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Injection of total saponins from Panax notoginseng (ISPN) is a modern preparation derived from traditional Chinese medicine (TCM) and is widely applied in the treatment of cardiovascular, cerebrovascular, ophthalmology, and endocrine system diseases. With the increase in the clinical application of ISPN, its adverse drug reactions (ADRs) and related safety issues have attracted much attention. In the present study, a data-independent acquisition (DIA) strategy was proposed to comprehensively characterize the saponins contained in ISPN based on the ultra-high-performance liquid chromatography/quadrupole-Orbitrap MS (UHPLC/Q-Orbitrap MS) platform. As many as 276 saponins were detected, and 250 compounds were identified or tentatively identified based on the retention times and MS/MS data. Furthermore, a metabolomic strategy was utilized to discover the discriminative saponins between normal and ADR batches. The results showed that six saponins, including ginsenoside Rh4, ginsenoside Rk3, ginsenoside Rg5, ginsenoside Rk1, ginsenoside Rg6, and 20(S)-ginsenoside Rh2, were significantly different between the two groups. According to cytotoxicity analysis and degranulation detection of RBL-2H3 cells, ginsenoside Rg5, ginsenoside Rk1, and 20(S)-ginsenoside Rh2 were considered the potential compounds responsible for clinical ADRs, ultimately. In addition, the quantitative analysis showed that the content of these three compounds in ISPN samples with ADRs was generally higher than that in samples without ADRs. This study demonstrated that it is advisable to screen out potential markers related to ADRs for developing the quality standard of ISPN by the integration of untargeted metabolomic analysis and cell biology study, and thus reduce its ADRs in the clinic.

Drought stress caused by water deficit reduces plant productivity in many regions of the world. In plants, basic helix–loop–helix (bHLH) transcription factors regulate a wide range of cellular activities related to growth, development and stress response; however, the role of tomato SlbHLHs in drought stress responses remains elusive. Here, we used reverse genetics approaches to reveal the function of SlbHLH96, which is induced by drought and abscisic acid (ABA) treatment. We found that SlbHLH96 functions as a positive regulator of drought tolerance in tomato. Overexpression of SlbHLH96 in tomato improves drought tolerance by stimulating the expression of genes encoding antioxidants, ABA signaling molecules and stress-related proteins. In contrast, silencing of SlbHLH96 in tomato reduces drought tolerance. SlbHLH96 physically interacts with an ethylene-responsive factor, SlERF4, and silencing of SlERF4 in tomato also decreases drought tolerance. Furthermore, SlbHLH96 can repress the expression of the ABA catabolic gene, SlCYP707A2, through direct binding to its promoter. Our results uncover a novel mechanism of SlbHLH96-mediated drought tolerance in tomato plants, which can be exploited for breeding drought-resilient crops.

A novel dual-response fluorescent sensor based on a diarylethene photoswitching unit and a dansylhydrazine functional group has been synthesized. The compound exhibited high selectivity for Fe3+ and Cu2+ with independent fluorescence signal outputs. In the presence of Fe3+, the sensor formed a 1:1 metal complex, resulting in a remarkable "turn-off" fluorescence signal. On the other hand, its fluorescence intensity was notably enhanced (turn-on) and a color change from bright yellow to bright blue was observed when the sensor interacted with Cu2+, which was due to the hydrolysis reaction of the dansyl acid dye, as confirmed by high-resolution mass spectrometry-electrospray ionization and infrared spectrum. The detection limits were 9.73 × 10-8 mol L-1 for Fe3+ and 3.49 × 10-7 mol L-1 for Cu2+, respectively. From the unimolecular platform, two molecular logic circuits were constructed using the fluorescence emission intensity at 557/494 nm (Fe3+/Cu2+) as the outputs and the combined stimuli of Fe3+/ethylenediaminetetraacetic acid, Cu2+, and UV/vis as the inputs. In addition, the sensor was successfully used to determine Fe3+ in water samples from Ganjiang River and soil samples from Nanchang fields.

