Defining the Activated Fibroblast Population in Lung Fibrosis Using Single-Cell Sequencing.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disorder driven by unrelenting extracellular matrix deposition. Fibroblasts are recognized as the central mediators of extracellular matrix production in IPF; however, the characteristics of the underlying fibroblast cell populations in IPF remain poorly understood. Here, we use an unbiased single-cell RNA sequencing analysis of a bleomycin-induced pulmonary fibrosis model to characterize molecular responses to fibrotic injury. Lung cells were isolated on Day 11 to capture emerging fibrosis and gene expression was analyzed by three complementary techniques, which, together, generated a 49-gene signature that defined an activated subpopulation of fibroblasts. However, none of the identified genes were specific to the activated cells or to the disease setting, implying that the activated fibroblasts are not uniquely defined, but exhibit a similar, yet amplified, gene expression pattern to control cells. Our findings have important implications for fibrosis research, including: 1) defining myofibroblasts with any single marker will fail to capture much of the underlying biology; 2) fibroblast activation is poorly correlated with expression of transforming growth factor-ß pathway genes; 3) single-cell analysis provides insight into the mechanism of action of effective therapies (nintedanib); 4) early events in lung fibrosis need not involve significant changes in fibroblast number; populations that do increase in number, such as macrophages, dendritic cells, and proliferating myeloid cells, may merit closer examination for their role in pathogenesis.

[Significance and Expressions of MMP-1, TIMP-1 and TGF-ß1 in Valve Tissue of Rheumatic Heart Disease].

OBJECTIVES: To explore expressions of matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinases-1 (TIMP-1) and transforming growth factor-ß1 (TGF-ß1) in valve tissue of rheumatic heart disease (RHD), and to analyzed their roles in RHD. METHODS: The expressions of MMP-1, TIMP-1 and TGF-ß1 proteins and mRNAs were tested by Western blot and RT-PCR methods in valve tissues in participants with (experimental group, n=30) and without RHD (control group, n=15). Collagen fibers were detected by Masson staining, and collagen volume fraction (CVF) was caculated. The correlations of CVF and the expressions of MMP-1, TIMP-1 and TGF-ß1 were analyzed. RESULTS: The collagen fibers, CVF, and the protein and mRNA expressions of MMP-1 and TGF-ß1 in experimental group were higher than those in control group, while the protein and mRNA expressions of TIMP-1 in experimental group were lower than those in control group. The expression of TIMP-1 was negatively correlated with TGF-ß1 and CVF in valve tissues, while MMP-1 was positively correlated with them. The expression of TGF-ß1 was positively correlated with CVF in valve tissues. CONCLUSIONS: MMP-1, TIMP-1 and TGF-ß1 contribute to the progression of fibrosis in RHD.

Analysis of apoptosis induced by perfluorooctane sulfonates (PFOS) in mouse Leydig cells in vitro.

To explore the possible mechanism of perfluorooctane sulfonates (PFOS's) reproductive toxicity, mouse Leydig cells cultured in vitro were exposed to a serial concentration of PFOS for four more days of culture. Apoptosis during the process was checked. After 24 h, apoptosis occurred to all of the groups ≥ 50 µg/mL PFOS. After 72 h, 37.5 µg/mL dose also showed apoptosis, and the most apoptosis signals, averagely 18 per well, were observed in 62.5 µg/mL dose group. An increase in ROS (p < 0.05) and a decrease of mitochondrial membrane potential (p < 0.01) was confirmed in those groups with ≥ 12.5 µg/mL dose. ROS levels peaked in 50 µg/mL and 62.5 µg/mL groups, nearly two-folds higher than control. PFOS was also observed to down-regulate the protein expression of Bcl-2 and to up-regulate that of Bax. The apoptosis induced by PFOS in mouse Leydig cells was shown to be related to mitochondrially mediated pathways and to involve oxidative stress.

Exposure to bisphenol-A affects fear memory and histone acetylation of the hippocampus in adult mice.

