Rhodiola sachalinensis is an endangered species with important medicinal value. We used inter-simple sequence repeat (ISSR) and methylation-sensitive amplified polymorphism (MSAP) markers to analyze genetic and epigenetic differentiation in different populations of R. sachalinensis, including three natural populations and an ex situ population. Chromatographic fingerprint was used to reveal HPLC fingerprint differentiation. According to our results, the ex situ population of R. sachalinensis has higher level genetic diversity and greater HPLC fingerprint variation than natural populations, but shows lower epigenetic diversity. Most genetic variation (54.88%) was found to be distributed within populations, and epigenetic variation was primarily distributed among populations (63.87%). UPGMA cluster analysis of ISSR and MSAP data showed identical results, with individuals from each given population grouping together. The results of UPGMA cluster analysis of HPLC fingerprint patterns was significantly different from results obtained from ISSR and MSAP data. Correlation analysis revealed close relationships among altitude, genetic structure, epigenetic structure, and HPLC fingerprint patterns (R2 = 0.98 for genetic and epigenetic distance; R2 = 0.90 for DNA methylation level and altitude; R2 = –0.95 for HPLC fingerprint and altitude). Taken together, our results indicate that ex situ population of R. sachalinensis show significantly different genetic and epigenetic population structures and HPLC fingerprint patterns. Along with other potential explanations, these findings suggest that the ex situ environmental factors caused by different altitude play an important role in keeping hereditary characteristic of R. sachalinensis.
Mesenchymal stromal cells (MSCs) are multipotential adult cells present in all tissues. Paracrine effects and differentiating ability make MSCs an ideal cell source for tissue regeneration. However, little is known about how interactions between implanted MSCs and native cells influence cellular growth, proliferation, and behaviour. By using an in vitro three-dimensional (3D) co-culture assay of normal or scarred human vocal fold fibroblasts (VFFs) and bone marrow-derived MSCs (BM-MSCs) in a uniquely suited hyaluronan hydrogel (HyStem–VF), we investigated cell morphology, survival rate, proliferation and protein and gene expression of VFFs and BM-MSCs. BM-MSCs inhibited cell proliferation of both normal and scarred VFFs without changes in VFF morphology or viability. BM-MSCs demonstrated decreased proliferation and survival rate after 7 days of co-culture with VFFs. Interactions between BM-MSCs and VFFs led to a significant increase in protein secretion of collagen I and hepatocyte growth factor (HGF) and a decrease of vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6). In particular, BM-MSCs significantly upregulated matrix metalloproteinase 1 (MMP1) and HGF gene expression for scarred VFFs compared to normal VFFs, indicating the potential for increases in extracellular matrix remodelling and tissue regeneration. Application of BM-MSCs-hydrogels may play a significant role in tissue regeneration, providing a therapeutic approach for vocal fold scarring.
BM-MSCs; VFFs; three-dimensional co-culture; cell regulation; hydrogel
Growing evidence indicates that intestinal microbiota regulate our metabolism. Probiotics confer health benefits that may depend on their ability to affect the gut microbiota. The objective of this study was to examine the effect of supplementation with the probiotic strain, Lactobacillus rhamnosus hsryfm 1301, on the gut microbiota in a hyperlipidemic rat model, and to explore the associations between the gut microbiota and the serum lipids.
The hyperlipidemic rat model was established by feeding rats a high-fat diet for 28 d. The rats’ gut microbiota were analyzed using high-throughput sequencing before and after L. rhamnosus hsryfm 1301 supplementation or its fermented milk for 28 d. The serum lipids level was also tested.