Photochromic asymmetrical diarylethene derivatives 1o–4o bearing different benzo-five-membered heterocyclic rings were synthesized and characterized with single-crystal X-ray diffraction microscopy. The effects of heterocyclic ring on their properties including the photochromic behaviors in solution, solid state and crystalline phase were systematically investigated. The diarylethenes bearing benzothiophene or indole ring exhibited high cyclization quantum yields in solution, and evidently enhanced the fluorescent modulation efficiency in solid state, while those containing benzofuran or thiophene ring exhibited the opposite behaviors. In addition, indole ring red-shifted the absorption maximum, and notably enhanced the fatigue resistance of the diarylethene. These results indicate that heteroaryl ring played an important role in the photoisomerization of these diarylethenes, causing the heterocyclic effect.

The pathogenesis of hepatocellular carcinoma (HC) is unclear. It is suggested that psychological stress associates with the pathogenesis of liver cancer. Bcl2-like protein 12 (Bcl2L12) suppresses p53 protein. This study tests a hypothesis that the major stress hormone, cortisol, inhibits the expression of p53 in HC cells (HCC) via up regulating the expression of Bcl2L12. Peripheral blood samples were collected from patients with HC to be analyzed for the levels of cortisol. HCC were cultured to assess the role of cortisol in the regulation of the expression of Bcl2L12 and p53 in HCC. We observed that the serum cortisol levels were higher in HC patients. Expression of Bcl2L12 in HCC was correlated with serum cortisol. Cortisol enhanced the Bcl2L12 expression in HCC. Bcl2L12 binding to the TP53 promoter was correlated with p53 expression in HCC. Cortisol increased the Bcl2L12 expression in HCC to inhibit p53 expression. Stress hormone cortisol suppresses p53 in HCC via enhancing Bcl2L12 expression in HCC. The results suggest that cortisol may be a therapeutic target for the treatment of HC.

Four centrosymmetric 9,10-diheteroarylanthracene (DHA) derivatives, including 9,10-dithienylanthracene (DTA), 9,10-difurylanthracene (DFA), 9,10-di-(N-t-butyloxycarboryl-2-pyrryl)anthracene (DBPA), and 9,10-dipyrrylanthracene (DPA) have been synthesized and characterized. All of these DHA derivatives displayed distinct aggregation-induced emission (AIE) behaviors except for DBPA, which showed typical aggregation-caused quenching (ACQ) properties. Their crystal structures exhibited nonplanar conformations on account of the intramolecular torsional effects and intramolecular interactions in rigid molecules. The investigation of the effects of the anthracene core and the side heterocyclic units on the AIE properties demonstrated that the heterocycle moiety is the key factor for the AIE features. These DHA AIEgens exhibited excellent bioimaging performance under physiological conditions.

Two diarylethenes containing the hybrid structures of benzothiophene and pyrrole backbone were synthesized and their structures and properties including photochromism, fatigue resistance, and fluorescence were systematically investigated. Both of them exhibit good photochromisms and can function as fluorescence switches in both solution and solid state upon the irradiations of UV/vis lights. Compared with that bearing cyano group (1O), the carboxamide group containing diarylethene (2O) exhibits a higher molar absorption coefficient in solution, and its maximum absorption in solid state red-shifts by 30 nm, as compared with that in solution. In addition, the application potentials of 2O in bioimaging are demonstrated with excellent performances under physiological conditions.