Bisphenol-A (BPA), an environmental endocrine disruptor, has been reported to possess weak estrogenic, anti-estrogenic, and anti-androgen properties. Previous evidence indicates that perinatal exposure to low levels of BPA affects anxiety-like and cognitive behaviors in adult rodents. The present study aims to investigate the effect of BPA on emotional memory using the contextual fear conditioning of male mice in adulthood exposed to BPA for 90days. The results indicated that exposure to BPA increased the freezing time 1h and 24h after fear conditioning training. Furthermore, western blot analyses showed that BPA exposure decreased the level of N-methyl-d-aspartic acid (NMDA) receptor subunit NR1 and increased the expression of histone deacetylase 2 (HDAC2) before fear conditioning training in the hippocampus of male mice. One and twenty-four hours after fear conditioning training, BPA enhanced the changes of the expressions of NR1, phosphorylated extracellular regulated protein kinases (ERK1/2), and histone acetylation induced by contextual fear conditioning in the hippocampus. These results suggest that long term exposure to BPA enhanced fear memory by the concomitant increased level of NMDA receptor and/or the enhanced histone acetylation in the hippocampus, which may be associated with activation of ERK1/2 signaling pathway.

Sex-specific effects of bisphenol-A on memory and synaptic structural modification in hippocampus of adult mice.

Humans are routinely exposed to low levels of bisphenol A (BPA), a synthetic xenoestrogen widely used in the production of polycarbonate plastics. The effects of long-term exposure to BPA on memory and modification of synaptic structure in hippocampus of adult mice were investigated in the present study. The adult mice were exposed to BPA (0.4, 4, and 40 mg/kg/day) or arachis oil for 12 weeks. In open field test, BPA at 0.4, 4, or 40 mg/kg/day increased the frequency of rearing and time in the central area of the males, while BPA at 0.4 mg/kg/day reduced the frequency of rearing in the females. Exposure to BPA (0.4 or 40 mg/kg/day) extended the average escape pathlength to the hidden platform in Morris water maze task and shortened the step-down latency 24 h after footshock of the males, but no changes were found in the females for these measures. Meanwhile, BPA induced a reduced numeric synaptic density and a negative effect on the structural parameters of synaptic interface, including an enlarged synaptic cleft and the reduced length of active zone and PSD thickness, in the hippocampus of the male mice. Western blot analyses further indicated that BPA down-regulated expressions of synaptic proteins (synapsin I and PSD-95) and synaptic NMDA receptor subunit NR1 and AMPA receptor subunit GluR1 in the hippocampus of the males. These results suggest that long-term exposure to low levels of BPA in adulthood sex-specifically impaired spatial and passive avoidance memory of mice. These effects may be associated with the higher susceptibility of the hippocampal synaptic plasticity processes, such as remodeling of spinal synapses and the expressions of synaptic proteins (e.g. synapsin I and PSD-95) and NMDA and AMPA receptors, to BPA in the adult male mice.

Myocardial Injury in Rats Exposed to High-Intensity Exercise Evaluated by 2-D Speckle Tracking Imaging.

OBJECTIVE: The aim of the work described here was to evaluate strain and morphological change of the left ventricle in Sprague Dawley (SD) rats at different exercise intensities by 2-D speckle tracking imaging (STI). METHODS: Seventy-two 8-wk-old SD rats were divided into four groups on the basis of exercise intensity: sedentary (SED), low-intensity running, medium-intensity running (MIR) and high-intensity running (HIR). Each group was further sub-divided into three groups of different exercise lengths: 1, 4 and 8 wk. The structural measurements of the left ventricle and left ventricular ejection fraction (LVEF) were obtained by echocardiography. Systolic peak values of global longitudinal, circumferential and radial strains (GLS, GCS and GRS) were obtained. Histopathological results of the cross-sectional area (CSA) of myocardial cells, collagen volume fraction (CVF) of the myocardium and perivascular collagen area (PVCA) were also observed. RESULTS: Structural measurements of the left ventricle and LVEF did not change with different exercise intensities or lengths. GLS of the HIR8 wk sub-group was significantly lower than those of the SED8 wk and MIR8 wk sub-groups. Conversely, the GLS and GCS of the HIR8 wk sub-group were lower than those of the HIR1 wk and HIR4 wk sub-groups. Histopathologically, the CSA of myocardial cells significantly increased across all HIR sub-groups and the MIR4 wk and MIR8 wk sub-groups. CVFendo and PVCA were also significantly increased in the HIR4 wk and HIR8 wk sub-groups. The HIR8 wk group also had regional swelling and ill-defined boundaries of myocardial cells. CONCLUSION: Prolonged, high-intensity exercise may lead to exercise-induced injury of the myocardium. Two-dimensional STI can be used as a non-invasive early detection method for exercise-induced injury of myocardial function, compared with LVEF.

