Chromosome-level genome assembly of Prunella vulgaris L. provides insights into pentacyclic triterpenoid biosynthesis.

Prunella vulgaris is one of the bestselling and widely used medicinal herbs. It is recorded as an ace medicine for cleansing and protecting the liver in Chinese Pharmacopoeia and has been used as the main constitutions of many herbal tea formulas in China for centuries. It is also a traditional folk medicine in Europe and other countries of Asia. Pentacyclic triterpenoids are a major class of bioactive compounds produced in P. vulgaris. However, their biosynthetic mechanism remains to be elucidated. Here, we report a chromosome-level reference genome of P. vulgaris using an approach combining Illumina, ONT, and Hi-C technologies. It is 671.95 Mb in size with a scaffold N50 of 49.10 Mb and a complete BUSCO of 98.45%. About 98.31% of the sequence was anchored into 14 pseudochromosomes. Comparative genome analysis revealed a recent WGD in P. vulgaris. Genome-wide analysis identified 35 932 protein-coding genes (PCGs), of which 59 encode enzymes involved in 2,3-oxidosqualene biosynthesis. In addition, 10 PvOSC, 358 PvCYP, and 177 PvUGT genes were identified, of which five PvOSCs, 25 PvCYPs, and 9 PvUGTs were predicted to be involved in the biosynthesis of pentacyclic triterpenoids. Biochemical activity assay of PvOSC2, PvOSC4, and PvOSC6 recombinant proteins showed that they were mixed amyrin synthase (MAS), lupeol synthase (LUS), and ß-amyrin synthase (BAS), respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of pentacyclic triterpenoids in P. vulgaris.

Chromosome-level genome assembly of Salvia miltiorrhiza with orange roots uncovers the role of Sm2OGD3 in catalyzing 15,16-dehydrogenation of tanshinones.

Salvia miltiorrhiza is well known for its clinical practice in treating heart and cardiovascular diseases. Its roots, used for traditional Chinese medicine materials, are usually brick-red due to accumulation of red pigments, such as tanshinone IIA and tanshinone I. Here we report a S. miltiorrhiza line (shh) with orange roots. Compared with the red roots of normal S. miltiorrhiza plants, the contents of tanshinones with a single bond at C-15,16 were increased, whereas those with a double bond at C-15,16 were significantly decreased in shh. We assembled a high-quality chromosome-level genome of shh. Phylogenomic analysis showed that the relationship between two S. miltiorrhiza lines with red roots was closer than the relationship with shh. It indicates that shh could not be the mutant of an extant S. miltiorrhiza line with red roots. Comparative genomic and transcriptomic analyses showed that a 1.0 kb DNA fragment was deleted in shh Sm2OGD3m. Complementation assay showed that overexpression of intact Sm2OGD3 in shh hairy roots recovered furan D-ring tanshinone accumulation. Consistently, in vitro protein assay showed that Sm2OGD3 catalyzed the conversion of cyptotanshinone, 15,16-dihydrotanshinone I and 1,2,15,16-tetrahydrotanshinone I into tanshinone IIA, tanshinone I and 1,2-dihydrotanshinone I, respectively. Thus, Sm2OGD3 functions as tanshinone 15,16-dehydrogenase and is a key enzyme in tanshinone biosynthesis. The results provide novel insights into the metabolic network of medicinally important tanshinone compounds.

Transcriptional Factor-Mediated Regulation of Active Component Biosynthesis in Medicinal Plants.

Plants produce thousands of chemically diverse secondary metabolites, many of which have valuable pharmaceutical properties. There is much interest in the synthesis of these pharmaceuticallyvaluable compounds, including the key enzymes and the transcription factors involved. The function and regulatory mechanism of transcription factors in biotic and abiotic stresses have been studied in depth. However, their regulatory roles in the biosynthesis of bioactive compounds, especially in medicinal plants, have only begun. Here, we review what is currently known about how transcription factors contribute to the synthesis of bioactive compounds (alkaloids, terpenoids, flavonoids, and phenolic acids) in medicinal plants. Recent progress has been made in the cloning and characterization of transcription factors in medicinal plants on the genome scale. So far, several large transcription factors have been identified in MYB, WRKY, bHLH, ZIP, AP2/ERF transcription factors. These transcription factors have been predicted to regulate bioactive compound production. These transcription factors positively or negatively regulate the expression of multiple genes encoding key enzymes, and thereby control the metabolic flow through the biosynthetic pathway. Although the research addressing this niche topic is in its infancy, significant progress has been made, and advances in high-throughput sequencing technology are expected to accelerate the discovery of key regulatory transcription factors in medicinal plants. This review is likely to be useful for those interested in the synthesis of pharmaceutically- valuable plant compounds, especially those aiming to breed or engineer plants that produce greater yields of these compounds.

Local Treatment of Osteoporotic Sheep Vertebral Body With Calcium Sulfate for Decreasing the Potential Fracture Risk: Microstructural and Biomechanical Evaluations.