The rats’ primary gut microbiota were composed of Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetes and Verrucomicrobia. The abundance and diversity of the gut microbiota generally decreased after feeding with a high-fat diet, with a significant decrease in the relative abundance of Bacteroidetes, but with an increase in that of Firmicutes (P < 0.05). Administration of L. rhamnosus hsryfm 1301 or its fermented milk for 28 d, could recover the Bacteroidetes and Verrucomicrobia abundance and could decrease the Firmicutes abundance, which was associated with a significant reduction in the serum lipids’ level in the hyperlipidemic rats with high-fat diet induced. The abundance of 22 genera of gut bacteria was changed significantly after probiotic intervention for 28 d (P < 0.05). A positive correlation was observed between Ruminococcus spp. and serum triglycerides, Dorea spp. and serum cholesterol (TC) and low-density lipoprotein (LDL-C), and Enterococcus spp. and high-density lipoprotein. The Butyrivibrio spp. negatively correlated with TC and LDL-C.
Our results suggest that the lipid metabolism of hyperlipidemic rats was improved by regulating the gut microbiota with supplementation of L.rhamnosus hsryfm 1301 or its fermented milk for 28 d.
Electronic supplementary material
The online version of this article (doi:10.1186/1472-6882-14-386) contains supplementary material, which is available to authorized users.
A novel glycopeptide (Cs-GP1) with an average molecular weight (Mw) of 6.0 kDa was isolated and purified by column chromatography from the lower Mw fraction of exopolysaccharide (EPS) produced by a medicinal fungus Cordyceps sinensis Cs-HK1. Its carbohydrate moiety was mainly composed of glucose and mannose at 3.2:1.0 mole ratio, indicating an O-linked glycopeptide. The peptide chain contained relatively high mole ratios of aspartic acid, glutamic acid and glycine (3.3–3.5 relative to arginine) but relatively low ratios of tyrosine and histidine. The peptide chain sequence analyzed after trypsin digestion by LC-MS was KNGIFQFGEDCAAGSISHELGGFREFREFLKQAGLE. Cs-GP1 exhibited remarkable antioxidant capacity with a Trolox equivalent antioxidant capacity of 1183.8 μmol/g and a ferric reducing ability of 611.1 μmol Fe(II)/g, and significant protective effect against H2O2-induced PC12 cell injury at a minimum dose of 10 μg/mL. This is the first report on the structure and bioactivity of an extracellular glycopeptide from the Cordyceps species.
Cordyceps sinensis; glycopeptide; structure; antioxidant; cell protection
Postural tachycardia syndrome (POTS) is a heterogeneous disorder that creates challenges for treatment. Beta-blocker was one of the most commonly used drugs, but it is inconsistently effective. The purpose of this study is to explore whether orthostatic plasma norepinephrine level could be an indicator of therapeutic effectiveness of metoprolol for POTS in children.
Twenty-seven children with POTS were enrolled in our study. They received metoprolol treatment, and their orthostatic plasma norepinephrine levels were measured by high-performance liquid chromatography method. Three months after rmetoprolol treatment, 25 patients were followed up. A receiver-operating characteristic (ROC) curve was used to explore the predictive value of orthostatic plasma norepinephrine level.
The symptom severity and increment of heat rate from supine position to upright of patients positively correlated with their orthostatic plasma norepinephrine level (r = 0.599, P < 0.001; r = 0.633, P <0.001, respectively). Orthostatic plasma norepinephrine level in responders to metoprolol was significantly higher than that of nonresponders (P = 0.028). A ROC curve on the predictive value of orthostatic plasma norepinephrine level showed that the area under the curve was 0.785. Using a cutoff value for orthostatic plasma norepinephrine level of 3.59 pg/ml yielded both sensitivity (76.9%) and specificity (91.7%) in predicting the efficacy of metoprolol therapy for POTS.
Orthostatic plasma norepinephrine level of > 3.59 pg/ml was an indicator of the effectiveness of metoprolol therapy for POTS in children and adolescents.