Association of complement pathways with COVID-19 severity and outcomes.

OBJECTIVES: Complement activation has been implicated in COVID-19 pathogenesis. This study aimed to assess the levels of complement activation products and full-length proteins in hospitalized patients with COVID-19, and evaluated whether complement pathway markers are associated with outcomes. METHODS: Longitudinal measurements of complement biomarkers from 89 hospitalized adult patients, grouped by baseline disease severity, enrolled in an adaptive, phase 2/3, randomized, double-blind, placebo-controlled trial and treated with intravenous sarilumab (200 mg or 400 mg) or placebo (NCT04315298), were performed. These measurements were then correlated with clinical and laboratory parameters. RESULTS: All complement pathways were activated in hospitalized patients with COVID-19. Alternative pathway activation was predominant earlier in the disease course. Complement biomarkers correlated with multiple variables of multi-organ dysfunction and inflammatory injury. High plasma sC5b-9, C3a, factor Bb levels, and low mannan-binding lectin levels were associated with increased mortality. Sarilumab treatment showed a modest inhibitory effect on complement activation. Moreover, sera from patients spontaneously deposited C5b-9 complex on the endothelial surface ex vivo, suggesting a microvascular thrombotic potential. CONCLUSION: These results advance our understanding of COVID-19 disease pathophysiology and demonstrate the importance of specific complement pathway components as prognostic biomarkers in COVID-19.

Value of segmental myocardial strain by 2-dimensional strain echocardiography for assessment of scar area induced in a rat model of myocardial infarction.

OBJECTIVES: Two-dimensional strain echocardiography (2DSE) technique has enabled accurate quantification of regional myocardial function. This experimental study was aimed to investigate the value of 2DSE in detection of segmental regional myocardial dysfunction induced by fibrosis following myocardial infarction in a small animal (rat) model. METHODS: A rat model of myocardial infarction was established by ligation of the proximal left anterior descending coronary artery in 17 SD rats. Regional myocardial function was detected by 2DSE at baseline and 4-weeks post-infarction, including end-systolic radial strain and strain rate (SR and SrR) and end-systolic circumferential strain and strain rate (SC and SrC) of each of six segments at papillary level. According to the size of scar found by histologic Masson staining, the optimal cutoff points of parameters for detecting scar area were analyzed and the sensitivity and specificity of every parameter to detect myocardial scar were obtained using ROC. RESULTS: (1) Comparing with parameters measured at baseline, there were significant decreases in SR, SrR, SC and SrC of each segment at 4 weeks post-infarction, with the worst in the infarct area (32.90 ± 8.79 vs 11.18 ± 3.89, 6.28 ± 1.35 vs 3.18 ± 0.47, -14.46 ± 2.21 vs -6.30 ± 2.17 and 4.93 ± 0.95 vs 2.59 ± 1.16, respectively) (all P < 0.05). (2)By 4 weeks, the myocardium of infarct area (anteroseptum, anterior and anterolateral) had fibrosis (31.33 ± 9.89, 73.42 ± 13.21 and 13.99 ± 3.24%, respectively) with minimal fibrosis in inferoseptal segment (0.32 ± 0.19%), no fibrosis was found in the inferior and inferolateral segments. (3)Significant negative correlations were found between the size of segmental scar and 2DSE parameters (r-value -0.61 ~ -0.80, all P < 0.01) with the strongest correlation in SR. SR less than 10% has 84% sensitivity and 98% specificity for detecting segments of scar area greater than 30% with AUC = 0.97. CONCLUSIONS: 2DSE is able to assess regional myocardial dysfunction in a rat model of myocardial infarction and has high accuracy in detecting infarct segments with scar area greater than 30%.