Background : Therapeutic angiogenesis is a novel strategy for the treatment of ischemic diseases that involves promotion of angiogenesis in ischemic tissues via the use of proangiogenic agents. However, effective proangiogenic drugs that activate the Ang2/Tie2 signaling pathway remain scarce. Purpose : We aimed to investigate the proangiogenic activity of notoginsenoside R1 (NR1) isolated from total saponins of Panax notoginseng with regard to activation of the Ang2/Tie2 signaling pathway. Methods : We examined the proangiogenic effects of NR1 by assessing the effects of NR1 on the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). An aortic ring assay and vascular endothelial growth factor receptor inhibitor (VRI)-induced vascular regression in a zebrafish model were used to confirm the proangiogenic effects of NR1 ex vivo and in vivo. Furthermore, the molecular mechanism was investigated by Western blot analysis. Results : We found that NR1 promoted the proliferation, mobility and tube formation of HUVECs in vitro. It also increased the number of sprouting vessels in rat aortic rings and rescued VRI-induced vascular regression in zebrafish. NR1-induced angiogenesis was dependent on Tie2 receptor activation mediated by increased autocrine Ang2 in HUVECs, and inhibition of the Ang2/Tie2 pathway abrogated the proangiogenic effects of NR1. Conclusions : Our results suggest that NR1 promotes angiogenesis by activating the Ang2/Tie2 signaling pathway. Thus, NR1-induced activation of the Ang2/Tie2 pathway is an effective proangiogenic approach. NR1 may be useful agent for the treatment of ischemic diseases.

The effect of Mo and Al addition on the microstructure as well as creep rupture properties at 760 °C/850 MPa was investigated by transmission electron microscopy (TEM) in a Ni-based single-crystal (SC) alloy with the composition of Ni–6.5Al–8.0Mo–2.4Cr–6.2Ta–4.9Co–1.5Re–(0.01–0.05)Y (wt%). The microstructure analysis shows that 0.5 wt% Al addition induces rapid decrease in creep rupture life, and this can be attributed to the formation of dense stacking faults cutting into γ′ precipitates, which can be explained by the increase in Orowan stress caused by the narrower γ channel width and the decrease in stacking faults energy. Besides, 1.5 wt% Mo addition increases the anti-phase boundary energy and decreases the stacking faults energy, resulting in fewer stacking faults and thus a slight decrease in the creep rupture life.

The additional 1.5 wt% Mo was added in a Ni-base single crystal (SC) alloy with the composition of Ni–6.5Al–8.0Mo–2.4Cr–6.2Ta–4.9Co–1.5Re–(0.01–0.05)Y (wt%) to study the effect of Mo content on the microstructure and stress-rupture properties. The creep and stress-rupture tests under the conditions of 850 °C/500 MPa and 1100 °C/130 MPa were conducted, and the microstructure of as-cast, heat treated and stress ruptured specimens were analyzed. It was found that the 1.5 wt% Mo addition enhanced the stress-rupture lives at both intermediate (850 °C) and high (1100 °C) temperatures. The microstructure analysis showed that adding 1.5 wt% Mo in the basic alloy affected the microstructure dramatically, i.e., the Mo-rich phases formed in the specimens of as-cast and stress-ruptured specimens. It is considered that the improvement of the stress-rupture lives is duo to the strengthening effect of Mo to both γ and γ′ phases and the decrease of stacking fault energy, diffusion constant and dislocation spacing. The Mo-rich phases precipitated under condition of 1100 °C/130 MPa did not affect the creep and stress-rupture properties obviously in the present study.

Ni3Al (Ni75Al25) and Ni3Al–2Mo/Ta (Ni73.5Al24.5Mo/Ta2) single crystals were prepared by the Bridgman method under high temperature gradient (~170 °C/cm) at the withdrawal rates of 5 and 20 μm/s. The experimental results showed that the addition of Mo decreased the liquidus and solidus temperatures, and the addition of Ta increased the liquidus and solidus temperatures. Meanwhile, both Mo and Ta additions were found to increase the solidification range of experimental alloy. It has been found that for solidification of stoichiometric Ni3Al at low withdrawal rate (5 μm/s), β/γ′ eutectic first solidified from liquid (L→β+γ′) and then β phase completely transformed into pure γ′ (β→γ′) after the completion of solidification. However, at high withdrawal rate of 20 μm/s, the remaining β phase was found in the as-cast microstructure of Alloy Ni3Al. Different from stoichiometric Ni3Al, the solidification sequence of Alloy Ni3Al–2Mo was identified as L→β+γ′+Mo-rich phase (ternary eutectic reaction). In addition, the addition of Ta led to the formation of primary γ′ phase and then subsequent intercellular/interdendritic β/γ′ eutectic microstructure. Based on above study, a new Ta and Mo containing Ni3Al based single crystal alloy with superior tensile strength at ultra-high temperature (>1100 °C) was designed.

