The Regulatory Role and Mechanism of Circadian Rhythm in Hemoglobin Co-cultured Neurovascular Unit.

Objective: Intracranial hemorrhage (ICH), the second most common subtype of stroke, exacerbates the disruption of the blood-brain barrier (BBB), leading to vasogenic edema, plasma protein extravasation, and infiltration of neurotoxic substances. The clearance capacity of the brain plays a crucial role in maintaining BBB homeostasis and facilitating patient recovery after hemorrhage. This study aimed to investigate the effect of circadian rhythms on BBB function, neuronal damage, and clearance capabilities. Methods: The transwell model and hemoglobin were co-cultured to simulate the BBB environment after ICH. After intervention with different light groups, neuronal apoptosis was determined, glial phagocytosis was analyzed, the expression of endogenous clearing-related proteins aquaporin 4 (AQP4) and low-density lipoprotein receptor-related protein 1 (LRP1) was detected by western blotting and immunofluorescence dual standard method, and the expression of the tight junction protein occludin and melatonin receptor 1A (MTNR1A) was quantitatively analyzed. Results: Circadian rhythms play a key role in maintaining the integrity of the BBB, reducing oxidative stress-induced neuronal damage, and improving microglial phagocytosis. Meanwhile, the expression of occludin and MTNR1A in neurovascular unit (NVU) co-cultured with hemoglobin improved the expression of AQP4 and LRP1, the key proteins in the NVU's endogenous brain clearance system. Conclusion: Circadian rhythm (alternating black and white light) protects the NVU BBB function after ICH, promotes the expression of proteins related to the clearance of the hematoma, provides new evidence for the clinical treatment of patients recovering from ICH, and improves the circadian rhythm to promote brain metabolism and hematoma clearance.

Surface molecularly imprinted polymers based on magnetic multi-walled carbon nanotubes for the highly selective purification of resveratrol from crude extracts of Vitis vinifera, Arachis hypogaea, and Polygonum cuspidatum.

In this work, surface molecularly imprinted polymers based on magnetic multi-walled carbon nanotubes were prepared for the specific recognition and adsorption of resveratrol. The functionalization of magnetic multi-walled carbon nanotubes and the synthesis process of surface molecularly imprinted polymers were optimized. Characterizations were performed to demonstrate the successful synthesis of the imprinted materials. The imprinted materials showed satisfactory adsorption capacity of resveratrol (45.73 ± 1.72 mg/g) and excellent selectivity (imprinting factor 2.89 ± 0.15). In addition, the imprinted materials were used as adsorbents in molecularly imprinted solid-phase extraction for the purification of resveratrol from crude extracts of some food and medicinal resources, achieving recoveries of 93.69%-95.53% with high purities of 88.37%-92.33%. Moreover, the purified products exhibited extremely strong free radical scavenging activity compared with crude extracts. Overall, this work provided a promising approach for the highly selective purification of resveratrol from natural resources, which would contribute to the application of this valuable compound in the food/nutraceutical fields.

Blue LED light promoting the growth, accumulation of high-value isoflavonoids and astragalosides, antioxidant response, and biosynthesis gene expression in Astragalus membranaceus (Fisch.) Bunge hairy root cultures.

The root of Astragalus membranaceus (Fisch.) Bunge is one of the most frequently used herbs in traditional Chinese medicine (TCM) formulae for fighting COVID-19 infections, due to the presence of isoflavonoids and astragalosides associated with antiviral and immune-enhancing activities. For the first time, the exposure of A. membranaceus hairy root cultures (AMHRCs) to different colors of LED lights i.e., red, green, blue, red/green/blue (1/1/1, RGB), and white, was conducted to promote the root growth and accumulation of isoflavonoids and astragalosides. LED light treatment regardless of colors was found beneficial for root growth, which might be a result of the formation of more root hairs upon light stimulation. Blue LED light was found most effective for enhancing phytochemical accumulation. Results showed that the productivity of root biomass in blue-light grown AMHRCs with an initial inoculum size of 0.6% for 55 days was 1.40-fold higher than that in dark (control), and yields of high-value isoflavonoids and astragalosides including calycosin, formononetin, astragaloside IV, and astragaloside I increased by 3.17-fold, 2.66-fold, 1.78-fold, and 1.52-fold relative to control, respectively. Moreover, the photooxidative stress together with transcriptional activation of biosynthesis genes might contribute to the enhanced accumulation of isoflavonoids and astragalosides in blue-light grown AMHRCs. Overall, this work offered a feasible approach for obtaining higher yields of root biomass and medicinally important compounds in AMHRCs via the simple supplementation of blue LED light, which made blue-light grown AMHRCs industrially attractive as plant factory in controlled growing systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s11240-023-02486-7.