Chlorpyrifos exposure reduces reproductive capacity owing to a damaging effect on gametogenesis in the nematode Caenorhabditis elegans.

Previous studies have revealed that chlorpyrifos exposure adversely affects the reproductive capacity of male rodents. The present study investigated the reproductive toxicity of chlorpyrifos exposure and possible related mechanisms using the nematode Caenorhabditis elegans. L4 nematode larvae were exposed to chlorpyrifos at concentrations of 0.003, 0.03, 0.3 and 3.0 mg l(-1) for different durations. In addition to decreased brood size, reduced spermatid size, increased percentage of abnormal spermatids, suppressed spermatid activation and motility of sperm, damaged oocyte morphology, increased numbers of apoptotic cells and unfertilized oocytes were observed in nematodes exposed to various concentrations of chlorpyrifos. Moreover, expression patterns of the genes spe-10, spe-15, fer-1, prg-1, glp-1, mlh-1, cyb-3, ced-3, ced-4 and ced-9 (which are associated with spermatid size, spermatid activation and morphology, oocyte morphology, oocyte function, and apoptosis) were altered after chlorpyrifos exposure. Therefore, chlorpyrifos exposure may adversely affect fertility in nematodes by influencing both spermatogenesis and oogenesis. Alterations in the expression patterns of genes involved in gametogenesis may explain the corresponding changes in gametogenesis in nematodes exposed to chlorpyrifos. Hence, the model organism Caenorhabditis elegans is recommended for assessment of reproductive toxicity relating to gametogenesis.

[Adverse cardiovascular effects of antiretrovirals in female mice during gestation].

Objective: To evaluate the effects of antiretrovirals on cardiovascular function and some biochemical indexes in gestational female rats. Methods: Nineteen 9-week-old female and six 10-week-old male SD rats were divided into normal control group (CON) and highly active antiretroviral therapy group (HARRT), 9/10 female rats and 3 male rats were combined into one cage, totally 2 cages. Female rats in CON group were intragastrically given with normal saline (NS, 10 ml/kg) every morning and evening, while female rats in HARRT group were treated with equal volume antiretrovirals (AZT 31.25 mg/kg + 3TC 15.63 mg/kg + LPV/r (41.67/10.42) mg/kg) for 3 months. The body weight and survival rate of female rats were recorded. Echocardiography and multichannel physiological recorder were used to detect arterial blood pressure and cardiac hemodynamic parameters. The levels of blood glucose, blood lipids, myocardial enzymes and liver enzymes were detected by corresponding kits. Myocardial collagen fibers were observed by Masson staining and the ultrastructure of myocardial cells were observed by transmission electron microscopy. Results: All female rats in CON group survived (9/9), while only 6 rats in HARRT group survived (6/10). Compared with CON group, the body weight of female rats in HAART group was decreased significantly(P＜0.01); the levels of left ventricular end diastolic diameter (LVDd), interventricular septal thickness (IVST), thickness of left ventricular posterior wall (LVPWT) , left atrial diameter (LAD) and arterial diastolic pressure were increased significantly (P＜0.05); the level of LVP+dP/dtmax was decreased (P＜0.01). The levels of triglyceride, creatine kinase, and glutamic oxaloacetic transaminase were decreased (P＜0.05 or P＜0.01), while the level of glucose was increased (P＜0.05). The collagen fibers were increased in myocardial tissue, and ultrastructure of myocardial cells was abnormal. Conclusion: Antiretrovirals during gestation can cause cardiovascular diseases in female rats.

Activin A directly impairs human cardiomyocyte contractile function indicating a potential role in heart failure development.