A new asymmetric perfluordiarylethene (1O) was synthesized using 4, 5-[bis-(5-ethylacetate-yl)-2-thienyl]-1H-imidazole as a functional group. 1O exhibited favorable reversible cyclization and cycloreversion reactions upon alternating irradiation with UV and visible light. Both of its open- and closed-ring isomers were found to be highly selective towards Cu2+ with significant absorption and color changes, which could be used as a ‘naked-eye’ colorimetric sensor for Cu2+ detection. Upon exposure to acid, its fluorescence dramatically enhanced by 14-fold with a color change from dark to bright cyan due to the formation of the protonated compound. Moreover, 1O showed obvious fluorescence “turn-on” signal response towards Al3+, and the detection limit for Al3+ was determined to be 4.8 × 10−9 mol L−1. Based on the fluorescence signals of 1O, a combinational stimuli logic circuit were designed by using the fluorescence intensity as the output signal with the inputs of lights, Al3+ and EDTA. Finally, 1O could be used as a biological probe for detecting intracellular Al3+ in a physiological environmental.

Fifteen strains of mAb specific for HA of the A/Hong Kong/482/97 (H5N1) influenza virus were generated. The HA antigenic sites of the human A/Hong Kong/482/97 (H5N1) influenza virus were defined by using yeast cell surface-displaying system and anti-H5 HA mAb. Evolution analysis of H5 HA identified residues that exhibit diversifying selection in the antigenic sites and demonstrated surprising differences between residue variation of H5 HA and H3 HA. A conserved neutralizing epitope in the H5 HA protein recognized by mAb H5M9 was found using viruses isolated from 1997-2006. Seven single amino acid substitutions were introduced into the HA antigenic sites, respectively, and the alteration of antigenicity was assessed. The structure obtained by homology-modeling and molecular dynamic methods showed that a subtle substitution at residue 124 propagates throughout its nearby loop (152-159). We discuss how the structural changes caused by point mutation might explain the altered antigenicity of the HA protein. The results demonstrate the existence of immunodominant positions in the H5 HA protein, alteration of these residues might improve the immunogenicity of vaccine strains.

To warrant potential clinical testing, the equine anti-SARS-CoV F(ab')(2) requires evaluation in as many animal models as possible and a safety test in a primate model. In this study, we evaluated the pharmacokinetics, tolerance and immunity of this kind of antibody in macaques and rats. Results showed that the F(ab')(2) fragments had a normal metabolism in injected animals. The general physiological indexes did not differ between animals injected with anti-SARS-CoV F(ab')(2) or saline. However, a mild inflammatory response in local injection site and a moderate immune response against this antibody in the successively injected animals were observed, which however recovered 3 weeks after the last injection. The antibody titring from 1:100 to 400 against the equine anti-SARS-CoV F(ab')(2) in the inoculated hosts could be detected at week 2 during the successive injections of the equine F(ab')(2). The considerable safety of this antibody used in primates and the fact that the immune system of the host can be motivated by post-injection of the F(ab')(2) indicate that this type of anti-SARS-CoV antibody can be used for prevention and treatment of SASR, especially at the early stage of this virus infection. In addition, it can also provide the precious time for the combined use of other anti-SARS-CoV agents such as antiviral drug and vaccine.