Cocultivation of pigeon pea hairy root cultures and Aspergillus for the enhanced production of cajaninstilbene acid.

Pigeon pea hairy root cultures (PPHRCs) have been proven to be a promising alternative for the production of health-beneficial phenolic compounds, such as the most important health-promoting compound, i.e., cajaninstilbene acid (CSA). In this study, PPHRCs were cocultured with live Aspergillus fungi for further improving phenolic productivity via biological elicitation. Aspergillus oryzae CGMCC 3.951 (AO 3.951) was found to be the optimal fungus that could achieve the maximum increment of CSA (10.73-fold increase) in 42-day-old PPHRCs under the inoculum size of mycelia 0.50% and cocultivation time 36 h. More precisely, the contents of CSA in hairy roots and culture media after fungal elicitation increased by 9.87- and 62.18-fold over control, respectively. Meanwhile, the contents of flavonoid glycosides decreased, while aglycone yields increased upon AO 3.951 elicitation. Moreover, AO 3.951 could trigger the oxidative stress and pathogen defense response thus activating the expression of biosynthesis- and ABC transporter-related genes, which contributed to the intracellular accumulation and extracellular secretion of phenolic compounds (especially CSA) in PPHRCs. And PAL2, 4CL2, STS1, and I3'H were likely to be the potential key enzyme genes regulating the biosynthesis of CSA, and ABCB11X1-1, ABCB11, and ABCG24X2 were closely related to the transmembrane transport of CSA. Overall, the cocultivation approach could make PPHRCs more commercially attractive for the production of high-value phenolic compounds such as CSA and flavonoid aglycones in nutraceutical/medicinal fields. And the elucidation of crucial biosynthesis and transport genes was important for systematic metabolic engineering aimed at increasing CSA productivity. KEY POINTS: • Cocultivation of PPHRCs and live fungi was to enhance CSA production and secretion. • PPHRCs augmented CSA productivity 10.73-fold when cocultured with AO 3.951 mycelia. • Several biosynthesis and transport genes related to CSA production were clarified.

Health-Promoting Phenolic Compound Accumulation, Antioxidant Response, Endogenous Salicylic Acid Generation, and Biosynthesis Gene Expression in Germinated Pigeon Pea Seeds Treated with UV-B Radiation.

Germinated pigeon pea seeds (GPPSs) are good dietary supplements with satisfactory nutritional and medicinal values. In this study, UV-B treatment was used to promote the accumulation of health-promoting phenolic compounds (10 flavonoids and 1 stilbene) in GPPS. The total yield of 11 phenolic compounds (235 839.76 ± 17 118.24 ng/g DW) significantly improved (2.53-fold increase) in GPPSs exposed to UV-B radiation (3 W/m2) for 8 h, whereas free amino acid and reducing sugar contents exhibited a decreasing tendency during UV-B exposure. Meanwhile, the positive response in the antioxidant activities of enzymes and nonenzymatic extracts was noticed in UV-B-treated GPPSs. Moreover, UV-B radiation could cause tissue damages in hypocotyls and cotyledons of the GPPSs and enhance the generation of endogenous salicylic acid, thus activating the expression of biosynthesis genes (especially CHS and STS1). Overall, the simple UV-B supplementation strategy makes GPPSs more attractive as functional foods/nutraceuticals in diet for promoting human health.

Identification of genes associated with biosynthesis of bioactive flavonoids and taxoids in Taxus cuspidata Sieb. et Zucc. plantlets exposed to UV-B radiation.

