Hormonal Regulation and Stimulation Response of Jatropha curcas L. Homolog Overexpression on Tobacco Leaf Growth by Transcriptome Analysis.

The Flowering locus T (FT) gene encodes the florigen protein, which primarily regulates the flowering time in plants. Recent studies have shown that FT genes also significantly affect plant growth and development. The FT gene overexpression in plants promotes flowering and suppresses leaf and stem development. This study aimed to conduct a transcriptome analysis to investigate the multiple effects of Jatropha curcas L. homolog (JcFT) overexpression on leaf growth in tobacco plants. The findings revealed that JcFT overexpression affected various biological processes during leaf development, including plant hormone levels and signal transduction, lipid oxidation metabolism, terpenoid metabolism, and the jasmonic-acid-mediated signaling pathway. These results suggested that the effects of FT overexpression in plants were complex and multifaceted, and the combination of these factors might contribute to a reduction in the leaf size. This study comprehensively analyzed the effects of JcFT on leaf development at the transcriptome level and provided new insights into the function of FT and its homologous genes.

Integrated transcriptome and physiological analysis revealed core transcription factors that promote flavonoid biosynthesis in apricot in response to pathogenic fungal infection.

MAIN CONCLUSION: Integrated transcriptome and physiological analysis of apricot leaves after Fusarium solani treatment. In addition, we identified core transcription factors and flavonoid-related synthase genes which may function in apricot disease resistance. Apricot (Prunus armeniaca) is an important economic fruit species, whose yield and quality of fruit are limited owing to its susceptibility to diseases. However, the molecular mechanisms underlying the response of P. armeniaca to diseases is still unknown. In this study, we used physiology and transcriptome analysis to characterize responses of P. armeniaca subjected to Fusarium solani. The results showed increasing malondialdehyde (MDA) content, enhanced peroxidase (POD) and catalase (CAT) activity during F. solani infestation. A large number of differentially expressed genes (DEGs), which included 4281 upregulated DEGs and 3305 downregulated DEGs, were detected in P. armeniaca leaves exposed to F. solani infestation. Changes in expression of transcription factors (TFs), including bHLH, AP2/ERF, and WRKY indicated their role in triggering pathogen-responsive genes in P. armeniaca. During the P. armeniaca response to F. solani infestation, the content of total flavonoid was changed, and we identified enzyme genes associated with flavonoid biosynthesis. Ectopic overexpression of PabHLH15 and PabHLH102 in Nicotiana benthamiana conferred elevated resistance to Fspa_1. Moreover, PabHLH15 and PabHLH102 positively interact with the promoter of flavonoid biosynthesis-related genes. A regulatory network of TFs regulating enzyme genes related to flavonoid synthesis affecting apricot disease resistance was constructed. These results reveal the potential underlying mechanisms of the F. solani response of P. armeniaca, which would help improve the disease resistance of P. armeniaca and may cultivate high-quality disease-resistant varieties in the future.

Membrane Dynamics Regulated by Cytoskeleton in Plant Immunity.

The plasma membrane (PM), which is composed of a lipid layer implanted with proteins, has diverse functions in plant responses to environmental triggers. The heterogenous dynamics of lipids and proteins in the plasma membrane play important roles in regulating cellular activities with an intricate pathway that orchestrates reception, signal transduction and appropriate response in the plant immune system. In the process of the plasma membrane participating in defense responses, the cytoskeletal elements have important functions in a variety of ways, including regulation of protein and lipid dynamics as well as vesicle trafficking. In this review, we summarized how the plasma membrane contributed to plant immunity and focused on the dynamic process of cytoskeleton regulation of endocytosis and exocytosis and propose future research directions.

Genome-wide analysis of TPX2 gene family in Populus trichocarpa and its specific response genes under various abiotic stresses.