The high susceptibility of elderly to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) indicates how crucial it is to protect the elderly by various strategies. Aged BALB/c mice displayed a high susceptibility to SARS-CoV and have been a valuable platform for evaluation of strategies against SARS-CoV infection. In this study, we confirmed the validity of this model using various methods, and verified that equine anti-SARS-CoV F(ab')(2) can prevent aged animals from SARS-CoV infection. In a therapeutic setting, treatment with anti-SARS-CoV F(ab')(2) decreased viral load more than several thousand folds in the lungs. Thus, this antibody should be a potential candidate for treatment of elderly patients suffering from SARS.

It has been shown that exogenous antigens can access the MHC class I pathway of professional antigen-processing cells. However, details as to how the MHC class I-peptide complex forms in the presentation pathway are still poorly understood. Here we used MHC class I-peptide-specific antibodies to investigate the formation and intracellular location of class I-peptide complexes in macrophages. We observed that the formation of class I-peptide complexes occurs within a few hours and lasts for another few hours on the cell surface of macrophages following loading with filamentous phage particles. The class I-peptide complexes in the process were co-localized with MHC class II molecules and endocytic system markers. Moreover, endosomal compartments containing class I-peptide complexes were found within intracellular organelles stained by DiOC6 and calnexin. In addition, the cross-presentation of phage particles was transporter associated with antigen processing (TAP)-dependent and sensitive to proteasome inhibitors and NH(4)Cl. These data suggest that endocytosed phage particles may be processed and cross-presented in organelles positive for phagosome and endoplasmic reticulum (ER) markers via a classical ER MHC class I loading mechanism.

Fifteen strains of mAb specific for HA of the A/Hong Kong/482/97 (H5N1) influenza virus were generated. The HA antigenic sites of the human A/Hong Kong/482/97 (H5N1) influenza virus were defined by using yeast cell surface-displaying system and anti-H5 HA mAb. Evolution analysis of H5 HA identified residues that exhibit diversifying selection in the antigenic sites and demonstrated surprising differences between residue variation of H5 HA and H3 HA. A conserved neutralizing epitope in the H5 HA protein recognized by mAb H5M9 was found using viruses isolated from 1997-2006. Seven single amino acid substitutions were introduced into the HA antigenic sites, respectively, and the alteration of antigenicity was assessed. The structure obtained by homology-modeling and molecular dynamic methods showed that a subtle substitution at residue 124 propagates throughout its nearby loop (152-159). We discuss how the structural changes caused by point mutation might explain the altered antigenicity of the HA protein. The results demonstrate the existence of immunodominant positions in the H5 HA protein, alteration of these residues might improve the immunogenicity of vaccine strains.

To warrant potential clinical testing, the equine anti-severe acute respiratory syndrome coronavirus (SARS-CoV) F(ab')(2) requires evaluation in as many animal models as possible. In this study, we established a new animal model, the Chinese hamster, susceptible to SARS-CoV infection. SARS-CoV could propagate effectively and sustain high levels for 1 wk in animal lungs. All animals were protected from SARS-CoV infection in preventive settings. Further, when used therapeutically this antibody led to an approximately 4-log(10) decrease in viral burden in infected animal lungs. The pathological changes in lungs correlated closely with the dose of antibody administered. The excellent preventive and therapeutic roles of equine anti-SARS-CoV F(ab')(2) in several animal models, including the novel Chinese hamster model described in this study, have provided exciting data concerning its potential clinical study.

It is known that macrophage scavenger receptor A (SR-A) can protect mice from endotoxemia. In addition, Escherichia coli O111:B4 LPS from Sigma (sLPS), which contains both TLR4 and TLR2 agonists, was previously reported to be able to induce SR-A expression on murine macrophage cell line RAW264.7. However, the relative role of both TLR4 and TLR2 agonists from Sigma (sLPS) in the up-regulation of SR-A on RAW264.7 is still undefined. Here, we found that sLPS could only slightly up-regulate SR-A on RAW264.7 following removing its TLR4 and TLR2 agonists, respectively. In contrast, the combination of TLR4 agonist uLPS (re-extracted sLPS) and TLR2 agonist Pam3CSK4 dramatically induced SR-A expression, and synergistically promoted RAW264.7 to bind and internalize FITC-LPS specifically through SR-A. The combination had no such effect either on TLR2 or TLR4 expression, and incubation with IL-6, IL-10, IL-12 or TNF-alpha alone could not induce SR-A expression on RAW264.7. In addition, treatment with a NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) could only weakly suppress the up-regulation of SR-A by the combination. However, the combination synergistically promoted MAPK p38 phosphorylation, and p38 specific inhibitor SB203580 completely suppressed its inducible effect on SR-A expression. Hence, we demonstrated that up-regulation of SR-A by sLPS was resulted from the cooperation of its TLR4 and TLR2 agonists through p38, and we also presented a novel synergy effect of TLR2 and TLR4 agonists.