UV-B radiation is a typical environmental stressor that can promote phytochemical accumulation in plants. Taxus species are highly appreciated due to the existence of bioactive taxoids (especially paclitaxel) and flavonoids. However, the effect of UV-B radiation on taxoid and flavonoid biosynthesis in Taxus cuspidata Sieb. et Zucc. is largely unknown. In the present work, the accumulation of taxoids and flavonoids in T. cuspidata plantlets was significantly induced by 12 and 24 h of UV-B radiation (3 W/m2), and a large number of significantly differentially expressed genes were obtained via transcriptomic analysis. The significant up-regulation of antioxidant enzyme- and flavonoid biosynthesis-related genes (phenylalanine ammonia lyase 1, chalcone synthase 2, flavonol synthase 1, and flavonoid 3', 5'-hydroxylase 2), suggested that UV-B might cause the oxidative stress thus promoting flavonoid accumulation in T. cuspidata. Moreover, the expression of some genes related to jasmonate metabolism and taxoid biosynthesis (taxadiene synthase, baccatin III-3-amino 3-phenylpropanoyltransferase 1, taxadiene-5α-hydroxylase, and ethylene response factors 15) was significantly activated, which indicated that UV-B might initiate jasmonate signaling pathway that contributed to taxoid enhancement in T. cuspidata. Additionally, the identification of some up-regulated genes involved in lignin biosynthesis pathway indicated that the lignification process in T. cuspidata might be stimulated for defense against UV-B radiation. Overall, our findings provided a better understanding of some potential key genes associated with flavonoid and taxoid biosynthesis in T. cuspidata exposed to UV-B radiation.

Bi-Basal Ganglia Involvement with Signal Evolution in a Case of Anti-LGI1 Encephalitis.

Anti-leucine-rich glioma-inactivated 1 (LGI1) encephalitis is a rare type of non-paraneoplastic limbic encephalitis (LE) mainly characterized by seizures, cognitive disorder, faciobrachial dystonic seizures (FBDS), hyponatremia, insomnia, and autonomic dysfunction. Here, we report the case of an elderly female patient who tested positive for antibodies against LGI1 and was initially thought to have Hashimoto encephalopathy (HE) due to its similar clinical features and the patient's high titers of antithyroid antibody. Interestingly, during the course of the disease, the patient exhibited typical FBDS and brain magnetic resonance imaging (MRI) showed a hyperintense signal evolution from T2/Fluid attenuated inversion recovery (FLAIR) to T1-weighted image in the bilateral basal ganglia (BG), which have rarely been reported previously.

Application of UV-B radiation for enhancing the accumulation of bioactive phenolic compounds in pigeon pea [Cajanus cajan (L.) Millsp.] hairy root cultures.

UV-B radiation is an ideal elicitation strategy for promoting phytochemical accumulation in plant in vitro cultures, associated with various advantages of easy manipulation, cost-effectiveness, no residue, and instantaneous termination. For the first time, UV-B radiation was used to enhance the production of bioactive phenolic compounds (flavonoids and stilbenes) in pigeon pea hairy root cultures (PPHRCs). The total yield of eight flavonoids (414.95 ± 50.68 µg/g DW) in 42-day-old PPHRCs exposed to 4 h of UV-B radiation increased by 1.49-fold as against control, whereas the yield of cajaninstilbene acid (6566.01 ± 702.14 µg/g DW) in PPHRCs undergoing 10 h of UV-B radiation significantly increased by 2.31-fold over control. UV-B radiation was found to induce the oxidative stress in PPHRCs and cause the tissue damage to hairy roots, which improved the levels of endogenous salicylic acid thus triggering the expression of genes involved in phenylpropanoid biosynthesis pathway. And, a regulation competition in metabolic flow dominated by CHS and STS was responsible for the difference in accumulation trends of flavonoids and cajaninstilbene acid. Results of this study not only provide a feasible and simple UV-B supplementation strategy for the enhanced production of bioactive phenolic compounds (especially the high-value cajaninstilbene acid) in PPHRCs, but also contributed to the understanding of photobiological responses related to secondary metabolism.

Regulatory effects of miR-138 and RUNX3 on Th1/Th2 balance in peripheral blood of children with cough variant asthma. / 咳嗽变异性哮喘患儿外周血miR-138及RUNX3对Th1/Th2平衡的调节作用.