Microtubules are essential for regulating cell morphogenesis, plant growth, and the response of plants to abiotic stresses. TPX2 proteins are the main players determining the spatiotemporally dynamic nature of the MTs. However, how TPX2 members respond to abiotic stresses in poplar remains largely unknown. Herein, 19 TPX2 family members were identified from the poplar genome and analyzed the structural characteristics as well as gene expression patterns. All TPX2 members had the conserved structural characteristics, but exhibited different expression profiles in different tissues, indicating their varying roles during plant growth. Additionally, several light, hormone, and abiotic stress responsive cis-acting regulatory elements were detected on the promoters of PtTPX2 genes. Furthermore, expression analysis in various tissues of Populus trichocarpa showed that the PtTPX2 genes responded differently to heat, drought and salt stress. In summary, these results provide a comprehensive analysis for the TPX2 gene family in poplar and an effective contribution to revealing the mechanisms of PtTPX2 in the regulatory network of abiotic stress.

The Cytoskeleton in Plant Immunity: Dynamics, Regulation, and Function.

The plant cytoskeleton, consisting of actin filaments and microtubules, is a highly dynamic filamentous framework involved in plant growth, development, and stress responses. Recently, research has demonstrated that the plant cytoskeleton undergoes rapid remodeling upon sensing pathogen attacks, coordinating the formation of microdomain immune complexes, the dynamic and turnover of pattern-recognizing receptors (PRRs), the movement and aggregation of organelles, and the transportation of defense compounds, thus serving as an important platform for responding to pathogen infections. Meanwhile, pathogens produce effectors targeting the cytoskeleton to achieve pathogenicity. Recent findings have uncovered several cytoskeleton-associated proteins mediating cytoskeletal remodeling and defense signaling. Furthermore, the reorganization of the actin cytoskeleton is revealed to further feedback-regulate reactive oxygen species (ROS) production and trigger salicylic acid (SA) signaling, suggesting an extremely complex role of the cytoskeleton in plant immunity. Here, we describe recent advances in understanding the host cytoskeleton dynamics upon sensing pathogens and summarize the effectors that target the cytoskeleton. We highlight advances in the regulation of cytoskeletal remodeling associated with the defense response and assess the important function of the rearrangement of the cytoskeleton in the immune response. Finally, we propose suggestions for future research in this area.

[Historical evolution of Salviae Miltiorrhizae Radix et Rhizoma processing methods].

China has a long history of Salviae Miltiorrhizae Radix et Rhizoma processing with multiple methods available. The pre-sent study collated and summarized the Salviae Miltiorrhizae Radix et Rhizoma processing methods recorded in 23 related herbal medicine books, all editions of Chinese Pharmacopoeia, the 1988 edition of National Regulations for Processing of Chinese Medicine, and 20 current local processing specifications and standards. The results demonstrated various processing methods of Salviae Miltiorrhizae Radix et Rhizoma, such as removing residual part of stem, plantlet, or soil, smashing, filing, cutting, decocting, washing with wine, soaking in wine, and stir-frying with wine or blood from pig heart, while raw and wine-processed products are mainly used in modern times. Due to the lack of unified standards, the phenomena of multiple methods adopted in one place and different methods in different places have led to uneven quality of Salviae Miltiorrhizae Radix et Rhizoma pieces, even affecting the safety and effectiveness of its clinical medication. This study is expected to provide a reference for the development of Salviae Miltiorrhizae Radix et Rhizoma processing and its rational medication.

Plant multiscale networks: charting plant connectivity by multi-level analysis and imaging techniques.

In multicellular and even single-celled organisms, individual components are interconnected at multiscale levels to produce enormously complex biological networks that help these systems maintain homeostasis for development and environmental adaptation. Systems biology studies initially adopted network analysis to explore how relationships between individual components give rise to complex biological processes. Network analysis has been applied to dissect the complex connectivity of mammalian brains across different scales in time and space in The Human Brain Project. In plant science, network analysis has similarly been applied to study the connectivity of plant components at the molecular, subcellular, cellular, organic, and organism levels. Analysis of these multiscale networks contributes to our understanding of how genotype determines phenotype. In this review, we summarized the theoretical framework of plant multiscale networks and introduced studies investigating plant networks by various experimental and computational modalities. We next discussed the currently available analytic methodologies and multi-level imaging techniques used to map multiscale networks in plants. Finally, we highlighted some of the technical challenges and key questions remaining to be addressed in this emerging field.

