Tetrandrine, a bioactive active compound mainly found in the roots of Stephania tetrandra, exhibits various pharmacological properties. In vitro hairy root (HR) culture may serve as a promising solution for the extraction of tetrandrine, overcoming the limitations of natural cultivation. The present study describes the consistent production of tetrandrine from S. tetrandra hairy roots induced by different strains of Agrobacterium rhizogenes. Cultivation in woody plant medium (WPM) resulted in the highest HR biomass (0.056 g/petri-dish) and tetrandrine content (7.28 mg/L) as compared to other media. The maximum HR biomass (6.95 g dw/L) and tetrandrine production (68.69 mg/L) were obtained in the fifth week of cultivation. The presence of ammonium nitrate (800 mg/L), calcium nitrate (1156 mg/L), sucrose (20 g/L) and casein (2 g/L) enhanced the tetrandrine production. Moreover, the fed-batch cultivation demonstrated that the NH4NO3 (1200 mg/L) was an important growth limiting factor that yielded the highest tetrandrine amount (119.59 mg/L). The cultivation of hairy roots in a mist trickling bioreactor for eight weeks was less (26.24 mg/L) than in the flask. Despite a lower tetrandrine yield observed in bioreactors compared to flask cultures, refining the growth medium and fine-tuning bioreactor operations hold promise for boosting tetrandrine yield.

Aromatic compounds persist as hazardous contaminants in both aquatic and terrestrial environments, needing rapid and effective remediation strategies. This study evaluated toluene and benzene biodegradation under sulfate and nitrate-reducing conditions in column experiments, utilizing aquifer sediments from a contaminated site. Over a period of 36 weeks, four glass columns were operated simultaneously in an alternating flow-batch regime. Each column received either nitrate or sulfate as an electron acceptor while being exposed to different substrate compositions in varied exposure orders. A redox dependent contaminant removal efficiency was observed, with toluene removal efficiency at 81% under sulfate and 55% under nitrate-reducing conditions, and benzene removal efficiency approximately at 44% and 59%, respectively, within 4 to 6 weeks. The rapid removal under anaerobic conditions was attributed to the alternating flow-batch regime, allowing biomass growth in batch mode, and applying selection pressure to non-specific biodegraders during flow regime. Toluene removal remained unaffected by benzene\'s presence but exhibited slight inhibition in the presence of an aromatic mixture composed of BTEX, indene, indane, and naphthalene. Benzene removal efficiency dropped to 8% in the presence of toluene but remained unaffected by the mixture. Pre-exposure to a single compound enhanced breakdown efficiency when further faced with a more complex mixture. Additionally, beta-diversity analysis conducted on the four columns revealed distinct microbial community clustering between sulfate and nitrate-reducing conditions, emphasizing the determining role of redox conditions. Findings of this study can be used to develop more effective pollution cleanup strategies, specifically targeting parameters like redox conditions, substrate interactions, and pollution history, thus improving our ability to mitigate contamination across diverse environments.

This study aims to develop an ex vivo organ-on-a-chip model, intervertebral Disc-on-a-ChipMF, to investigate integrated effects of mechanical loading and nutrition on disc health. The system consists of a detachable multilayer microfluidic chip, a Computer-Arduino-based control system, and a mechanical loading unit, which were optimized for accurate axial force measurement and the maintenance of a 21-day ex vivo disc culture. To ensure accuracy of axial force, we optimized the axial mechanical loading regimen, used the Computer-Arduino-based system and low-profile force sensors (LPFS) to control the mechanical loading unit, and modeled the force distribution by using computational simulation. A 21-day ex vivo disc culture was demonstrated using the Disc-on-a-ChipMF system, with optimized mechanical loading (0.02 MPa at 1Hz, 1.5 hr/day) and flow rate (1 μL/min). The structural integrity, collagen breakdown, catabolic enzyme activities, and disc cell and collagen alignment revealed that the on-chip cultured discs exhibited a preferred disc health similar to that of native discs for up to 21 days, while discs in a static culture showed detrimental degenerative changes. The mouse Disc-on-a-ChipMF system mimics in vivo disc microenvironment and provides a valuable platform for studying the effects of various factors on disc health and degeneration and testing new therapies.

Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1-184) and a hydrophobic C-terminal domain (residues 185-243). When a recombinant fusion protein construct [bacterial pelB leader sequence - human apoA-I (1-243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1-184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1-184) was secreted from the bacteria. When the pelB-apoA-I (1-184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1-184) was the sole major protein present. Incubation of apoA-I (1-184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.