Activin A has been linked to cardiac dysfunction in aging and disease, with elevated circulating levels found in patients with hypertension, atherosclerosis, and heart failure. Here, we investigated whether Activin A directly impairs cardiomyocyte (CM) contractile function and kinetics utilizing cell, tissue, and animal models. Hydrodynamic gene delivery-mediated overexpression of Activin A in wild-type mice was sufficient to impair cardiac function, and resulted in increased cardiac stress markers (N-terminal pro-atrial natriuretic peptide) and cardiac atrophy. In human-induced pluripotent stem cell-derived (hiPSC) CMs, Activin A caused increased phosphorylation of SMAD2/3 and significantly upregulated SERPINE1 and FSTL3 (markers of SMAD2/3 activation and activin signaling, respectively). Activin A signaling in hiPSC-CMs resulted in impaired contractility, prolonged relaxation kinetics, and spontaneous beating in a dose-dependent manner. To identify the cardiac cellular source of Activin A, inflammatory cytokines were applied to human cardiac fibroblasts. Interleukin -1ß induced a strong upregulation of Activin A. Mechanistically, we observed that Activin A-treated hiPSC-CMs exhibited impaired diastolic calcium handling with reduced expression of calcium regulatory genes (SERCA2, RYR2, CACNB2). Importantly, when Activin A was inhibited with an anti-Activin A antibody, maladaptive calcium handling and CM contractile dysfunction were abrogated. Therefore, inflammatory cytokines may play a key role by acting on cardiac fibroblasts, causing local upregulation of Activin A that directly acts on CMs to impair contractility. These findings demonstrate that Activin A acts directly on CMs, which may contribute to the cardiac dysfunction seen in aging populations and in patients with heart failure.

Perinatal exposure to bisphenol-A changes N-methyl-D-aspartate receptor expression in the hippocampus of male rat offspring.

Bisphenol-A (BPA) is one of the most common environmental endocrine disrupters with mixed estrogen agonist/antagonist properties. The toxicity of BPA has been extensively evaluated in a variety of tests in rodents, including developmental and reproductive toxicity, and carcinogenicity. The objective of the present study is to evaluate whether or not perinatal maternal exposure to BPA at 0.05, 0.5, 5, 50, and 200 mg/kg/d affects N-methyl-D-aspartate (NMDA) receptor (NMDAR) subunits NR1, NR2A, 2B, estrogen receptor beta (ERbeta), and aromatase cytochrome P450 (P450arom) protein expressions of hippocampus in male rat offspring during postnatal development. Western-blotting analyses showed that perinatal exposure to BPA significantly affected the expression of NMDAR subunits. At the lower doses of 0.05 to 50 mg/kg/d, BPA concentration dependently inhibited the expression of NMDAR subunits. However, at the higher dose (200 mg/kg/d), the effects of BPA on these subunits were different, with a stronger inhibition of NR1 expression and a slighter inhibition of NR2A, 2B expression when compared with those at the lower dosage of BPA. In addition, perinatal exposure to BPA inhibited the expression of ERbeta protein, but increased P450arom protein expression in a concentration-dependent manner, especially during the early postnatal period (the first 1-3 postnatal weeks). No significant influence of BPA on P450arom was observed at postnatal week 8. These data suggest that environmental BPA exposure may affect the development of the brain, enhancing the local biosynthesis of estrogen in the brain, inhibiting ERbeta and NMDAR expressions.

Effects and Molecular Mechanism of L-Type Calcium Channel on Fluoride-Induced Kidney Injury.

This study aimed to investigate the role and molecular mechanism of L-type calcium channel (LTCC) on fluoride exposure-induced kidney injury. Subchronic and chronic fluoride exposures were included in the experiment. Each part contained 140 ICR male mice. They were randomly divided into 7 groups: control group, high-fluoride group (NaF 30 mg/L), low-fluoride group (NaF 5 mg/L), high/low-fluoride + agonist (FPL64176) group, high/low-fluoride + inhibitor (nifedipine) group. One week before the end of fluoride exposure, each mouse in the fluoride exposure group was injected intraperitoneally with LTCC agonist (FPL64176) or inhibitor (nifedipine) (5 mg/kg day). The apoptosis of kidney cell was observed by TUNEL, and the protein expression levels of Cav1.2 and CaM, CaMKII, Bcl-2, and Bax were detected by Western blot. Compared with the control group, the protein expression levels of Cav1.2, CaM, and Bax significantly increased, and those of CaMKII and Bcl-2 significantly decreased, the ratio of Bax/Bcl-2 also significantly increased, and the number of apoptotic kidney cells significantly increased in the high/low-fluoride group and in the high/low-fluoride + agonist group. The above indicators and fluoride exposure concentrations showed in time- and dose-dependent changes. Compared with the high/low-fluoride + agonist group, the protein expression level of the molecular in the kidney cells above mentioned was significantly opposite and the number of apoptotic kidney cells significantly decreased in the high/low-fluoride + inhibitor group. In conclusion, LTCC mediates the kidney injury induced by fluoride exposure in mice. Fluoride exposure induced abnormal expression of the Cav1.2 protein, Ca2+ signal transduction pathway, and apoptosis-regulated proteins, which is one of the molecular mechanisms. Nifedipine may be a new and effective anti-fluoride drug.