Regulation of cytoskeleton-associated protein activities: Linking cellular signals to plant cytoskeletal function.

The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in plant cells, including trafficking and exocytosis of membrane and wall components during cell expansion, and regulation of hypocotyl elongation in response to light. Cytoskeletal remodeling also has key functions in disease resistance and abiotic stress responses. Many stimuli result in altered activity of cytoskeleton-associated proteins, microtubule-associated proteins (MAPs) and actin-binding proteins (ABPs). MAPs and ABPs are the main players determining the spatiotemporally dynamic nature of the cytoskeleton, functioning in a sensory hub that decodes signals to modulate plant cytoskeletal behavior. Moreover, MAP and ABP activities and levels are precisely regulated during development and environmental responses, but our understanding of this process remains limited. In this review, we summarize the evidence linking multiple signaling pathways, MAP and ABP activities and levels, and cytoskeletal rearrangements in plant cells. We highlight advances in elucidating the multiple mechanisms that regulate MAP and ABP activities and levels, including calcium and calmodulin signaling, ROP GTPase activity, phospholipid signaling, and post-translational modifications.

Finding a resveratrol analogue as potential anticancer agent with apoptosis and cycle arrest.

Resveratrol has been extensively studied as the anti-cancer agent. A variety of resveratrol analogues have been developed with structural modification to improve its bioactivity. In this work, resveratrol analogues, compound 1-4, were designed and synthesized with the Stille-Heck reaction. These results showed compound 1-4 had better anticancer effect than that of parent resveratrol. Especially compound 1 ((E)-4,4'-(ethene-1,2-diyl)bis(3-methylphenol)) displayed the excellent cytotoxicity and high selectivity. The mechanism research indicated compound 1 inhibited cell proliferation by binary paths of cell cycle arrest in S phase regulated by cyclin A1/A2 and apoptosis induction mediated by Bax/Bcl2 in a prooxidant manner.

Finding an efficient tetramethylated hydroxydiethylene of resveratrol analogue for potential anticancer agent.

With the improvement and advance in cancer diagnosis and treatment, the cancer is still a major cause of morbidity and mortality throughout the world. Obviously, new breakthroughs in therapies remain be urgent needed. In this work, we designed and synthesized the compound 1-4, namely resveratrol analogues with methylation of hydroxy distyrene, to further explore its new anti-cancer potential. Encouragingly, compound 1 ((E)-4,4'-(ethene-1,2-diyl)bis(3,5-dimethylphenol)) exhibited cytotoxicity superior to resveratrol in MCF 7 cells. More importantly, the compound 1 showed greater toxicity to tumor cells than that to normal cells, which proved that it could selectively kill tumor cells. The favorable results encouraged us to explore the inhibitory mechanism of compound 1 on MCF 7 cells. The research finding indicated the compound 1 inhibited tumor cell proliferation by both arresting cell cycle in S phase and apoptosis via a prooxidant manner. In addition, the results further verified compound 1 caused cell cycle arrest in S phase and apoptosis by down-regulation of the cycling A1/cycling A2 expression and the rise of Bax/Bcl-2 ratio in a p21-dependant pathway in MCF 7 cells. Therefore, these results are helpful for the effective design of anticancer reagents and the better understanding of their mechanism of action.

Atorvastatin suppresses vascular hypersensitivity and remodeling induced by transient adventitial administration of lipopolysaccharide in rats.