Norepinephrine; Postural tachycardia syndrome; Metoprolol
The aim of the present study was to investigate the antioxidant and anti-inflammatory effects of Paeonia and Schisandra extracts in asthmatic rats. An ethanol extraction method was used to prepare the Schisandra and Paeonia extracts, and the levels of hydroxyl radical, total antioxidant activity and total phenolic content were detected. The rats were divided into three groups: Treatment (group A), model (group B) and control (group C). The treatment group received traditional Chinese antiasthmatic medicine (mixed extract, 2 ml/day) for 10 days. Levels of malondialdehyde (MDA), Cu-Zn-superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected in the serum, while interleukin (IL)-4, IL-6, interferon (IFN)-γ, IL-13 and IL-22 levels were analyzed in the serum, bronchoalveolar lavage fluid and lung tissue homogenates of the three groups. In addition, pathological changes of the tracheal tissues were observed via biopsies and the NF-κB p65 level was measured in the lung tissue using immunohistochemistry. Total antioxidant activity, hydroxyl radical levels and total phenolic content in the mixed herbal extracts were higher than those in the single herbal extracts. At day 5 following the treatment, the number of eosinophils was significantly reduced in the tracheal tissues. At day 10 following the treatment, the mucosa was significantly repaired. In vivo antioxidant levels revealed that the serum and erythrocyte SOD activity and GSH-Px were higher in group A as compared with group B, while the level of MDA in group A was lower than that in group B (P<0.05). The levels of serum and erythrocyte SOD activity and GSH-Px in group B were lower than those in group C, while the level of MDA in group B was higher than that in group C (P<0.05). IL-4, IL-6 and IL-13 levels in the serum, bronchoalveolar lavage fluid and lung tissue in group A were not significantly different from those in group B (P>0.05). However, IFN-γ levels in group A significantly increased as compared with the level in group B, while IL-22 levels decreased significantly in group A as compared with group B (P<0.05). IL-4, IL-6, IL-13 and IL-22 levels in the lung tissue, bronchoalveolar lavage fluid and serum in group B were significantly higher than those in group C. In addition, the IFN-γ level decreased significantly in group B as compared with the level in group C (P<0.05). Immunohistochemical analysis revealed that the protein expression of NF-κB p65 in group A was significantly lower compared with group B (P<0.05). Therefore, Paeonia and Schisandra extracts may be used to treat asthma through their in vivo antioxidant and anti-inflammatory effects.
Schisandra; Paeonia; asthma; antioxidant; anti-inflammatory
The regulatory mechanism of Indian hedgehog (IHH) in colorectal carcinogenesis has not been elucidated. In the current study, the expression of IHH were investigated in 7 digestive tract cancer cell lines, and in 10 normal colorectal mucosas (NCs), 30 hyperplastic polyps (HPs), 35 colorectal adenomas (ADs), and 40 colorectal adenocarcinomas (CAs) by semi-quantitative RT-PCR and immunohistochemical staining. Moreover, the mutational status of adenomatous polyposis coli (APC) and β-catenin in these tumors were analyzed by direct sequencing. IHH mRNA was lost in the 4 colon cancer cell lines harboring APC mutation. IHH mRNA was significantly decreased in CAs (0.17 ± 0.22), compared with that in ADs (0.38 ± 0.35) and HPs (0.56 ± 0.38, P < 0.05). IHH protein was expressed at a very low level or absent in both ADs (7.51 ± 11.92) and CAs (5.15 ± 9.21) in comparison to that in HPs (19.47 ± 17.91) and NCs (42.40 ± 13.67, P < 0.05). Moreover, APC mutations were negatively correlated with IHH mRNA expression (Spearman’s R = -0.636, P < 0.01) and IHH protein expression (Spearman’s R = -0.426, P < 0.01). In conclusion, down-regulation of IHH expression might be an early event during the carcinogenesis of colorectal cancer. The activation of Wnt signaling by APC mutation might contribute to the down-regulation or loss of IHH expression in colorectal tumors.