Background: The epidemic outbreak of severe acute respiratory syndrome (SARS) posed a worldwide threat to public health and economic stability. Although the pandemic has been contained, concerns over its recurrence remain. It is essential to identify specific diagnostic agents and antiviral vaccine candidates to fight this highly contagious disease. Methods: We generated 14 monoclonal antibodies (mAbs) specific to the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein and used these to thoroughly map the N protein antigenic determinants. We identified the immunodominant antigenic sites responsible for the antibodies in sera from SARS patients and antisera from small animals and differentiated the linear from the conformational antibody-combining sites comprising the natural epitopes by use of yeast surface display. Results: We identified 5 conformational and 3 linear epitopes within the entire N protein; 3 conformational and 3 linear epitopes were immunodominant. The antibody responses to the N protein fragments in mammalian sera revealed that 3 regions of the N protein are strong antigenic domains. We expanded the specificity of the N protein epitope and identified 4 novel conformational epitopes (amino acids 1–69, 68–213, 212–341, and 337–422). Conclusion: The antigenic structures identified for the SARS-CoV N protein, the epitope-specific mAbs, and the serum antibody profile in SARS patients have potential use in the clinical diagnosis and understanding of the protective immunity to SARS-CoV.

In this paper, the stress-rupture tests of a low Re-containing single crystal alloy IC21 before and after thermal exposure at 1100 °C for various periods of time were conducted under the test condition of 1100 °C/137 MPa, and the microstructure of the tested specimens was characterized by SEM and TEM. The experimental results showed that the stress rupture life of this alloy was over 150 h after the standard heat treatment of 1320 °C, 10 h/AC+870, 32 h/AC, however the stress rupture life decreased with the increase of exposure time due to the microstructure degradation. The TEM analysis revealed that the interface mismatch dislocation networks were well established. It was observed that these mismatch networks could form at 1100 °C even after thermal exposure for 1 h without the external stress, which is quite different from that in the traditional single crystal superalloys.

Background MicroRNAs are 22–24 nt non-coding RNAs that bind to the 3′ UTR of target mRNAs, thereby inducing mRNA degradation or inhibiting mRNA translation. Due to their implication in the regulation of post-transcriptional processes, the role of miRNAs in hepatocellular carcinoma (HCC) has been extensively studied. However, the function of miR-7 in HCC remains to be demonstrated. Methods 50 paired HCC tissues and matched peritumor tissues from patients were collected. The mRNA level of miR-7 was detected by qRT-PCR. The protein level of Kruppel-like factor 4 (KLF-4) was determined by western blot. Cell proliferation and invasive ability were measured using MTT and transwell invasion assay, respectively. Results We demonstrated that miR-7 was downregulated in 50 HCC tissues and the low expression of miR-7 was significantly correlate with tumour size. Moreover, overexpression of miR-7 significantly inhibited the proliferation and invasion of HCC cells. Over 100 target genes of miR-7 were predicted by Targetscan, and KLF-4 was indicated as the most promising candidate. Luciferase report assay showed that KLF-4 could be silenced by miR-7, so as to restore the impairment of cell proliferation and invasion in HCC cells. Conclusions In summary, we revealed a role of miR-7-KLF-4 axis in HCC cells, and the combination of both biomarkers might improve HCC diagnosis.