Influence of cytokinins and yeast extract on growth and flavone production in hairy root cultures of Scutellaria baicalensis

Hairy roots produce various bioactive chemical compounds than wild-type roots which offer a promising in vitro approach for synthesizing important nutraceutical compounds. The purpose of this study is to increase the production of flavone compounds in hairy root cultures of Scutellaria baicalensis by the addition of elicitors such as cytokinins and yeast extract. Cytokinins such as kinetin (KIN), 6-benzylaminopurine (BAP), and Thidiazuron (TDZ) were utilized at 0.1, 0.5, and 1.0 mg/L, whereas for yeast extract treatment 50, 100, and 150 mg/L concentrations were added to the ½ SH medium. Effects of elicitors were measured in terms of dry biomass and flavone contents (baicalin, baicalein, and wogonin) using high-performance liquid chromatography (HPLC). The highest dry weight was achieved in the control hairy root than that of all cytokinins-treated hairy root cultures. In all the cytokinin-treated hairy root cultures, as the concentration increased the dry weight of the hairy root decreased. In contrast, in all the yeast extract-treated hairy root cultures as the concentration increases the dry weight of the hairy root increased, whereas the highest dry weight was achieved in 150 mg/L of yeast extract. Moving to the flavone content, baicalin was detected highest content in all the hairy root cultures supplied with cytokinin and yeast extract. The highest total flavone content was achieved in the hairy root culture treated with 1.0 mg/L of TDZ and 50 mg/L of yeast extract. This result might help the commercial agronomic sector by facilitating the in vitro mass production of nutraceuticals using S. baicalensis hairy root cultures

Assessment of organic acid and sugar composition in apricot, plumcot, plum, and peach during fruit development

Variation in content of organic acids and soluble sugars, and in physical characteristics was evaluated in apricot (P. armeniaca L. cv. Harcot), plumcot (plum-apricot hybrid, P. salicina ⅹ P. armeniaca L. cv. Harmony), plum (P. salicina Lindl. cv. Formosa), and peach (P. persica L. Batsch cv. Jinmi). The content of organic acids and sugars, as well as parameters of fruit quality (weight, dimensions, firmness, total soluble solids, and total acidity) in Prunus fruits during fruit development were determined. Organic acids, including oxalic acid, quinic acid, malic acid, shikimic acid, citric acid, and quinic acid, sugars, including sucrose, fructose, glucose, and sugar alcohol (sorbitol), were identified and quantified using HPLC. Organic acid mostly increased during the early stages of fruit growth (30 - 60 days after full bloom) and decreased until fruits were fully ripened. In general, plum was the highest in most organic acids compared with the other fruits, while apricot contained the lowest acid content except for citric acid. Sucrose, fructose, and glucose content increased with fruit development, unlike content of sorbitol. Plumcot contained the highest fructose, and peach showed the maximum content of sucrose at full maturation stages. Total soluble solids averaged 17.5, 14.8, 11.9, and 10.6 ºBrix in apricot, plumcot, plum, and peach, respectively, whereas total acidity was 0.9, 1.4, 0.5, and 0.3% in four Prunus cultivars at ripened stages. Shikimic acid was significantly correlated with oxalic acid in apricot, plumcot, and plum, but not in peach. Fructose and glucose were highly correlated in plumcot, plum, and peach.

Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide1, 2. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis3, 4, 5, several of which are currently in clinical trials6, 7, 8. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis

Practical magnetic bead-based capillary electrophoresis with laser-induced fluorescence for detecting endogenous miRNA in plasma