Indian hedgehog; Wnt signaling pathway; colorectal cancer; APC mutation
Measurement of the changes in intracellular Ca2+ levels is an important assay for drug discovery. In this report, we describe a novel Ca2+ indicator, dCys-GCaMP, based on the green fluorescent protein and the development of a rapid and simple cell-based functional assay using this new Ca2+ indicator. We demonstrated the sensitivity and reliability of the assay by measuring the cellular responses to the agonists, antagonists, channel blockers, and modulators of the ionotropic N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. HEK293 cells coexpressing the NMDA receptor and dCys-GCaMP displayed a strong increase in fluorescence intensity when stimulated with the agonist glutamate. This increase in the fluorescence signal was agonist concentration dependent and could be blocked by NMDAR antagonists and channel blockers. The pharmacological parameters measured with the dCys-GCaMP assay are in close agreement with those derived from conventional assays with synthetic dye fluo-4 and literature values. In addition, we showed that this assay could be used on G protein-coupled receptors as well, as exemplified by studies on the α1A adrenergic receptor. A limited scale evaluation of the assay performance in a 96-well compound screening format suggests that the dCys-GCaMP assay could be easily adapted to a high-throughput screening environment. The most important advantage of this new assay over the conventional fluo-4 and aequorin assays is the elimination of the dye-loading or substrate-loading process.
AIM: To investigate the gastric muscle injury caused by endoplasmic reticulum (ER) stress in rats with diabetic gastroparesis.
METHODS: Forty rats were randomly divided into two groups: a control group and a diabetic group. Diabetes was induced by intraperitoneal injection of 60 mg/kg of streptozotocin. Gastric emptying was determined at the 4th and 12th week. The ultrastructural changes in gastric smooth muscle cells (SMCs) were investigated by transmission electron microscopy. TdT-mediated dUTP nick end labeling (TUNEL) assay was performed to assess apoptosis of SMCs. Expression of the ER stress marker, glucose-regulated protein 78 (GRP78), and the ER-specific apoptosis mediator, caspase-12 protein, was determined by immunohistochemistry.
RESULTS: Gastric emptying was significantly lower in the diabetic rats than in the control rats at the 12th wk (40.71% ± 2.50%, control rats vs 54.65% ± 5.22%, diabetic rats; P < 0.05). Swollen and distended ER with an irregular shape was observed in gastric SMCs in diabetic rats. Apoptosis of gastric SMCs increased in the diabetic rats in addition to increased expression of GRP78 and caspase-12 proteins.
CONCLUSION: ER stress and ER stress-mediated apoptosis are activated in gastric SMCs in diabetic rats with gastroparesis.
Diabetic gastroparesis; Apoptosis; Endoplasmic reticulum stress; glucose-regulated protein 78 kD; Caspase-12
For cells seeded in scaffolds, transplanted cell survival rate plays an important role for cell transplantation efficiency, and is essential for successful cell transplantation. Fibroblast viability in HyStem-C was examined by a double staining Live/Dead Viability/Cytotoxicity assay, and cell images were analyzed using MetaMorph software for calculating live cell percentage for fresh and cryopreserved cells at different incubation time points, delivery methods, differing DMSO and cell concentrations. The results of this research demonstrated that in HyStem-C, the viability of cryopreserved cells (85%) was significantly lower than fresh collected cells (96.7%). In addition, the physical force from a 27 gauge needle significantly decreased frozen cell survival rates to 83-85% compared to pipette delivered cells. Higher DMSO concentration (1.0%) and higher cell density (2 × 107 per milliliter) also significantly decreased cell survival to 73%. Cryopreserved cell viability in three dimensional scaffolding can be maintained over 80% with cell density of 1 × 107 per milliliter, total DMSO concentration of 0.5%, and passed through a 27-gauge needle. These results demonstrate the viability of cells seeded in hyaluronan hydrogel with commonly used storage and delivery methods can bring rather satisfactory cell transplantation efficiency.