OBJECTIVES: To study the expression levels of microRNA-138 (miR-138) and Runt-related transcription factor 3 (RUNX3) in peripheral blood of children with cough variant asthma (CVA) and their regulatory effects on Th1/Th2 balance. METHODS: Sixty-five children with CVA (CVA group) and 30 healthy children (control group) were enrolled. Peripheral venous blood samples were collected for both groups, and CD4+ T cells were isolated and cultured. Enzyme-linked immunosorbent assay was used to measure the levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-5, and IL-13 that were secreted by CD4+ T cells. Flow cytometry was used to determine the percentages of Th1 and Th2 cells. Quantitative real-time PCR was used to measure the level of RUNX3 mRNA in CD4+ T cells and the level of miR-138 in peripheral blood. Western blot was used to determine the protein expression of RUNX3 in CD4+ T cells. The dual-luciferase reporter assay was used to determine the targeting effects of miR-138 and RUNX3. The RUNX3-mimic plasmid was transfected into CD4+ T cells, and the effects on the levels of IFN-γ, IL-2, IL-4, IL-5, and IL-13 and the percentages of Th1 and Th2 cells were measured. RESULTS: Compared with the control group, the CVA group showed significantly decreased levels of IFN-γ and IL-2 from CD4+ T cells, significantly increased levels of IL-4, IL-5, and IL-13 from CD4+ T cells, significantly decreased Th1 cell percentage and Th1/Th2 ratio, and a significantly increased Th2 cell percentage (P<0.05). The CVA group showed significantly lower relative expression levels of RUNX3 mRNA and protein in CD4+ T cells in peripheral blood than the control group (P<0.001). The relative expression level of miR-138 was significantly higher in the CVA group than in the control group (P<0.001). MiR-138 could target the expression of RUNX3. Upregulating the expression of RUNX3 in CD4+ T cells induced significantly increased levels of IFN-γ and IL-2, significantly decreased levels of IL-4, IL-5, and IL-13, significantly increased Th1 cell percentage and Th1/Th2 ratio, and a significantly decreased Th2 cell percentage (P<0.05). CONCLUSIONS: MiR-138 regulates Th1/Th2 balance by targeting RUNX3 in children with CVA, providing a new direction for the treatment of CVA.

Intraoperative Neurophysiology and Transcranial Doppler for Detection of Cerebral Ischemia and Hyperperfusion During Carotid Endarterectomy.

OBJECTIVE: To evaluate and compare efficacy of intraoperative neurophysiological monitoring (IONM) and intraoperative transcranial Doppler (TCD) techniques for identification of hypoperfusion during carotid artery clamp and hyperperfusion after release of occlusion during carotid endarterectomy. METHODS: This was a retrospective, consecutive case series of 152 patients undergoing carotid endarterectomy between June 2018 and March 2020. Somatosensory evoked potentials, motor evoked potentials, electroencephalogram, and TCD were obtained. RESULTS: Three patient cohorts were observed after clamping the carotid artery: A, in 132 of 152 patients (87%), TCD blood flow velocity decreased by <50% and there were no changes in IONM; B, in 5 of 152 (3%) patients, TCD blood flow rate was reduced 50%-100% with no changes in IONM; C, in 15 patients (10%), blood flow velocity was reduced by 50%-100% and all IONM modalities met warning criteria. With increased blood pressure, IONM and blood flow velocities improved to less than warning criteria in 8 of 15 patients. In 6 of the 7 remaining patients, IONM modalities recovered to baseline immediately after clamps were removed from the carotid artery. The 1 patient with persistent motor evoked potential deterioration experienced postoperative proximal muscle weakness, which recovered 48 hours later. In 22 patients, TCD detected hyperperfusion at the moment of clamp release. CONCLUSIONS: TCD blood flow velocity is correlated with motor evoked potential and somatosensory evoked potential amplitude changes after clamping. After declamping, TCD can detect hyperperfusion and help regulate blood pressure to prevent hyperperfusion.

Role of TREM-1 in the development of early brain injury after subarachnoid hemorrhage.