MicroRNAs (miRNAs) are small, non-coding RNAs crucial for gene regulation and implicated in various human diseases. Their potential as clinical prognostic and diagnostic biomarkers in biological fluids necessitates reliable detection methods. In this study, a combination of streptavidin-coupled magnetic beads and capillary electrophoresis with laser-induced fluorescence (CE-LIF) was used to extract and analyze plasma miRNAs. Specifically, miRNAs hybridized with a biotinylated fluorescent DNA probe were isolated from plasma using magnetic beads. These hybridized miRNAs were then directly injected into the CE-LIF system for analysis, eliminating the need for additional processing steps. Both the hybridization and bead-to-probe binding were executed concurrently, regulated by temperature and time. Through the optimization of magnetic bead extraction and CE-LIF conditions, we developed a highly sensitive assay for miR-21 quantification in plasma. The assay displayed remarkable linearity (R2 = 0.9975) within a 0.1–5 pM range and exhibited favorable precision (0.22–1.26 %) and accuracy (98.31–111.19 %). Importantly, we successfully detected endogenous miR-21 in plasma samples from both a lung cancer patient and healthy adults, revealing a 1.7-fold overexpression of miR-21 in lung cancer plasma relative to normal samples. Our findings suggest that this developed system offers a simple and sensitive approach for detecting endogenous miRNAs in plasma, showing its potential utility in disease diagnostics. To our knowledge, this is the first study to utilize CE-LIF for plasma miRNA detection

Analysis of the curriculum for special needs classes : point of view inclusive education

近年、インクルーシブ教育を推進していく上で、特別支援学級の役割が重要視されてきている。しかしながら、特別支援学級の教育課程については、通常の小・中学校に準ずることが表記されているのみである。また、特別支援学級においては、様々な授業実践がなされているものの、インクルーシブ教育の現合、を取り入れた特別支援学級の教育課程の編成に関する研究は見当たらない。そこで本研究では、インクルーシブ、教育制面指標(lEAI)を用い、インクルーシブ教育の観点から、日本の特別支援学級における制度・政策の分析を行った。また、特別支援学級で行われている実践事例を分析し、インクルーシプ教育の観点に基づ、いた日本の特別支援学級における教育課程編成の課題を明らかにすることを目的とする。IEAIの領域と実践事例を対応分析した結果、IEAIの観点である「権利の保障」領域においては、「教科外活動の保障」や「公平性の確保」、「人的・物的環境整備J領域においては、「共に学ぶ場の設定」や「多職種及び保護者との連携」、「教育課程の改善」領域においては、「地域社会への参加促進」や「障害理解の促進」、「リーダー育成」の観点を踏まえ、教育課程を編成する必要がある事が明らかになった

LED Lights Influenced Phytochemical Contents and Biological Activities in Kale (<i>Brassica oleracea</i> L. var. <i>acephala</i>) Microgreens

Light-emitting diodes (LEDs) are regarded as an effective artificial light source for producing sprouts, microgreens, and baby leaves. Thus, this study aimed to investigate the influence of different LED lights (white, red, and blue) on the biosynthesis of secondary metabolites (glucosinolates, carotenoids, and phenolics) and the biological effects on kale microgreens. Microgreens irradiated with white LEDs showed higher levels of carotenoids, including lutein, 13-cis-β-carotene, α-carotene, β-carotene, and 9-cis-β-carotene, than those irradiated with red or blue LEDs. These findings were consistent with higher expression levels of carotenoid biosynthetic genes (BoPDS and BoZDS) in white-irradiated kale microgreens. Similarly, microgreens irradiated with white and blue LEDs showed slightly higher levels of glucosinolates, including glucoiberin, progoitrin, sinigrin, and glucobrassicanapin, than those irradiated with red LEDs. These results agree with the high expression levels of BoMYB28-2, BoMYB28-3, and BoMYB29 in white- and blue-irradiated kale microgreens. In contrast, kale microgreens irradiated with blue LEDs contained higher levels of phenolic compounds (gallic acid, catechin, ferulic acid, sinapic acid, and quercetin). According to the total phenolic content (TPC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition assays, the extracts of kale microgreens irradiated with blue LEDs had slightly higher antioxidant activities, and the DPPH inhibition percentage had a positive correlation with TPC in the microgreens. Furthermore, the extracts of kale microgreens irradiated with blue LEDs exhibited stronger antibacterial properties against normal pathogens and multidrug-resistant pathogens than those irradiated with white and red LEDs. These results indicate that white-LED lights are suitable for carotenoid production, whereas blue-LED lights are efficient in increasing the accumulation of phenolics and their biological activities in kale microgreens

Polyamine and EIF5A hypusination downstream of c-Myc confers targeted therapy resistance in BRAF mutant melanoma

Abstract Background BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms. Methods CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts. Results We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings. Conclusions Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors