Vitexicarpin Induces Apoptosis and Inhibits Metastatic Properties via the AKT-PRAS40 Pathway in Human Osteosarcoma.

Osteosarcoma, which has poor prognosis after metastasis, is the most common type of bone cancer in children and adolescents. Therefore, plant-derived bioactive compounds are being actively developed for cancer therapy. Artemisia apiacea Hance ex Walp. is a traditional medicinal plant native to Eastern Asia, including China, Japan, and Korea. Vitexicarpin (Vitex), derived from A. apiacea, has demonstrated analgesic, anti-inflammatory, antitumour, and immunoregulatory properties; however, there are no published studies on Vitex isolated from the aerial parts of A. apiacea. Thus, this study aimed to evaluate the antitumour activity of Vitex against human osteosarcoma cells. In the present study, Vitex (>99% purity) isolated from A. apiacea induced significant cell death in human osteosarcoma MG63 cells in a dose- and time-dependent manner; cell death was mediated by apoptosis, as evidenced by the appearance of cleaved-PARP, cleaved-caspase 3, anti-apoptotic proteins (Survivin and Bcl-2), pro-apoptotic proteins (Bax), and cell cycle-related proteins (Cyclin D1, Cdk4, and Cdk6). Additionally, a human phosphokinase array proteome profiler revealed that Vitex suppressed AKT-dependent downstream kinases. Further, Vitex reduced the phosphorylation of PRAS40, which is associated with autophagy and metastasis, induced autophagosome formation, and suppressed programmed cell death and necroptosis. Furthermore, Vitex induced antimetastatic activity by suppressing the migration and invasion of MMP13, which is the primary protease that degrades type I collagen for tumour-induced osteolysis in bone tissues and preferential metastasis sites. Taken together, our results suggest that Vitex is an attractive target for treating human osteosarcoma.

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors.

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.

A New Perspective on Metabolites and Bioactive Compounds from Fungi.

Fungi play an important role in the solution to important global problems. Making use of processes and goods that are based on fungi can help promote sustainability by making the most efficient use of natural resources. Fungi stand apart from other organisms due to their extraordinary capacity to generate organic compounds. They are necessary for the psychological and physiological well-being of people worldwide. They are excellent producers of vitamins, pigments, hydrolytic enzymes, biofuels, organic acids, polysaccharides, and secondary metabolites such as antibiotics, anticancer treatments, hypocholesterolemic pharmaceuticals, and immunosuppressants. Other secondary metabolites include biofuels. In addition, polysaccharides are produced by them. We provide a condensed explanation of the significance of secondary metabolites in a variety of industries, such as the pharmaceutical industry, the food industry, the textile industry, and the transportation industry. In addition to providing a better understanding of biosynthetic regulation and the possibilities of genetic engineering, improved laboratory processes for the selection of nontoxigenic fungal strains have permitted the manufacture of larger quantities of safe commercial items. The significance of fungi in industrial settings is the topic that will be investigated in this review.

Vitamin D status and clinical implications in the adult population of Malaysia: a position paper by the Malaysian Vitamin D Special Interest Group.

PURPOSE: Vitamin D deficiency and insufficiency is common among populations globally, and in Asia and Malaysia. The purpose of this Position Paper is to propose recommendations for both clinicians and non-clinicians to promote vitamin D sufficiency in Malaysian adults. Formation of a national multisector, multidisciplinary alliance is also proposed to progress initiatives relating to safe sun exposure, adequate vitamin D intake through food fortification, and vitamin D supplementation for high-risk groups. METHODS: Literature reviews were undertaken to inform summaries of the following: vitamin D status globally and in Asian and Malaysian populations, vitamin D status among individuals with common medical conditions, and current recommendations to achieve vitamin D sufficiency through sun exposure, food intake and supplementation. Recommendations were based on the findings of the literature reviews, recent European guidance on vitamin D supplementation, the 2018 road map for action on vitamin D in low- and middle-income countries, and research recommendations proposed by the Malaysian Ministry of Health in 2017. RESULTS: Recommendations on assessment of vitamin D in the adult Malaysian population include using serum or plasma 25-hydroxyvitamin D concentration as a biomarker, widespread participation by Malaysian laboratories in the Vitamin D Standardization Program, adoption of the US Endocrine Society definitions of vitamin D deficiency and insufficiency, and development of a comprehensive nationwide vitamin D status study. Specific high-risk groups are identified for vitamin D assessment and recommendations relating to loading doses and ongoing management are also made. CONCLUSION: This Position Paper provides individual clinicians and national stakeholder organisations with clear recommendations to achieve vitamin D sufficiency in the adult population of Malaysia.