BACKGROUND: The phenotypic transition of vascular smooth muscle cells (VSMCs) from a contractile to a proliferative state markedly affects the pathophysiology of cardiovascular diseases. The adventitial inflammation can promote neointimal formation and vascular remodeling. We used direct administration of lipopolysaccharide (LPS) into the periphery of the carotid artery to investigate the influence of transient adventitial inflammation on vascular remodeling and its potential mechanism. METHODS: Male 15-week-old Wistar rats were randomly assigned to four groups with six rats in each group. The rats of groups I and II were administered distilled water, and group III and IV were treated with fasudil and atorvastatin respectively. All treatments were given daily for 11 days. On day 8, the adventitia in group I was injected with 5 µL sterile saline, and the group II-IV were injected with 5 µL sterilized LPS. The carotid blood flow and femoral blood pressure were measured in vivo, and the thickness of vascular intima and middle layer was measured in vitro. Serum interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were determined using enzyme-linked immunosorbent assay (ELISA) assay. And the Rho-associated coiled-coil-containing protein kinase 2 (ROCK2), myosin phosphatase target subunit 1 (MYPT1), myosin light chain (MLC), myocardin, SM-α actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected by western blot. The comparisons were made by one-way analysis of variance with Bonferroni's post hoc test. A value of P<0.05 was considered to represent a statistically significant difference. RESULTS: Transient adventitial inflammation induced by LPS caused no obvious change in basal blood flow, but did lead to vascular hypersensitivity to serotonin. Morphological examinations revealed that the medial layer was the only domain affected, and showed VSMC proliferation and rearrangement. LPS increased serum IL-6 and TNFα contents, ROCK2 expression and activity, and caused changes in the expression levels of some stereotypical VSMC genes. Similar to the Rho-kinase inhibitor fasudil, atorvastatin completely restored the morphological alterations, even increased blood flow. CONCLUSIONS: Our study confirms the beneficial effect of atorvastatin on the vascular system in terms of morphology and function.

Hop2 Interacts with ATF4 to Promote Osteoblast Differentiation.

Activating transcription factor 4 (ATF4) is a member of the basic leucine zipper (bZip) transcription factor family required for the terminal differentiation of osteoblasts. Despite its critical importance as one of the three main osteoblast differentiation transcription factors, regulators of osteoblast terminal maturation remain poorly defined. Here we report the identification of homologous pairing protein 2 (Hop2) as a dimerization partner of ATF4 in osteoblasts via the yeast two-hybrid system. Deletional mapping revealed that the Zip domain of Hop2 is necessary and sufficient to bind ATF4 and to enhance ATF4-dependent transcription. Ectopic Hop2 expression in preosteoblasts increased endogenous ATF4 protein content and accelerated osteoblast differentiation. Mice lacking Hop2 (Hop2-/- ) have a normal stature but exhibit an osteopenic phenotype similar to the one observed in Atf4-/- mice, albeit milder, which is associated with decreased Osteocalcin mRNA expression and reduced type I collagen synthesis. Compound heterozygous mice (Atf4+/- :Hop2+/- ) display identical skeletal defects to those found in Hop2-/- mice. These results indicate that Hop2 plays a previous unknown role as a determinant of osteoblast maturation via its regulation of ATF4 transcriptional activity. Our work for the first time reveals a function of Hop2 beyond its role in guiding the alignment of homologous chromosomes. © 2019 American Society for Bone and Mineral Research.

COP1 mediates dark-specific degradation of microtubule-associated protein WDL3 in regulating Arabidopsis hypocotyl elongation.