Environmental enrichment induces synaptic structural modification after transient focal cerebral ischemia in rats.

Environmental enrichment (EE), where animals are exposed to a complex novel environment, has been shown to induce synaptic plasticity in both intact and injured animals. The purpose of this study was to investigate the effects of EE on spatial memory and structural modifications of synaptic junctions in rats following transient focal cerebral ischemia. Adult male Sprague-Dawley rats underwent right middle cerebral artery occlusion (MCAO) for 40 min and reperfusion. On day 3 after MCAO or sham surgery, rats were randomly assigned for 14 days to enriched or standard environmental housing. Spatial memory was then tested by the Morris water maze. Parietal cortex and the CA1 region of hippocampus were processed for electron microscopy and stereological techniques were used to evaluate plasticity of synaptic junctions. EE after MCAO improved spatial memory, with shortened escape length, increased frequency of crossings at the location of the platform, and increased percentage of time spent in the quadrant where the platform was previously located. Synaptic ultrastructural analysis showed that EE after MCAO increased numeric synaptic density in parietal cortex, and induced structural changes in synaptic junctions, with a decreased width of synaptic clefts and increased thickness of postsynaptic densities (PSD) in parietal cortex and hippocampus, accompanying improved performance on the spatial memory task. Using Western blot analysis, we determined the expression of glutamate receptor NMDAR1, and PSD-95, the best characterized protein member of the PSD-95 family, that was abundantly expressed in the PSD of excitatory synapses. The results showed that the content of NMDAR1 was not altered in MCAO rats of EE; however, the phosphorylated NMDAR1 increased significantly when compared with the standard environment housing MCAO rats. In addition, EE inhibited the impaired expression of PSD-95 induced by MCAO in parietal cortex and hippocampus. These data suggest that improved spatial memory of cerebral ischemic rats by EE is associated with structural modifications of synaptic junctions in several brain regions.

Evaluation of pesticide toxicities with differing mechanisms using Caenorhabditis elegans.

The aim of this study was to (1) determine whether model organism Caenorhabditis elegans was sensitive to pesticides at the maximum concentration limits regulated by national agency standards, and (2) examine the multi-biological toxicities occurring as a result of exposure to pesticides. Five pesticides, namely, chlorpyrifos, imibacloprid, buprofezin, cyhalothrin, and glyphosate, with four different mechanisms of action were selected for the investigation. In accordance with national agency requirements, 4 exposed groups were used for each tested pesticide with the concentration scales ranging from 1.0 x 10(-3) to 1 mg/L. L4 larvae were exposed for 24 and 72 h, respectively. Endpoints of locomotion, propagation, and development were selected for the assay as parameters of toxicity. After exposure for 24 h, both the body bend frequency and head thrash frequency of nematodes exposed to chlorpyrifos, imibacloprid, and cyhalothrin decreased in a concentration-dependent manner, and there were significant differences between exposed groups at maximum concentration level (MCL) compared to control. The generation time of nematodes exposed to buprofezin 24 h significantly increased in a concentration-dependent manner in the highest exposed group. When exposed for 72 h, the body bend frequency and head thrash frequency of nematodes exposed to cyhalothrin markedly decreased at MCL. The generation time and brood size of nematodes exposed to buprofezin were reduced in a concentration-dependent manner. The behavior of nematodes was sensitive to pesticides with neurotoxic properties, while pesticides affecting insect growth modified the reproductive system. The effects of pesticides on nematodes exposed for 24 h appeared more sensitive than with exposure for 72 h. Caenorhabditis elegans may thus be used for assessing the adverse effects of pesticide residues in aquatic environment.