The Association of Intravenous Iron Administered the Day before Total Knee Arthroplasty with Postoperative Anemia and Functional Recovery.

Background and Objectives: Total knee arthroplasty (TKA) involves blood loss, increasing the risk of postoperative anemia and delayed functional recovery. Intravenous (IV) iron supplementation limits postoperative anemia; however, the effectiveness of IV iron, administered one day before TKA, on postoperative anemia and functional recovery has scarcely been studied. Materials and Methods: We conducted a retrospective cohort study with propensity score matching using two consecutive groups of patients who underwent TKA using tranexamic acid: the iron group received 500 mg ferric derisomaltose intravenously one day before surgery (n = 46); the non-iron group did not (n = 46). Hemoglobin (Hb) level was determined at postoperative days (PODs) 2, 4, 6, 14, and 30. Ferritin, transferrin saturation (TSAT), and functional iron deficiency anemia (IDA) rate were measured at PODs 2, 4, 6, and 14. Length of hospital stay and transfusion rate were also evaluated. Results: The iron group had higher Hb levels at PODs 6, 14, and 30 and higher ferritin and TSAT at PODs 2, 4, 6, and 14. The functional IDA rate was significantly higher in the non-iron group at PODs 2, 4, 6, and 14. Length of hospital stay was significantly shorter in the iron group; however, the rate of transfusion did not differ between the two groups. Conclusions: IV iron administered one day before TKA was associated with postoperative anemia recovery and length of hospital stay; however, it did not lower the postoperative transfusion rate.

Development and authentication of Panax ginseng cv. Sunhong with high yield and multiple tolerance to heat damage, rusty roots and lodging.

Ginseng (Panax ginseng) has been used as a valuable medicinal plant in Asia, and the demand for ginseng production for health functional food is increasing worldwide after the COVID-19 crisis. Although a number of cultivars have been developed to increase ginseng production, none of them were widely cultivated in Korea because they could not resist various environmental stresses while being grown in one place for at least 4 years. To address this, Sunhong was developed as a ginseng cultivar with high yield and multiple stress tolerance by pure line selection. Sunhong showed high yield and heat tolerance comparable to Yunpoong, a representative high-yielding cultivar, and exhibited 1.4 times lower prevalence of rusty roots than Yunpoong, suggesting that Sunhong can keep its high yield and quality during long-term cultivation. In addition, distinct color and lodging resistance were expected to increase the convenience of cultivation. To supply pure seeds to farmers, we also established a reliable high-throughput authentication system for Sunhong and seven ginseng cultivars through genotyping-by-sequencing (GBS) analysis. The GBS approach enabled to identify a sufficient number of informative SNPs in ginseng, a heterozygous and polyploid species. These results contribute to the improvement of yield, quality, and homogeneity, and therefore promote the ginseng industry. Supplementary Information: The online version contains supplementary material available at 10.1007/s13580-023-00526-x.

Neck Symptoms and Associated Clinical Outcomes in Patients Following Concussion.

OBJECTIVE: To examine the frequency and association of neck pain symptoms in patients with a concussion. STUDY SETTING AND PARTICIPANTS: Three-hundred and thirty-one consecutively enrolled patients aged 9 to 68 years with a diagnosed concussion 1 to 384 days post-injury were enrolled at a concussion clinic from a single integrated healthcare system in Western Pennsylvania between 2019 and 2021. DESIGN: Retrospective cohort analysis of prospectively collected concussion screening tool intake survey responses and clinical outcomes data. The primary outcome was self-reported neck pain or difficulty with neck movement on the Concussion Clinical Profiles Screening (CP Screen) tool, recovery time, and incidence of treatment referral. Immediate Post-concussion Assessment and Cognitive Testing (ImPACT) composite scores, Vestibular/Ocular Motor Screening (VOMS) item scores, type and severity of neck symptoms, mechanism of injury, time from injury to clinic presentation, medical history, and concussion symptom profile were secondary outcomes. RESULTS: Of the 306 consecutively enrolled eligible patients in the registry, 145 (47%) reported neck pain, 68 (22.2%) reported difficulty moving their neck, and 146 (47.7%) reported either symptom. A total of 47 (15.4%) participants reported more severe neck symptoms, and this group took longer to recover (40 ± 27 days) than those not reporting neck symptoms (30 ± 28 days; U = 8316, P < .001). Stepwise logistic regression predicting more severe neck symptoms was significant (Nagelkerke R2 = 0.174, χ 2 = 9.315, P = .316) with older age ( P = .019) and mechanism of injury including motor vehicle collisions (MVCs) ( P = .047) and falls ( P = .044) as risk factors. MVCs and falls were associated with over 4 times and 2 times greater risk, respectively, for reporting more severe neck symptoms. CONCLUSION: Neck pain and stiffness symptoms are common in patients with a concussion following high-energy mechanisms of injury including MVCs or falls from height. These symptoms are associated with prolonged recovery. Providers should evaluate neck symptoms and consider targeted treatment strategies to limit their effects in patients with a concussion.

Neuroprotective effects of bornyl acetate on experimental autoimmune encephalomyelitis via anti-inflammatory effects and maintaining blood-brain-barrier integrity.

BACKGROUND: Bornyl acetate (BA), a chemical component of essential oil in the Pinus family, has yet to be actively studies in terms of its therapeutic effect on numerous diseases, including autoimmune diseases. PURPOSE: This study aimed to investigate the pharmacological effects and molecular mechanisms of BA on myelin oligodendrocyte glycoprotein (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE) mice in an animal model of multiple sclerosis (MS), a representative autoimmune disease in central nervous system. METHODS: BA (100, 200, or 400 mg/kg) was orally treated to EAE mice once daily for 30 days after immunization for the behavioral test and for the 16th-18th days for the histopathological and molecular analyses, from the onset stage (8th day) of EAE symptoms. RESULTS: BA mitigated behavioral dysfunction (motor disability) and demyelination in the spinal cord that were associated with the down-regulation of representative pro-inflammatory cytokines (interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha), enzymes (cyclooxygenase-2 and inducible nitric oxide synthase), and chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein-1 alpha, and regulated on activation), and decreased infiltration of microglia (CD11b+/CD45+(low)) and macrophages (CD11b+/CD45+(high)). The anti-inflammatory effect of BA was related to the inhibition of mitogen-activated protein kinases and nuclear factor-kappa B pathways. BA also reduced the recruitment/infiltration rates of CD4+ T, Th1, and Th17 cells into the spinal cords of EAE mice, which was related to reduced blood-spinal cord barrier (BSCB) disruption. CONCLUSION: These findings strongly suggest that BA may alleviate EAE due to its anti-inflammatory and BSCB protective activities. This indicates that BA is a potential therapeutic agent for treating autoimmune demyelinating diseases including MS.

Suffruticosol B Is an Osteogenic Inducer through Osteoblast Differentiation, Autophagy, Adhesion, and Migration.

Suffruticosol B (Suf-B) is a stilbene found in Paeonia suffruticosa ANDR., which has been traditionally used in medicine. Stilbenes and their derivatives possess various pharmacological effects, such as anticancer, anti-inflammatory, and anti-osteoporotic activities. This study aimed to explore the bone-forming activities and mechanisms of Suf-B in pre-osteoblasts. Herein, >99.9% pure Suf-B was isolated from P. suffruticosa methanolic extracts. High concentrations of Suf-B were cytotoxic, whereas low concentrations did not affect cytotoxicity in pre-osteoblasts. Under zero levels of cytotoxicity, Suf-B exhibited bone-forming abilities by enhancing alkaline phosphatase enzyme activities, bone matrix calcification, and expression levels with non-collagenous proteins. Suf-B induces intracellular signal transduction, leading to nuclear RUNX2 expression. Suf-B-stimulated differentiation showed increases in autophagy proteins and autophagosomes, as well as enhancement of osteoblast adhesion and transmigration on the ECM. These results indicate that Suf-B has osteogenic qualities related to differentiation, autophagy, adhesion, and migration. This also suggests that Suf-B could have a therapeutic effect as a phytomedicine in skeletal disorders.

Phytotherapeutic applications of alkaloids in treating breast cancer.

Breast cancer is one of the major causes of mortality in women worldwide. The current treatments available are radiation therapy (RT), surgery, endocrine (hormone) therapy (ET), chemotherapy (CT), and targeted therapy. These treatments are associated with certain side effects that demand the use of natural compounds due to their lower to negligible side effects. One such category of natural compounds is alkaloids. Alkaloids are a group of natural compounds that have gained widespread attention due to their use as potential therapeutics. Alkaloids exert anti-inflammatory and antiviral properties along with antimicrobial activities. In the current review, 12 alkaloids are reviewed in detail for their potential in treating breast cancer. These alkaloids have been shown to induce apoptosis, decrease tumor volume, inhibit cell proliferation and migration, and induce autophagy and they can also be used as a component of combination therapy. This review provides comprehensive information on the in vitro and in vivo therapeutic abilities of alkaloids to counteract breast cancer.

Association of hypercalciuria with vitamin D supplementation in patients undergoing ketogenic dietary therapy.

Background: Ketogenic dietary therapy (KDT) is used as an effective treatment for epilepsy. However, KDT carries the risk of bone health deterioration; therefore, vitamin D supplementation is required. Vitamin D replacement therapy in KDT has not been established because it may be related to hypercalciuria/urolithiasis, which are common adverse effects of KDT. Hence, this study aimed to evaluate the dose-dependent association between vitamin D3 and hypercalciuria/urolithiasis in patients undergoing KDT and dose optimization for renal complications. Materials and methods: Overall, 140 patients with intractable childhood epilepsy started 3:1 KDT (lipid to non-lipid ratio) at the Severance Children's Hospital from January 2016 to December 2019. Regular visits were recommended after KDT initiation. Participants were assessed for height, weight, serum 25-hydroxyvitamin D (25-OH-D3) level, parathyroid hormone level, and ratio of urinary excretion of calcium and creatinine (Uca/Ucr). Kidney sonography was conducted annually. Patients who already had urolithiasis and were taking hydrochlorothiazide before KDT, failed to maintain KDT for 3 months, did not visit the pediatric endocrine department regularly, did not take prescribed calcium and vitamin D3 properly, or needed hospitalization for > 1°month because of serious medical illness were excluded. Data from patients who started diuretic agents, e.g., hydrochlorothiazide, were excluded from that point because the excretion of calcium in the urine may be altered in these patients. Result: In total, 49 patients were included in this study. Uca/Ucr ratio significantly decreased with increasing levels of 25-OH-D3 (p = 0.027). The odds ratio for hypercalciuria was 0.945 (95% confidence interval, 0.912-0.979; p = 0.002) per 1.0 ng/mL increment in 25-OH-D3 level. Based on findings of receiver operating characteristic curve analysis and Youden's J statistic, the cut-off 25-OH-D3 level for preventing hypercalciuria was > 39.1 ng/mL at 6 months. Furthermore, the vitamin D3 supplementation dose cut-off was > 49.5 IU/kg for hypercalciuria prevention. Conclusion: An inverse relationship between Uca/Ucr ratio and 25-OH-D3 level was noted, which means that vitamin D supplementation is helpful for preventing hypercalciuria related to KDT. We suggest that the recommended 25-OH-D3 level is > 40 ng/mL for hypercalciuria prevention and that KDT for children with epilepsy can be optimized by vitamin D3 supplementation at 50 IU/kg.

Effects of Scoparone on differentiation, adhesion, migration, autophagy and mineralization through the osteogenic signalling pathways.