Triggering receptor expressed on myeloid cells-1 (TREM-1) was found to be induced in the context of subarachnoid hemorrhage (SAH) before. This study further investigates its role in the development of SAH-induced early brain injury (EBI). Firstly, rats were randomly divided into Sham and SAH groups for analysis of temporal patterns and cellular localization of TREM-1. Secondly, TREM-1 intervention was administrated to produce Sham, vehicle, antagonist and agonist groups, for analyzing TREM-1, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and NF-κB expressions at 24 h post-modeling, and EBI assessment at 24 h and 72 h. Thirdly, TLR4 inhibitor (TAK-242) was exploited to produce Sham, Sham+TAK-242, SAH, and SAH + TAK-242 groups to analyze the effects of TLR4 inhibition on TREM-1 induction and EBI evaluation at 72 h. Fourthly, the relationship of soluble TREM-1 (sTREM-1) levels in cerebrospinal fluid of SAH patients with Hunt-Hess grades were explored. The results showed that TREM-1 increased in the brain after experimental SAH (eSAH) early at 6 h and peaked at 48 h, which was found to be located in microglia and endothelial cells. TREM-1 inhibition attenuated EBI associated with TLR4/MyD88/NF-κB suppression, while enhancement had the opposite effects. Contrarily, TLR4 inhibition prevented TREM-1 induction and ameliorated EBI. In addition, sTREM-1 levels in SAH patients positively correlated with Hunt-Hess grades. Overall, the present study provides new evidence that TREM-1 increases dynamically in the brain after eSAH and it is located in microglia and endothelial cells, which may aggravate EBI by interacting with TLR4 pathway. And sTREM-1 in patients might act as a monitoring biomarker of EBI, providing new insights for future studies.

Regulatory effects of miR-138 and RUNX3 on Th1/Th2 balance in peripheral blood of children with cough variant asthma / 中国当代儿科杂志

OBJECTIVES@#To study the expression levels of microRNA-138 (miR-138) and Runt-related transcription factor 3 (RUNX3) in peripheral blood of children with cough variant asthma (CVA) and their regulatory effects on Th1/Th2 balance.@*METHODS@#Sixty-five children with CVA (CVA group) and 30 healthy children (control group) were enrolled. Peripheral venous blood samples were collected for both groups, and CD4@*RESULTS@#Compared with the control group, the CVA group showed significantly decreased levels of IFN-γ and IL-2 from CD4@*CONCLUSIONS@#MiR-138 regulates Th1/Th2 balance by targeting RUNX3 in children with CVA, providing a new direction for the treatment of CVA.

Simultaneous quantification of eleven bioactive phenolic compounds in pigeon pea natural resources and in vitro cultures by ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS/MS).

Bioactive phenolics primarily contribute to versatile health benefits of pigeon pea. For the first time, an UPLC-QqQ-MS/MS method was developed for the quantitative analysis of eleven bioactive phenolic compounds in pigeon pea natural resources (seeds, leaves, and roots) and in vitro cultures (calli and hairy roots). The proposed method could be achieved within 6 min of running time, and displayed the satisfactory linearity, sensitivity, precision, accuracy, and stability. According to analytical results, the distribution of eleven target compounds in different organs of pigeon pea was clarified. Also, it was surprisingly found that pigeon pea in vitro cultures exhibited superiority in contents of genistin and cajaninstilbene acid as compared with natural resources. Overall, the present work provided a rapid and sensitive analysis approach, which could be useful not only for quality control of pigeon pea natural resources, but also for applicability and safety evaluation of pigeon pea in vitro cultures.

High detection efficiency silicon single-photon detector with a monolithic integrated circuit of active quenching and active reset.

Silicon single-photon detectors (SPDs) are key devices for detecting single photons in the visible wavelength range. Photon detection efficiency (PDE) is one of the most important parameters of silicon SPDs, and increasing PDE is highly required for many applications. Here, we present a practical approach to increase the PDE of silicon SPDs with a monolithic integrated circuit of active quenching and active reset (AQAR). The AQAR integrated circuit is specifically designed for thick silicon single-photon avalanche diodes (SPADs) with high breakdown voltage (250 V-450 V) and then fabricated via the process of high-voltage 0.35-µm bipolar-CMOS-DMOS. The AQAR integrated circuit implements the maximum transition voltage of ∼68 V with 30 ns quenching time and 10 ns reset time, which can easily boost PDE to the upper limit by regulating the excess bias up to a high enough level. By using the AQAR integrated circuit, we design and characterize two SPDs with the SPADs disassembled from commercial products of single-photon counting modules (SPCMs). Compared with the original SPCMs, the PDE values are increased from 68.3% to 73.7% and 69.5% to 75.1% at 785 nm, respectively, with moderate increases in dark count rate and afterpulse probability. Our approach can effectively improve the performance of the practical applications requiring silicon SPDs.