Production of large amounts of functional NK and CAR-NK cells represents one of the bottlenecks for NK-based immunotherapy. In this study, we developed a large-scale, reliable, and practicable NK and CAR-NK production using G-Rex 100M bioreactors, which depend on a gas-permeable membrane technology. This system holds large volumes of medium with enhanced oxygen delivery, creating conditions conducive to large-scale PBNK and CAR-NK expansions for cancer therapy. Both peripheral blood NK cells (PBNKs) and CAR-NKs expanded in these bioreactors retained similar immunophenotypes and exhibited comparable cytotoxicity towards hepatocellular carcinoma (HCC) cells akin to that of NK and CAR-NK cells expanded in G-Rex 6 well bioreactors. Importantly, cryopreservation minimally affected the cytotoxicity of NK cells expanded using the G-Rex 100M bioreactors, establishing a robust platform for scaled-up NK and CAR-NK cell production. This method is promising for the development of \"off-the-shelf\" NK cells, supporting the future clinical implementation of NK cell immunotherapy.

CO2-based poly(3-hydroxybutyrate) (PHB) can be produced by the versatile bacterium Cupriavidus necator through chemolithoautotrophic fermentation, using a gas mixture consisting of CO2, H2, and O2. Despite offering a propitious route for carbon-neutral bioplastic manufacturing, its adoption is currently hampered by the wide explosive range of the required gas mixture, as well as the limited gas-to-liquid mass transfer rates. To address these challenges, pressure fermentation was applied as a robust and effective strategy, while ensuring safe operation by adhering to the limiting O2 concentration, utilizing state-of-the-art bioreactors. Consequently, exponential growth could be prolonged, boosting CO2-based PHB production from 10.8 g/L at 1.5 bar up to 29.6 g/L at 3 bar. The production gain closely aligns with the theoretical calculations, except for when the pressure was increased up to 4 bar. Overall, the demonstrated increase in PHB production underscores the potential of pressure fermentation to enhance aerobic gas fermentation.

Cardiovascular diseases remain a leading cause of mortality globally, with atherosclerosis representing a significant pathological means, often leading to myocardial infarction. Coronary artery bypass surgery, a common procedure used to treat coronary artery disease, presents challenges due to the limited autologous tissue availability or the shortcomings of synthetic grafts. Consequently, there is a growing interest in tissue engineering approaches to develop vascular substitutes. This review offers an updated picture of the state of the art in vascular tissue engineering, emphasising the design of scaffolds and dynamic culture conditions following a biomimetic approach. By emulating native vessel properties and, in particular, by mimicking the three-layer structure of the vascular wall, tissue-engineered grafts can improve long-term patency and clinical outcomes. Furthermore, ongoing research focuses on enhancing biomimicry through innovative scaffold materials, surface functionalisation strategies, and the use of bioreactors mimicking the physiological microenvironment. Through a multidisciplinary lens, this review provides insight into the latest advancements and future directions of vascular tissue engineering, with particular reference to employing biomimicry to create systems capable of reproducing the structure-function relationships present in the arterial wall. Despite the existence of a gap between benchtop innovation and clinical translation, it appears that the biomimetic technologies developed to date demonstrate promising results in preventing vascular occlusion due to blood clotting under laboratory conditions and in preclinical studies. Therefore, a multifaceted biomimetic approach could represent a winning strategy to ensure the translation of vascular tissue engineering into clinical practice.

OBJECTIVE: To investigate the cytotoxicity on human dental pulp cells (HDPCs) and Streptococcus mutans (S.mutans) biofilm formation on universal resin composite cements (UCs).
METHODS: Three UCs (RelyX Universal, 3 M Oral Care - RXU; Panavia SA Cement Universal, Kuraray Noritake - PSAU; SoloCem, Coltene - SCM) and one \'gold-standard\' multi-step cement (Panavia V5, Kuraray Noritake - PV5) were used following two polymerization protocols (light-cured - LC; self-cured - SC). Cytotoxicity (MTT) tests were performed after 1, 3 and 7 days of direct contact. Carboxy-2\',7\'-dichlorodihydrofluorescein diacetate was used to detect the release of reactive oxygen species (ROS), and interleukin 6 (IL-6) expression was analyzed by IL-6 proquantum high sensitivity immunoassay. S. mutans biofilms were grown on UCs samples in a bioreactor for 24 h, then adherent viable biomass was assessed using MTT assay. For microbiological procedures, half of UCs samples underwent accelerated aging. Data were statistically analyzed (α = 0.05).
RESULTS: The highest cytotoxicity was observed for PSAU SC, RXU SC, and PV5 SC at day 1, then for SC RXU after 3 days, and SC PSAU, LC PV5 and SCM after 1-week (p < 0.05). There was no increase in IL-6 expression after 1 day, while it increased depending on the group at 3 and 7 days. The highest ROS expression after 12 h was recorded for PSAU SC, PV5 SC and PV5 LC. Biofilm formation was as follows: RXU > > PSAU = PV5 > SCM, while light-curing systematically decreased biofilm formation (≈-33 %). Aging leveled out differences between UCs and between polymerization protocols.
CONCLUSIONS: The choice of cement brand, rather than category, and polymerization protocol influence cell viability and microbiological behavior. Light-curing is beneficial for reducing the harmful pulpal effect that UCs may possess.