Scoparone (SCOP), an active and efficient coumarin compound derived from Artemisia capillaris Thunb, has been used as a traditional Chinese herbal medicine. Herein, we investigated the effects of SCOP on the osteogenic processes using MC3T3-E1 pre-osteoblasts in in vitro cell systems. SCOP (C11 H10 O4 , > 99.17%) was purified and identified from A. capillaries. SCOP (0.1 to 100 µM concentrations) did not have cytotoxic effects in pre-osteoblasts; however, it promoted alkaline phosphatase (ALP) staining and activity, and mineralized nodule formation under early and late osteogenic induction. SCOP elevated osteogenic signals through the bone morphogenetic protein 2 (BMP2)-Smad1/5/8 pathway, leading to the increased expression of runt-related transcription factor 2 (RUNX2) with its target protein, matrix metallopeptidase 13 (MMP13). SCOP also induced the non-canonical BMP2-MAPKs pathway, but not the Wnt3a-ß-catenin pathway. Moreover, SCOP promoted autophagy, migration and adhesion under the osteogenic induction. Overall, the findings of this study demonstrated that SCOP has osteogenic effects associated with cell differentiation, adhesion, migration, autophagy and mineralization.

A Medicinal Halophyte Ipomoea pes-caprae (Linn.) R. Br.: A Review of Its Botany, Traditional Uses, Phytochemistry, and Bioactivity.

Ipomoea pes-caprae (Linn.) R. Br. (Convolvulaceae) is a halophytic plant that favorably grows in tropical and subtropical countries in Asia, America, Africa, and Australia. Even though this plant is considered a pan-tropical plant, I. pes-caprae has been found to occur in inland habitats and coasts of wider areas, such as Spain, Anguilla, South Africa, and Marshall Island, either through a purposeful introduction, accidentally by dispersal, or by spreading due to climate change. The plant parts are used in traditional medicine for treating a wide range of diseases, such as inflammation, gastrointestinal disorders, pain, and hypertension. Previous phytochemical analyses of the plant have revealed pharmacologically active components, such as alkaloids, glycosides, steroids, terpenoids, and flavonoids. These phytoconstituents are responsible for the wide range of biological activities possessed by I. pes-caprae plant parts and extracts. This review arranges the previous reports on the botany, distribution, traditional uses, chemical constituents, and biological activities of I. pes-caprae to facilitate further studies that would lead to the discovery of novel bioactive natural products from this halophyte.

Development of in vitro three-dimensional drug screening system for obesity-related metabolic syndrome.

Metabolic syndrome is increasingly common, and closely related with overweight or obesity. In the obese state, macrophages infiltrate to the adipose tissue (AT), resulting in chronic inflammation and insulin resistance in the AT cells. Recently, attention has been paid to the role of AT macrophages in metabolic disorders should be applied to the initial drug screening step, but it was difficult to mimic the inflammatory adipocytes using the traditional 2-dimensional (2D) culture. In this study, we developed the 3-dimensional (3D) culture system to overcome this limitation. After adipogenic differentiation, lipid droplets were highly accumulated in cells, and differentiation of preadipocytes was not declined by macrophage co-culture. However, only co-cultured cells expressed the insulin resistance features. Compare to mono-cultured adipocytes, co-cultured adipocytes showed reduced glucose uptake and GLUT4 did not translocated to cell membrane even though treatment of high concentration of insulin. Using 3D co-culture model, we develop a microwell-scale drug screening protocol to test anti-obesity effect. 3D cultured cells reacted more sensitive to drugs, and PPARγ antagonist GW9662 (10, 20 µM) repressed adipogenic differentiation in a concentration-dependent manner in 3D co-cultured cells.

Efficacy of the herbal pair, Radix Achyranthis Bidentatae and Eucommiae Cortex, in preventing glucocorticoid-induced osteoporosis in the zebrafish model.