[Studies on the Herba Plantaginis of different kinds and habitats by FTIR reflection spectroscopy-chemometrics analysis].

In the present paper, FTIR of different species obtained from different regions of sibling plantaginis were determined by Fourier transform infrared reflection spectroscopy (FTIRS), and thirty-five comparatively typical absorption peaks were selected and used to study genetic relationship, combined with chemometric methods. The phylogenetic cluster analysis revealed that three species could be divided into two groups based on the distance of 0.036, among which Plantago depressa Willd. was clustered with Plantago asiatica L. based on distance of 0.033, while clustered with Plantago virginica L. based on distance of 0.042, and the result was consistent with that of traditional taxology. The principal component analysis result revealed that the distances of Plantago asiatica L. in the similar environment are similar in three-dimensional FTIR chart, however, it is dispersive when obtained from different regions. This method is scientific, simple and direct, and has important theoretical and practical application value.

An efficient ICT-based ratio/colorimetric tripodal azobenzene probe for the recognition/discrimination of F-, AcO- and H2PO4- anions.

The tripodal probe L was readily prepared via introducing rhodamine and azobenzene groups in a two-step procedure. Studies of the recognition properties indicated that probe L exhibited high sensitivity and selectivity towards F-, AcO- and H2PO4- through a ratiometric colorimetric response with low detection limits of the order of 10-7 M. The complexation behaviour was fully investigated by spectral titration, 1H NMR spectroscopic titration and mass spectrometry. Probe L not only recognizes F-, AcO- and H2PO4-, but can also distinguish between these three anions via the different ratiometric behaviour in their UV-vis spectra (387/505 nm for L-H2PO4-, 387/530 nm for L-AcO- and 387/575 nm for L-F- complex) or via different colour changes (light coral for L-H2PO4-, light pink for L-AcO- and violet for the L-F- complex). Additionally, given the presence of NH and OH groups in probe L, which can be protonated and deprotonated, probe L further exhibited an excellent pH response over a wide pH range (pH 3 to pH 12).

A three-dimensional (time, wavelength and intensity) functioning fluorescent probe for the selective recognition/discrimination of Cu2+, Hg2+, Fe3+ and F- ions.

We have strategically incorporated three different fluorophores at tren to construct a multi-energy donor/acceptor "smart" probe L. This probe operates by using three-dimensional scales (response time, wavelength and fluorescence intensity) which allows for the selective recognition and discrimination of the Cu2+, Hg2+, Fe3+ and F- ions.

Identification and validation of reference genes for quantitative real-time PCR studies in Hedera helix L.

Reference gene evaluation and selection are necessary steps in gene expression analysis, especially in new plant varieties, through reverse transcription quantitative real-time PCR (RT-qPCR). Hedera helix L. is an important traditional medicinal plant recorded in European Pharmacopoeia. Research on gene expression in H. helix has not been widely explored, and no RT-qPCR studies have been reported. Thus, it is important and necessary to identify and validate suitable reference genes to for normalizing RT-qPCR results. In our study, 14 candidate protein-coding reference genes were selected. Their expression stability in five tissues (root, stem, leaf, petiole and shoot tip) and under seven abiotic stress conditions (cold, heat, drought, salinity, UV-C irradiation, abscisic acid and methyl jasmonate) were evaluated using geNorm and NormFinder. This study is the first to evaluate the stability of reference genes in H. helix. The results show that different reference genes should be chosen for normalization on the basis of various experimental conditions. F-box was more stable than the other selected genes under all analysis conditions except ABA treatment; 40S was the most stable reference gene under ABA treatment; in contrast, EXP and UBQ were the most unstable reference genes. The expressions of HhSE and Hhß-AS, which are two genes related to the biosynthetic pathway of triterpenoid saponins, were also examined for reference genes in different tissues and under various cold stress conditions. The validation results confirmed the applicability and accuracy of reference genes. Additionally, this study provides a basis for the accurate and widespread use of RT-qPCR in selecting genes from the genome of H. helix.