Salidroside, extracted from the root of Rhodiola rosea L, is known for its pharmacological properties, in particular its neuroprotective effects. 2-(4-Methoxyphenyl) ethyl-2-acetamido-2-deoxy-β-D- pyranoside (GlcNAc-Sal), an analog of salidroside, was recently synthesized and shown to possess neuroprotective properties. The purpose of the current study was to investigate the neuroprotective effects of GlcNAc-Sal against oxygen–glucose deprivation-reperfusion (OGD-R)-induced neurotoxicity in vitro and global cerebral ischemia-reperfusion (GCI-R) injury in vivo. Cell viability tests and Hoechst 33342 staining confirmed that GlcNAc-Sal pretreatment markedly attenuated OGD-R induced apoptotic cell death in immortalized mouse hippocampal HT22 cells. Western blot, immunofluorescence and PCR analyses revealed that GlcNAc-Sal pretreatment restored the balance of pro- and anti-apoptotic proteins and inhibited the activation of caspase-3 and PARP induced by OGD-R treatment. Further analyses showed that GlcNAc-Sal pretreatment antagonized reactive oxygen species (ROS) generation, iNOS-derived NO production and NO-related apoptotic cell death during OGD-R stimulation. GCI-R was induced by bilateral common carotid artery occlusion (BCCAO) and reperfusion in mice in vivo. Western blot analysis showed that GlcNAc-Sal pretreatment decreased the expression of caspase-3 and increased the expression of Bcl-2 (B-cell lymphoma 2)/Bax (Bcl-2-associated X protein) induced by GCI-R treatment. Our findings suggest that GlcNAc-Sal pretreatment prevents brain ischemia reperfusion injury by the direct or indirect suppression of cell apoptosis and GlcNAc-Sal could be developed as a broad-spectrum agent for the prevention and/or treatment of cerebral ischemic injury.
Mangiferin is a major bioactive ingredient in Mangifera indica Linn. (Anacardiaceae) leaves. Aqueous extract of such leaves have been used as an indigenous remedy for respiratory diseases like asthma and coughing in traditional Chinese medicine. However, underlying molecular mechanisms of mangiferin on anti-asthma remain unclear. In our present study, we investigated the anti-asthmatic effect of mangiferin on Th1/Th2 cytokine profiles and explored its underlying immunoregulatory mechanism in mouse model of allergic asthma. Mangiferin significantly reduced the total inflammatory cell counts and eosinophil infiltration, decreased the production of ovalbumin-specific IgE in serum and PGD2 in BALF. The antibody array analysis showed that mangiferin down-regulated the levels of one group of cytokines/chemokines including Th2-related IL-4, IL-5, IL-13, and others IL-3, IL-9, IL-17, RANTES, TNF-α, but simultaneously up-regulated Th1-related IFN-γ, IL-2 and IL-10 and IL-12 expression in serum. Thus it attenuates the imbalance of Th1/Th2 cells ratio by diminishing the abnormal mRNA levels of Th1 cytokines (IFN-γ and IL-12) and Th2 cytokines (IL-4, IL-5 and IL-13). Finally, mangiferin substantially inhibited the activation and expression of STAT-6 and GATA-3 in excised lung tissues. Our results suggest that mangiferin can exert anti-asthmatic effect. The underlying mechanism may attribute to the modulation of Th1/Th2 cytokine imbalance via inhibiting the STAT6 signaling pathway.
The gene for New Delhi metallo-β-lactamase 1 (NDM-1) has been reported to be transmitted via plasmids which are easily transferable and capable of wide distribution. We report the isolation of two NDM-1 producing strains and possible in vivo transfer of blaNDM-1 in a patient.
Clinical samples were collected for bacterial culture and antibiotic susceptibility testing from a patient during a 34-day hospitalization. The presence of blaNDM-1 was detected by PCR and sequencing. Plasmids of interest were sequenced. Medical records were reviewed for evidence of association between the administration of antibiotics and the acquisition of the NDM-1 resistance.