STUDY DESIGN: In this study, calcium sulfate (CS) was injected through pedicle into the osteoporotic vertebral body in vivo in sheep, and micro-computed tomography analysis, histologic observation, and biomechanical test were performed. OBJECTIVE: To investigate the improvement on microstructure and biomechanical performance of lumbar vertebrae augmented with CS in osteoporotic sheep. SUMMARY OF BACKGROUND DATA: The present treatments for osteoporosis relies on systemic medications intended to increase the bone mineral density (BMD). Although effective, these time-consuming medications provide little protection from fracture in the "early period" after initiation of therapy. In this regard, the strategy of local treatment is to target specific areas of the skeletal system that are prone to osteoporotic fractures. However, there is little or no research focused on local treatment of osteoporotic vertebrae with CS. METHODS: Eight female sheep were induced to osteoporosis with bilateral ovariectomy and methylprednisolone administration for 12 months. After successful establishment of an osteoporotic model, lumbar vertebrae (L1-L4) in every sheep were randomly divided into 2 groups: CS group and control group (2 vertebrae in each group in every sheep). CS was injected into the vertebral body transpedicularly in the CS group and no treatments were performed in the control group. Three months later, all sheep were killed and all L1-L4 vertebrae were harvested. Thereafter, microstructure and biomechanical performance of the cancellous bone of the vertebral body were assessed through micro-computed tomography analysis, histologic observation, and biomechanical test, respectively. RESULTS: After a 12-month induction with ovariectomy and methylprednisolone administration, the mean BMD of the sheep lumbar vertebrae significantly decreased (>25%) compared with the value before induction, which demonstrated successful establishment of osteoporosis. Three months after injection of CS, CS was completely degraded without any remains in bone tissue and the quality of bone tissue (amount and density of the bone tissue) in the CS group was significantly higher than that in the control group. The ultimate load, stiffness, and energy absorption in the CS group were all significantly higher than those in the control group. CONCLUSIONS: The preliminary data suggest that local injection of CS can significantly improve the amount, density, and biomechanical performance of the bone trabeculae in osteoporotic vertebra. The local injection of CS could also be used as a new method to improve the physical microstructure and augment the mechanical properties in "high-risk" vertebral bodies, decreasing the potential fracture risk of patients with osteoporosis. The strict inclusion and exclusion criteria should be performed before treatment.

Increased exposure of vitamin A by Chrysanthemum morifolium Ramat extract in rat was not via induction of CYP1A1, CYP1A2, and CYP2B1.

The aim of this study was to investigate the effect of Chrysanthemum morifolium Ramat (CM) extract on the pharmacokinetics of retinol and activities of cytochrome P450s (CYP450s) related to retinoid metabolism. Rats were treated with CM extract for 15 d. Plasma concentrations of retinol were measured following oral administration of retinol (45 mg/kg). Basal levels of retinol and retinoic acid in serum and liver were also measured. 7-Ethoxyresorufin-O-deethylase activity, phenacetin-O-deethylase activity, and 7-pentoxyresorufin-O-deethylase activities were used to assay the activities of CYP1A1, CYP1A2, and CYP2B1 in hepatic microsomes of rats, respectively. Protein expressions of the 3 CYP450s were measured by western blot. Our studies demonstrated that CM extract dose-dependently increased basal level of retinol in serum. In pharmacokinetic experiment, CM extract dose-dependently increased plasma concentrations of retinol after oral administration of retinol to rats treated with CM extract. But activities and expressions of CYP1A1, CYP1A2, and CYP2B1 in hepatic microsomes of rats were also induced by CM extract.

An experimental model of lacunar infarction: embolization of microthrombi.

OBJECTIVES: Microthrombi are undoubtedly the most common embolic material in the cerebral circulation, originating from even minor irregularities of the arterial wall, fibrillating atria, cardiac valves, and patent foramen ovale. Thrombus fragments are globular and likely to completely obstruct terminal vessels. In contrast, previous work with "atheroemboli" of needle-like cholesterol crystals rarely cause occlusions or infarctions instead creating small foci of inflammation. In this work, we asked if microthrombi would occlude terminal vessels and create lacunar type infarctions in the subcortical tissues of the rat brain where, as in human brain, collateral flow is limited relative to the cortex. METHODS: Three treatment groups of adult male Sprague-Dawley rats were studied. All groups underwent general anesthesia with monitoring of temperature and blood pressure during cannulation of the right internal carotid artery. In the group embolized with thrombus fragments (n = 12), animals had injections of 300 fragments of thrombus size 60 to 100 microns, the cholesterol group (n = 6) had injections of 300 cholesterol crystals of similar size, and the control group (n = 4) had injections of saline. Brains were harvested at 4 days with perfusion fixation and were examined by immunohistochemical staining for breaks in the blood brain barrier (BBB) (albumin), microglial activation (CD11b), astrocyte activation (GFAP), and infarction (loss of NeuN staining). Size and location of the areas of injury and infarction were recorded. RESULTS: Clot fragments caused discreet infarcts in 10/12 animals that were 0.1-1.7 mm in diameter and coincided with activation of microglia and astrocytes. In some areas, necrosis was already underway at this early time point. Consistent with our previous work, the infarcts caused by cholesterol crystals were smaller (P = .014). Foci of BBB disruption and microglial activation were distributed throughout the brain whereas areas of infarction were found almost exclusively in subcortical tissues (P = .029). CONCLUSIONS: Injecting microthrombi reproducibly caused areas of necrosis resembling lacunar type infarctions. These were primarily located in the striatum and thalamus presumably because these areas lack the branching, collateral network seen in the cortex. In addition, these data give further evidence that the extent of brain injury from emboli depends upon composition and shape as well as size.