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a well-known E3 ubiquitin ligase, functions as a central regulator of plant growth and photomorphogenic development in plants, including hypocotyl elongation. It has been well-established that, in darkness, COP1 targets many photomorphogenesis-promoting factors for ubiquitination and degradation in the nucleus. However, increasing evidence has shown that a proportion of COP1 is also localized outside the nucleus in dark-grown seedlings, but the physiological function of this localization remains largely unclear. In this study, we demonstrate that COP1 directly targets and mediates the degradation of WAVE-DAMPENED 2-LIKE 3 (WDL3) protein, a member of the microtubule-associated protein (MAP) WVD2/WDL family involved in regulating hypocotyl cell elongation of Arabidopsis seedlings. We show that COP1 interacts with WDL3 in vivo in a dark-dependent manner at cortical microtubules. Moreover, our data indicate that COP1 directly ubiquitinates WDL3 in vitro and that WDL3 protein is degraded in WT seedlings but is abundant in the cop1 mutant in the dark. Consistently, introduction of the wdl3 mutation weakened, whereas overexpression of WDL3 enhanced, the short-hypocotyl phenotype of cop1 mutant in darkness. Together, this study reveals a function of COP1 in regulating the protein turnover of a cytosol-localized MAP in etiolated hypocotyls, thus providing insights into COP1-mediated degradation of downstream factors to control seedling photomorphogenesis.

Constituents from the Roots of Actinidia chinensis and Their Cytochrome P450 Enzyme Inhibitory Activities.

A newly discovered triterpenoid, (2α,3ß)-2,3,23-trihydroxyurs-13(18)-en-28-oic acid (1), along with twelve known compounds (2 - 13), were isolated from the roots of Actinidia chinensis Planch (Actinidiaceae). Their chemical structures were determined by 1D- and 2D-NMR spectra and mass spectrometry (MS). The crude extracts and six main constituents (8 - 13) were tested for cytochrome P450 (CYPs) enzyme inhibitory activity. The results showed that, except for compound 8, compounds 9 - 13 had different inhibitory effects on the cytochrome P450 (CYPs) enzyme, and compound 9 significantly inhibited the catalytic activities of CYP3A4 to < 10% of its control activities.

Effect of long-term weightlessness on retina and optic nerve in tail-suspension rats.

AIM: To evaluate the effect of long-term weightlessness on retina and optic nerve in tail-suspension (TS) rats. METHODS: A stimulated weightlessness model was established by suspending rats' tail. After 12wk, the ultrastructure and the number of optic nerve axons were observed by transmission electron microscope. The number of survival retinal ganglion cells (RGCs) was calculated by fluorescent gold retrograde labeling. Retina cells apoptosis was detected by TUNEL staining. The function of optic nerve and retina was evaluated by the visual evoked potential (VEP) and oscillatory potentials (Ops). RESULTS: The optic nerve axons were swollen and sparsely aligned, and the lamellar separation and myelin disintegration occurred after 12wk in TS rats. The density of optic nerve axons was 32.23±3.92 (vs 37.43±4.13, P=0.0145), the RGCs density was 1645±46 cells/mm(2) (vs 1867±54 cells/mm(2) P=0.0000), the incidence rate of retinal cells apoptosis was 5.38%±0.53% (vs 4.75%±0.54%, P=0.0238), the amplitude of VEP-P100 was 15.43±2.14 µV (vs 17.67±2.17 µV, P=0.0424), the latency of VEP-P100 was 69.05±5.34ms (vs 62.43±4.87ms P=0.0143) and the sum amplitude of Ops was 81.05±8.34 µV (vs 91.67±10.21 µV, P=0.0280) in TS group and the control group, respectively. CONCLUSION: Long-term weightlessness can induce the ultrastructural changes and functional depress of the optic nerve, as well as retinal cell damages in TS rats.

As2 O3 combined with leflunomide prolongs heart xenograft survival via suppressing the response of Th1, Th2, and B cells in a rat model.

Xenotransplantation remits the severe shortage of human organs and tissues for transplantation, which is a problem that severely limits the application of transplantation to the treatment of human disease. However, severe immune rejection significantly limits the efficacy of xenotransplantation. In this study, we systematically investigated the immunosuppressive effect and mechanism of action of As2 O3 and leflunomide using a hamster-to-rat heart xenotransplantation model. We initially examined heart xenograft survival following As2 O3 and leflunomide treatment alone or combined treatment. We found that treatment with As2 O3 combined with leflunomide can significantly prolong the survival of heart xenograft by inhibiting Th1 and Th2 differentiation and reducing the production of IgG and IgM. Interestingly, As2 O3 and leflunomide showed low toxicity to the organs of the recipient. Taken together, these observations indicate that treatment with As2 O3 combined with leflunomide may be a promising immunosuppressive schedule for xenotransplantation.