Interaction of Microglia and Astrocytes in the Neurovascular Unit.

The interaction between microglia and astrocytes significantly influences neuroinflammation. Microglia/astrocytes, part of the neurovascular unit (NVU), are activated by various brain insults. The local extracellular and intracellular signals determine their characteristics and switch of phenotypes. Microglia and astrocytes are activated into two polarization states: the pro-inflammatory phenotype (M1 and A1) and the anti-inflammatory phenotype (M2 and A2). During neuroinflammation, induced by stroke or lipopolysaccharides, microglia are more sensitive to pathogens, or damage; they are thus initially activated into the M1 phenotype and produce common inflammatory signals such as IL-1 and TNF-α to trigger reactive astrocytes into the A1 phenotype. These inflammatory signals can be amplified not only by the self-feedback loop of microglial activation but also by the unique anatomy structure of astrocytes. As the pathology further progresses, resulting in local environmental changes, M1-like microglia switch to the M2 phenotype, and M2 crosstalk with A2. While astrocytes communicate simultaneously with neurons and blood vessels to maintain the function of neurons and the blood-brain barrier (BBB), their subtle changes may be identified and responded by astrocytes, and possibly transferred to microglia. Although both microglia and astrocytes have different functional characteristics, they can achieve immune "optimization" through their mutual communication and cooperation in the NVU and build a cascaded immune network of amplification.

Effective Production of Phenolic Compounds with Health Benefits in Pigeon Pea [Cajanus cajan (L.) Millsp.] Hairy Root Cultures.

Phenolic compounds in pigeon pea possess various biological properties beneficial to human health. In this study, pigeon pea hairy root cultures (PPHRCs) were developed as an effective in vitro platform for the production of phenolic compounds. A high-productive hairy root line was screened and characterized, and its culture conditions were optimized in terms of biomass productivity and phenolic yield. The comparative profiling of 10 phenolic compounds in PPHRCs and pigeon pea natural resources (seeds, leaves, and roots) was achieved by ultra-high-performance liquid chromatography-tandem mass spectrometry analysis. The total phenolic yield in PPHRCs (3278.44 µg/g) was much higher than those in seeds (68.86 µg/g) and roots (846.03 µg/g), and comparable to leaves (3379.49 µg/g). Notably, PPHRCs exhibited superiority in the yield of the most important health-promoting compound cajaninstilbene acid (2996.23 µg/g) against natural resources (4.42-2293.31 µg/g). Overall, PPHRCs could serve as promising potential alternative sources for the production of phenolic compounds with nutraceutical/medicinal values.

Simultaneous determination of taxoids and flavonoids in twigs and leaves of three Taxus species by UHPLC-MS/MS.

Taxus species are highly concerned due to the presence of anticancer taxoids (especially paclitaxel) and health beneficial flavonoids. For the first time, an UHPLC-MS/MS method was developed for the simultaneous determination of seven taxoids and seven flavonoids in twigs and leaves of three Taxus species. The satisfactory separation of fourteen target compounds was achieved within 5 min of running time on an Agilent ZORBAX Eclipse Plus C18 column (50 mm × 2.1 mm I.D., 1.8 µm) using an acetonitrile-water gradient elution program. Mass transitions of all analytes in selected reaction monitoring acquisition mode were systematically optimized for obtaining the highest signal intensities. Regression equations of all analytes exhibited excellent linearities with coefficients higher than 0.9990, and the lowest limits of quantification of all analytes ranged from 0.01 to 1.66 ng/mL. The intra- and inter-day precisions (relative standard deviations) of all analytes were less than 4.17% for retention time and less than 7.42% for peak area, and the spiking standard recoveries of all analytes ranged from 96.85%-104.77%. By the aid of the proposed method, the distribution of fourteen target compounds in twigs and leaves of Taxus chinensis, Taxus cuspidata, and Taxus media was clearly figured out. Overall, the present work provided a rapid and valid UHPLC-MS/MS approach, which could not only be useful for quality control and applicability assessment of twigs and leaves of the three Taxus species in pharmaceutical and nutraceutical industries, but also offer a good reference for the systematic analysis of taxoids and flavonoids in other Taxus species.

Chitosan promoting formononetin and calycosin accumulation in Astragalus membranaceus hairy root cultures via mitogen-activated protein kinase signaling cascades.