COVID-19, caused by the novel coronavirus SARS-CoV-2, has presented a significant challenge to global health, security, and the economy. Vaccination is considered a crucial measure in preventing virus transmission. The silkworm bioreactor has gained widespread usage in antigen presentation, monoclonal antibody preparation, and subunit vaccine development due to its safety, efficiency, convenience, and cost-effectiveness. In this study, we employed silkworm BmN cells and the silkworm MultiBac multigene co-expression system to successfully produce two prototype vaccines: a recombinant baculovirus vector vaccine (NPV) co-displaying the SARS-CoV-2 virus capsid protein and a capsid protein virus-like particle (VLP) vaccine. Following the purification of these vaccines, we immunized BALB/c mice to evaluate their immunogenicity. Our results demonstrated that both VLP and NPV prototype vaccines effectively elicited robust immune responses in mice. However, when equal inoculation doses between groups were compared, the recombinant NPV vaccine exhibited significantly higher serum antibody titers and increased expression of spleen cytokines and lymphocyte immune regulatory factors compared to the VLP group. These results suggested an increased immune efficacy of the recombinant NPV vaccine. Conversely, the VLP prototype vaccine displayed more pronounced effects on lymphocyte cell differentiation induction. This study successfully constructed two distinct morphological recombinant vaccine models and systematically elucidated their differences in humoral immune response and lymphocyte differentiation rate. Furthermore, it has fully harnessed the immense potential of silkworm bioreactors for vaccine research and development, providing valuable technical insights for studying mutated viruses like coronaviruses.

BACKGROUND: Trichoderma reesei is known for its ability to produce large amounts of extracellular proteins and is one of the most important industrially used filamentous fungus. Xylanase regulator 1 (XYR1) is the master regulator responsible for the activation of cellulase and hemicellulase gene expression under inducing conditions. It has been reported that strains with point mutations in certain areas of xyr1 bypass the need for inducing carbon source, allowing high (hemi)cellulase production even in the presence of glucose. These mutations also change the profile of produced proteins, shifting it more towards xylanase production, and increase the overall protein production in inducing conditions. However, how these mutations alter the metabolism and other cellular processes to cause these changes remains unclear.
RESULTS: In this study, we aimed to explore changes caused by a point mutation in xyr1 on transcriptomic and metabolic level to better understand the reasons behind the increased protein production in both repressing glucose and inducing lactose conditions. As expected, the expression of many carbohydrate-active enzyme (CAZy) genes was increased in the xyr1 mutant in both conditions. However, their induction was higher under inducing conditions. The xyr1 mutant strain built more biomass and produced more extracellular proteins during growth on lactose compared to the wild type xyr1 strain. Genes involved in oxidoreductive D-galactose catabolism pathway were upregulated in the xyr1 mutant strain, potentially contributing to the more efficient utilization of lactose. In addition to CAZy genes, clustering and enrichment analysis showed over-representation of mitochondria-related Gene Ontology terms in clusters where gene expression was higher in the xyr1 mutant, indicating that mitochondria play a role in the altered metabolic state associated with the xyr1 mutation. Metabolomics revealed that free tyrosine was more abundant in the xyr1 mutant strain in all measured timepoints, whereas multiple fatty acids were less abundant in the mutant strain on glucose.
CONCLUSIONS: The results contribute to more in-depth knowledge on T. reesei physiology growing under inducing and repressing carbon sources and gives new insights on the function of the master regulator XYR1. The vast data generated serve as a source for new targets for improved protein production.