OBJECTIVE: In traditional Chinese medicine, the herbal pair, Radix Achyranthis Bidentatae (RAB) and Eucommiae Cortex (EC), is widely used to treat osteoporosis. Herein, we determined whether this herbal pair can be used to ameliorate glucocorticoid (GC)-induced osteoporosis (GIOP) and find its optimal dosage in zebrafish. METHODS: The characteristics of the aqueous extract of RAB and EC were separately characterized using high-performance liquid chromatography. Osteoporosis was induced in 5-day post-fertilization zebrafish larvae by exposing them to 10 µmol/L dexamethasone (Dex) for 96 h. Seven combinations of different ratios of RAB and EC were co-administered. Treatment efficacy was determined by calculating zebrafish vertebral area and sum brightness, via alizarin red staining, and by detecting alkaline phosphatase (ALP) activity. Multiple regression analysis was conducted to test the optimal dosage ratio. RESULTS: According to the Chinese Pharmacopoeia (2015), ß-ecdysone (ß-Ecd) is a major bioactive marker in RAB extract, while pinoresinol diglucoside (PDG) is the major marker in EC extract. Both of ß-Ecd and PDG content values aligned with the Chinese Pharmacopoeia standards. Treatment with 10 µmol/L Dex reduced zebrafish vertebral area, sum brightness, and ALP activity, but RAB and EC attenuated these effects. Combining 50 µg/mL RAB and 50 µg/mL EC was optimal for preventing GIOP in zebrafish. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the mRNA expression of osteogenesis-related genes. A treatment of 10 µmol/L Dex decreased runt-related transcription factor 2 (Runx2), osteogenic protein-1 (OP-1), bone γ-carboxyglutamic acid-containing protein (BGLAP), and ß-catenin levels. This effect was counteracted by RAB and EC co-treatment (P < 0.05). Additionally, the effect of using the two herbal extracts together was better than single-herb treatments separately. These results demonstrated that RAB and EC preserve osteoblast function in the presence of GC. The best mass ratio was 1:1. CONCLUSION: RAB and EC herbal pair could ameliorate GC-induced effects in zebrafish, with 1:1 as the optimal dosage ratio.

Influence of Irradiation on Capsules of Silicone Implants Covered with Acellular Dermal Matrix in Mice.

BACKGROUND: In advanced breast cancer, radiotherapy is recommended as adjuvant therapy following breast reconstructive surgery. This inevitably led to growing concerns over possible complications of radiotherapy on implants. In this experimental animal study, we investigated the utility of acellular dermal matrix (ADM) wraps around implants as preventive management for radiotherapy complications. METHODS: Black mice (C57NL6; n = 32) were assigned to groups that either received radiation or did not: groups A and B underwent surgery using implants without radiotherapy; while groups C and D underwent surgery using implants with radiotherapy for one and three months, respectively. The hemispheric silicone implants with an 0.8-cm-diameter were inserted on the left back of each mouse, and implants wrapped by ADM were inserted on the right back. The Clinic 23EX LINAC model was used for irradiation at 10 Gy. The samples were evaluated by gross assessment, histological analysis, immunohistochemical analysis, and the Western blotting test. RESULTS: The H&E staining analysis showed that membrane thickness is smallest in group A, followed by groups C, D, and B. In a Masson trichrome histological analysis, collagen fibers became less dense and more widespread over time in the groups that received an ADM. Immunohistochemistry findings were similarly constant. However, the expression of TGF-ß1 was increased in the irradiated groups, whereas it was decreased in the non-irradiated groups as observed over time. CONCLUSIONS: Radiotherapy was shown to increase risk factors for capsular contracture, including inflammatory response, pseudoepithelium, thinning of membrane, and TGF-ß1 expression over time; however, the accompanying framework using an ADM as a barrier between implant and tissue was shown to be effective in alleviating these risks. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Antipathogenic Compounds That Are Effective at Very Low Concentrations and Have Both Antibiofilm and Antivirulence Effects against Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a human pathogen, causes both acute and chronic infections that are mediated by virulence factor production and biofilm formation. Since both characteristics of P. aeruginosa are regulated by quorum sensing (QS), we screened 126 synthetic chemicals for anti-QS activity and finally selected the compounds that have both antivirulence and antibiofilm activities. To efficiently screen the chemical library, the following reporter-based bioassay systems were used: the QS- or biofilm-specific promoter-lacZ fusions (lasIp- or PA1897p-lacZ for the QS activity and cdrAp-lacZ for measuring the intracellular c-di-GMP levels). We also measured the production of virulence factors and biofilm formation in P. aeruginosa. A small-animal infection model using mealworms was also used for virulence analysis. From this screening, MHY1383 and MHY1387 were found to have both antivirulence and antibiofilm activities in P. aeruginosa. Most importantly, MHY1383 and MHY1387 exhibited these activities at very low concentrations, showing a significant anti-QS effect at 100 pM and an antibiofilm effect at 1 to 10 pM. By treating P. aeruginosa with these compounds, the virulence factor production and biofilm formation of P. aeruginosa were significantly reduced. These compounds can be developed as promising antipathogenic and antibiofilm drugs that can be applied in situations where such compounds must be used in an extremely low concentration. Our findings also offer a significant advantage for developing therapeutic agents with few adverse side effects. IMPORTANCE Many antibiotics are increasingly losing their efficacy due to antibiotic resistance mediated by biofilm formation. In this study, we screened a synthetic chemical library and discovered several compounds that have both antivirulence and antibiofilm effects against Pseudomonas aeruginosa, a notorious human pathogen. Two of them had these effects at extremely low concentrations and are expected not to develop resistance, unlike conventional antibiotics, because they have no effect on the growth of bacteria. Our results strongly suggest that these compounds act on the target in a noncompetitive manner, indicating that they are distinct from other previously known quorum sensing inhibitors or biofilm inhibitors. Our findings offer a significant advantage for developing therapeutic agents with few adverse side effects.