Anti-inflammatory constituents from the root of Litsea cubeba in LPS-induced RAW 264.7 macrophages.

Context Litsea cubeba (Lour.) Pers. (Lauraceae) has long been used as a folk remedy in Traditional Chinese Medicine (TCM) for the treatment of rheumatic diseases. Previous studies from our laboratory indicated that L. cubeba extract showed anti-arthritic activity in rats. Objective To study L. cubeba chemically and biologically and to find the potential constituents responsible for its anti-arthritic effect. Materials and methods The compounds were isolated from the root of L. cubeba by column chromatography which eluted with PE:EtOAc gradient system, and the structures were elucidated by detailed spectroscopic data analysis; the anti-inflammatory activity of the isolated compounds was evaluated by lipopolysaccharide (LPS)-induced RAW 264.7 cells and the TNF-α and NO level were measured by ELISA (commercial kit); The iNOS and COX-2 mRNA expression were measured by RT-PCR and the phosphorylation of IκBα, IKKß, P38 and Akt were determined by western blots. Results A novel 9-fluorenone, 1-ethoxy-3,7-dihydroxy-4,6-dimethoxy-9-fluorenone (1), together with 4 known compounds, namely pinoresinol (2), syringaresinol (3), 9,9'-O-di-(E)-feruloyl-meso-5,5'-dimethoxysecoisolariciresinol (4) and lyoniresinol (5) were isolated from the root of L. cubeba for the first time. The IC50 for NO inhibition on compounds 1 and 4 were 56.1 ± 1.2 and 32.8 ± 2.3 µM, respectively. The IC50 for TNF-α inhibition were 28.2 ± 0.9 and 15.0 ± 1.0 µM, respectively. Both 1 and 4 suppress mRNA expression of iNOS, COX-2 and protein phosphorylation of IκBα, IKKß in LPS-induced RAW 264.7 cells. Discussion and conclusion Compounds 1 and 4 isolated from L. cubeba exhibited potent anti-inflammatory activity through the NF-κB signal pathway.

Effects of Foxp3 gene modified dendritic cells on mouse corneal allograft rejection.

OBJECTIVE: To investigate the effect of Foxp3 gene modified dendritic cells (Foxp3 + DC) on allogeneic T cells proliferation and to study the effect of Foxp3 + DC on corneal allograft rejection. METHODS: Lentivirus-Foxp3 was transfected into DC2.4 cells, as Foxp3 + DC cells. 42 BALB/c mice were randomly divided into: Group A (n = 6), normal group; Group B (n = 12), Group C (n = 12) and Group D (n = 12), allograft groups, were treated with normal saline, DC2.4, Foxp3 + DC by intraperitoneal injection, respectively. RESULTS: Compared with the control group, Foxp3 protein in the Foxp3 + DC cells increased significantly (P < 0.05); the expressions of CD80 and CD86 immunophenotypes of Foxp3 + DC cells decreased significantly (P < 0.05); IL-12 secretion reduced (P < 0.05), but IL-10 secretion was promoted (P < 0.05). The average transplant survival time in Group B was (14.833 ± 1.472) d, and Group C and Group D led to a statistically significant prolongation of transplant survival to (17.667 ± 1.366, 23.000 ± 2.000) d (P < 0.05) respectively. 14 d after transplantation, as compared with Group C and D, the expressions of IFN-γ in grafts markedly increased in Group B. 14 d after transplantation, as compared with Group B, the expressions of Foxp3 mRNA, IDO mRNA in grafts decreased remarkably in Group C and D (P < 0.05); as compared with Group C, the expressions of Foxp3 mRNA, IDO mRNA in grafts decreased remarkably in Group D (P < 0.05). CONCLUSION: Foxp3 + DC cells reduce the expression of costimulatory factors, reduce the secretion of IL-12, promote IL-10 production and inhibit the stimulation of alloreactive T cell proliferation response capacity. Foxp3 + DC cells play important roles in inhibiting corneal allograft immune response and prolonging graft survival time.