A NDM-1 positive Raoultella planticola was isolated from blood on the ninth day of hospitalization without administration of any carbapenem antibiotics and a NDM-1 positive Escherichia coli was isolated from feces on the 29th day of hospitalization and eight days after imipenem administration. The blaNDM-1 was carried by a 280 kb plasmid pRpNDM1-1 in R. planticola and a 58 kb plasmid pEcNDM1-4 in E. coli. The two plasmids shared a 4812 bp NDM-1-ISCR1 element which was found to be excisable from the plasmid as a free form and transferrable in vitro to a NDM-1 negative plasmid from E. coli.
blaNDM-1 was embedded in an ISCR1 complex class 1 integron as a novel 4812 bp NDM-1-ISCR1 element. The element was found to be able to self excise to become a free form, which may provide a new vehicle for NDM-1 dissemination. This mechanism could greatly accelerate the spread of NDM-1 mediated broad spectrum β-lactam resistance.
Rosai-Dorfman disease (RDD) is a rare, benign pseudolymphomatous condition, predominantly involving lymph nodes. Rosai-Dorfman disease (RDD) (sinus histiocytes with massive lymphadenopathy) rarely affects the intracranial region without involvement of other sites. It is a rare and idiopathic histoproliferation disorder characterized by painless lymphadenopathy. We report a case of 43-year-old male who presented with unconsciousness; MRI was done and right temporofrontal mass was found. Excision was done, and on histopathology it confirmed RDD.
Little is known about the clinical features and treatment of Chinese patients with Parkinson disease (PD).
A large cross-sectional survey of clinical features, medication use, and motor complications was conducted in 901 consecutive PD patients, from 42 randomly selected university-affiliated hospitals in four urban economic regions of China, between December 2006 and May 2007.
The 901 PD patients had age range 30 to 88, and median disease duration 50 months. Most (737, 81.8%) used L-dopa (median 375 mg/day), and often added low doses of other antiparkinsonian agents. Among L-dopa-treated patients, the prevalence of motor complications was low (dyskinesias: 8.5%; motor fluctuations: 18.6%), even among patients with disease duration ≥11 years (dyskinesias: 18.1%; motor fluctuations: 42.2%). Higher L-dopa use was associated with higher occurrence of dyskinesias (OR 2.44; 95% CI 1.20-5.13) and motor fluctuations (OR 2.48; 95% CI 1.49-4.14). Initiating PD treatment with L-dopa alone (OR 0.46; 95% CI 0.22-0.95) or in combination with other medications (OR 0.41; 95% CI 0.19-0.87) was associated with less dyskinesia than treatment initiated with non-L-dopa medication.
Many Chinese PD patients are treated with low-dose L-dopa and added low-dose antiparkinsonian agents, with a low prevalence of motor complications, which might be influenced by Chinese culture.
Parkinson disease; Treatment, Dyskinesias; Motor fluctuations, China
The title molecule, C24H25NOSi, is a hydrolysis product of the reaction between 9-trimethylsilyfluorenyl lithium and 2-methoxybenzonitrile. The fluorene ring system is substantially planar, with an r.m.s. deviation of 0.0288 Å from the best-fit plane through its 13 C atoms. This plane forms a dihedral angle of 58.07 (7)° with the 2-methoxybenzylamine ring plane. In the crystal, molecules are linked by N—H⋯π and C—H⋯π interactions, which leads to the formation of two-dimensional network lying parallel to the bc plane.