Biological Mechanisms of Paeonoside in the Differentiation of Pre-Osteoblasts and the Formation of Mineralized Nodules.

Paeonia suffruticosa is a magnificent and long-lived woody plant that has traditionally been used to treat various diseases including inflammatory, neurological, cancer, and cardiovascular diseases. In the present study, we demonstrated the biological mechanisms of paeonoside (PASI) isolated from the dried roots of P. suffruticosa in pre-osteoblasts. Herein, we found that PASI has no cytotoxic effects on pre-osteoblasts. Migration assay showed that PASI promoted wound healing and transmigration in osteoblast differentiation. PASI increased early osteoblast differentiation and mineralized nodule formation. In addition, PASI enhanced the expression of Wnt3a and bone morphogenetic protein 2 (BMP2) and activated their downstream molecules, Smad1/5/8 and ß-catenin, leading to increases in runt-related transcription factor 2 (RUNX2) expression during osteoblast differentiation. Furthermore, PASI-mediated osteoblast differentiation was attenuated by inhibiting the BMP2 and Wnt3a pathways, which was accompanied by reduction in the expression of RUNX2 in the nucleus. Taken together, our findings provide evidence that PASI enhances osteoblast differentiation and mineralized nodules by regulating RUNX2 expression through the BMP2 and Wnt3a pathways, suggesting a potential role for PASI targeting osteoblasts to treat bone diseases including osteoporosis and periodontitis.

Phenological growth stages of Korean ginseng (Panax ginseng) according to the extended BBCH scale.

BACKGROUND: Phenological studies are a prerequisite for accomplishing higher productivity and better crop quality in cultivated plants. However, there are no phenological studies on Panax ginseng that improve its production yield. This study aims to redefine the phenological growth stages of P. ginseng based on the existing Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie (BBCH) scale and proposes a disease control reference. METHODS: This study was conducted at the Korea Ginseng Corporation Experiment Station in Gyeonggi province, South Korea. Phenological observations were performed once weekly or twice monthly, based on the developmental stages. The existing BBCH scale with a three-digit code was used to redefine and update P. ginseng's phenological growth codes. RESULTS: The phenological description is divided into eight principal growth stages: three for vegetative growth (perennating bud, aerial shoot, and root development), four for reproductive growth (reproductive organ development, flowering, fruit development, and fruit maturation), and one for senescence according to the extended BBCH scale. A total of 58 secondary growth stages were described within the eight principal growth stages. Under each secondary growth stage, four mesostages are also taken into account, which contains the distinct patterns of the phenological characteristics in ginseng varieties and the process of transplanting seedlings. A practical management program for disease control was also proposed by using the BBCH code and the phenological data proposed in this work. CONCLUSION: The study introduces an extended BBCH scale for the phenological research of P. ginseng.

Paeonolide as a Novel Regulator of Core-Binding Factor Subunit Alpha-1 in Bone-Forming Cells.

Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 µM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 µM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 µM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 µM) increased the bone morphogenetic protein (BMP)-Smad1/5/8 and Wnt-ß-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 µM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.