*p < 0.05) under the above culture conditions. Second, NaBu has a time- and dose-dependent inhibitory effect on FCHO/IL-24 proliferation and induces G0/G1 phase arrest. Under 10% and 0.5% serum adherent culture, G0/G1 phase cells were increased by 11.3 ± 0.5% (**p < 0.01) and 15.0 ± 2.6% (**p < 0.01), respectively. No induction of apoptosis was observed under a high dosage of NaBu treatment. These results suggest that NaBu increases rhIL-24 secretion via inhibiting cell cycle progression, thereby trapping cells in the highly productive G0/G1 phase. Finally, with increasing NaBu dose, glucose concentration increased (**p < 0.01) while lactic acid and ammonia concentrations reduced significantly (**p < 0.01) in 10% and 0.5% serum adherent culture supernatant. RNA-seq showed that NaBu treatment affected multiple tumor and immune-related pathways. In conclusion, NaBu treatment dramatically promoted rhIL-24 production in engineered FCHO/IL-24 cells by altering downstream pathways and inducing G0/G1 cell arrest with little effect on apoptosis.

Biotechnological Approaches for Production of Artemisinin, an Anti-Malarial Drug from Artemisia annua L.

Artemisinin is an anti-malarial sesquiterpene lactone derived from Artemisia annua L. (Asteraceae family). One of the most widely used modes of treatment for malaria is an artemisinin-based combination therapy. Artemisinin and its associated compounds have a variety of pharmacological qualities that have helped achieve economic prominence in recent years. So far, research on the biosynthesis of this bioactive metabolite has revealed that it is produced in glandular trichomes and that the genes responsible for its production must be overexpressed in order to meet demand. Using biotechnological applications such as tissue culture, genetic engineering, and bioreactor-based approaches would aid in the upregulation of artemisinin yield, which is needed for the future. The current review focuses on the tissue culture aspects of propagation of A. annua and production of artemisinin from A. annua L. cell and organ cultures. The review also focuses on elicitation strategies in cell and organ cultures, as well as artemisinin biosynthesis and metabolic engineering of biosynthetic genes in Artemisia and plant model systems.