Correlation of in vivo and in vitro methods in measuring choroidal vascularization volumes using a subretinal injection induced choroidal neovascularization model.

BACKGROUND: In vivo quantification of choroidal neovascularization (CNV) based on noninvasive optical coherence tomography (OCT) examination and in vitro choroidal flatmount immunohistochemistry stained of CNV currently were used to evaluate the process and severity of age-related macular degeneration (AMD) both in human and animal studies. This study aimed to investigate the correlation between these two methods in murine CNV models induced by subretinal injection. METHODS: CNV was developed in 20 C57BL6/j mice by subretinal injection of adeno-associated viral delivery of a short hairpin RNA targeting sFLT-1 (AAV.shRNA.sFLT-1), as reported previously. After 4 weeks, CNV was imaged by OCT and fluorescence angiography. The scaling factors for each dimension, x, y, and z (µm/pixel) were recorded, and the corneal curvature standard was adjusted from human (7.7) to mice (1.4). The volume of each OCT image stack was calculated and then normalized by multiplying the number of voxels by the scaling factors for each dimension in Seg3D software (University of Utah Scientific Computing and Imaging Institute, available at http://www.sci.utah.edu/cibc-software/seg3d.html). Eighteen mice were prepared for choroidal flatmounts and stained by CD31. The CNV volumes were calculated using scanning laser confocal microscopy after immunohistochemistry staining. Two mice were stained by Hematoxylin and Eosin for observing the CNV morphology. RESULTS: The CNV volume calculated using OCT was, on average, 2.6 times larger than the volume calculated using the laser confocal microscopy. The correlation statistical analysis showed OCT measuring of CNV correlated significantly with the in vitro method (R 2 =0.448, P = 0.001, n = 18). The correlation coefficient for CNV quantification using OCT and confocal microscopy was 0.693 (n = 18, P = 0.001). CONCLUSIONS: There is a fair linear correlation on CNV volumes between in vivo and in vitro methods in CNV models induced by subretinal injection. The result might provide a useful evaluation of CNV both for the studies using CNV models induced by subretinal injection and human AMD studies.

Fingerprint analysis of Zhimu-Huangbai herb pair and simultaneous determination of its alkaloids, xanthone glycosides and steroidal saponins by HPLC-DAD-ELSD.

AIM: To develop and validate a high performance liquid chromatography (HPLC) coupled with diode array and evaporative light scattering detectors (DAD-ELSD) method for the quantitative determination and fingerprint analysis of ten active constituents in three chemical classes (namely, xanthone glycosides, steroidal saponins, and alkaloids) in Zhimu-Huangbai herb pair (ZB). METHOD: Chromatographic separation was performed on a Diamonsil C18 column (4.6 mm × 250 mm, 5 µm, Dikma) by gradient elution using acetic acid in acetonitrile solution at a flow rate of 1.0 mL·min(-1) at 260 nm. The drift tube temperature of ELSD was set to 60 °C and nebulizer gas pressure was 4.0 Bar. Method validation was performed to assure its linearity, limits of detection and quantification, precision, repeatability, stability, and accuracy. RESULTS: The HPLC-DAD-ELSD method allowed the quantification of ten compounds (phellodendrine, jatrorrhizine, palmatine, berberine, neomangiferin, mangiferin, timosaponin E-I, timosaponin B-II, timosaponin B, and timosaponin A-III), and was successfully applied to fingerprint analysis for ten batches of ZB samples. CONCLUSION: This was the first time to apply the combination of DAD and ELSD for the simultaneous determination of ten active ingredients in ZB. The results showed that the combination of quantitative analysis for marker ingredients and chemical fingerprint for the TCM herb pair provides a potentially powerful, widely introduced, and internationally accepted strategy for assessment of complex TCM formulas.