Chitosan, behaving as a potent biotic elicitor, can induce plant defense response with the consequent enhancement in phytoalexin accumulation. Accordingly, chitosan elicitation was conducted to promote the production of two phytoalexins, i.e. formononetin and calycosin (also known as health-promoting isoflavones), in Astragalus membranaceus hairy root cultures (AMHRCs). Compared with control, 12.45- and 6.17-fold increases in the yields of formononetin (764.19 ± 50.81 µg/g DW) and calycosin (611.53 ± 42.22 µg/g DW) were obtained in 34 day-old AMHRCs treated by 100 mg/L of chitosan for 24 h, respectively. Moreover, chitosan elicitation could cause oxidative burst that would induce the expression of genes (MPK3 and MPK6) related to mitogen-activated protein kinase signaling (MAPK) cascades, which contributed to the transcriptional activation of pathogenesis-related genes (ß-1,3-glucanase, Chitinase, and PR-1) and eight biosynthesis genes involved in the calycosin and formononetin pathway. Overall, the findings in this work not only highlight a feasible chitosan elicitation practice to enhance the in vitro production of two bioactive isoflavones for nutraceutical and food applications, but also contribute to understanding the phytoalexin biosynthesis in response to chitosan elicitation.

Remarkable enhancement of flavonoid production in a co-cultivation system of Isatis tinctoria L. hairy root cultures and immobilized Aspergillus niger.

The dried roots of Isatis tinctoria L. are highly traded in the pharmaceutical industry due to their notable anti-influenza efficacy. For the first time, I. tinctoria hairy root cultures (ITHRCs) were co-cultured with two immobilized live GRAS (Generally Recognized as Safe) fungi, i.e. Aspergillus niger and Aspergillus niger, for the elevated production of pharmacologically active flavonoids. Immobilized A. niger (IAN) was exhibited as the superior elicitor in the plant-fungus co-cultivation system. The highest flavonoid production (3018.31 ± 48.66 µg/g DW) were achieved in IAN-treated ITHRCs under the optimal conditions of IAN spore concentration ca.104 spores/mL, temperature 30 °C, initial pH value of media 7.0 and time 72 h, which remarkably increased 6.83-fold relative to non-treated control (441.91 ± 7.35 µg/g DW). Also, this study revealed that IAN elicitation could trigger the sequentially transient accumulation of signal molecules and intensify the oxidative stress in ITHRCs, which both contributed to the up-regulated expression of associated genes involved in flavonoid biosynthetic pathway. Moreover, IAN could be reused at least five cycles with satisfactory performance. Overall, the coupled culture of IAN and ITHRCs is a promising and effective approach for the enhanced production of flavonoids, which allows for the improved applicability of these valuable compounds in pharmaceutical fields.

Ultraviolet radiation for flavonoid augmentation in Isatis tinctoria L. hairy root cultures mediated by oxidative stress and biosynthetic gene expression.

Search of cost-effective strategies that can enhance the accumulation of phytochemicals of pharmaceutical interest in plant in vitro cultures is an essential task. For the first time, Isatis tinctoria L. hairy root cultures were exposed to ultraviolet radiation (ultraviolet-A, ultraviolet-B, and ultraviolet-C) in an attempt to promote the production of pharmacologically active flavonoids. Results showed that the maximum flavonoid accumulation (7259.12 ±â€¯198.19 µg/g DW) in I. tinctoria hairy root cultures treated by 108 kJ/m2 dose of UV-B radiation increased 16.51-fold as compared with that in control (439.68 ±â€¯8.27 µg/g DW). Additionally, antioxidant activity enhancement and cell wall reinforcement were found in the treated I. tinctoria hairy root cultures, indicating the positive-feedback responses to oxidative stress mediated by ultraviolet-B radiation. Moreover, the expression of chalcone synthase gene was tremendously up-regulated (up to 405.84-fold) in I. tinctoria hairy root cultures following ultraviolet-B radiation, which suggested chalcone synthase gene might play a crucial role in flavonoid augmentation. Overall, the present work provides a feasible approach for the enhanced production of biologically active flavonoids in I. tinctoria hairy root cultures via the simple supplementation of ultraviolet-B radiation, which is useful for the biotechnological production of these high-added value compounds to fulfil the ever-increasing demand in pharmaceutical fields.