Transforming growth factor-β1 (TGF-β1), an important cytokine with multiple functions, is secreted during wound healing. Previous studies have utilized two-dimensional (2D) cell culture to elucidate the functions of TGF-β1; however, 2D culture does not represent the complex three-dimensional (3D) in vivo environment. Using a synthetic hyaluronan (HA) extracellular matrix (ECM) hydrogel, we investigated the effect of TGF-β1 on fibroblasts cultured in three conditions—on tissue culture polystyrene (TCP), on HA (2D), and in HA (3D). After TGF-β1 treatment (0.1 to 20 ng/mL), morphological features and ECM regulation were analyzed by immunocytochemistry, Western blot, quantitative polymerase chain reaction, and zymogram assays. On TCP, cells showed the typical spindle shape with strong alpha smooth muscle actin (α-SMA) staining of cytoplasmic myofilaments along the cell axes after TGF-β1 treatment; on HA (2D), spindle-shape cells showed little α-SMA staining; in HA (3D), cells were smaller and rounded with less α-SMA deposition. The α-SMA gene and protein expression on TCP were significantly upregulated by TGF-β1, but TGF-β1 did not induce α-SMA expression in the presence of HA (both 2D and 3D). 3D HA culture significantly downregulated collagen I, III, and fibronectin expression, increased matrix metalloproteinase 1 and 2 (MMP1/MMP2) activity, upregulated MMP1 mRNA and downregulated TIMP3 mRNA expression. This study suggested that exogenous HA, particularly in 3D culture, appears to suppress ECM production, enhances ECM degradation and remodeling, and inhibits myofibroblast differentiation without decreasing TGF-β receptor expression.
In the social sciences, computer-based modeling has become an increasingly important tool receiving widespread attention. However, the derivation of the quantitative relationships linking individual moral behavior and social morality levels, so as to provide a useful basis for social policy-making, remains a challenge in the scholarly literature today. A quantitative measurement of morality from the perspective of complexity science constitutes an innovative attempt. Based on the NetLogo platform, this article examines the effect of various factors on social morality levels, using agents modeling moral behavior, immoral behavior, and a range of environmental social resources. Threshold values for the various parameters are obtained through sensitivity analysis; and practical solutions are proposed for reversing declines in social morality levels. The results show that: (1) Population size may accelerate or impede the speed with which immoral behavior comes to determine the overall level of social morality, but it has no effect on the level of social morality itself; (2) The impact of rewards and punishment on social morality levels follows the “5∶1 rewards-to-punishment rule,” which is to say that 5 units of rewards have the same effect as 1 unit of punishment; (3) The abundance of public resources is inversely related to the level of social morality; (4) When the cost of population mobility reaches 10% of the total energy level, immoral behavior begins to be suppressed (i.e. the 1/10 moral cost rule). The research approach and methods presented in this paper successfully address the difficulties involved in measuring social morality levels, and promise extensive application potentials.
Inhibition of BACE1 to prevent brain Aβ peptide formation is a potential disease-modifying approach to the treatment of Alzheimer’s disease. Despite over a decade of drug discovery efforts, the identification of brain-penetrant BACE1 inhibitors that substantially lower CNS Aβ levels following systemic administration remains challenging. In this report we describe structure-based optimization of a series of brain-penetrant BACE1 inhibitors derived from an iminopyrimidinone scaffold. Application of structure-based design in tandem with control of physicochemical properties culminated in the discovery of compound 16, which potently reduced cortex and CSF Aβ40 levels when administered orally to rats.
BACE1; inhibitor; Alzheimer’s disease; Aβ40; iminopyrimidinone; X-ray crystallography
Inhibition of BACE1 to prevent brain Aβ peptide formation
is a potential disease-modifying approach to the treatment of Alzheimer’s
disease. Despite over a decade of drug discovery efforts, the identification
of brain-penetrant BACE1 inhibitors that substantially lower CNS Aβ
levels following systemic administration remains challenging. In this
report we describe structure-based optimization of a series of brain-penetrant
BACE1 inhibitors derived from an iminopyrimidinone scaffold. Application
of structure-based design in tandem with control of physicochemical
properties culminated in the discovery of compound 16, which potently reduced cortex and CSF Aβ40 levels when administered
orally to rats.
BACE1; inhibitor; Alzheimer’s disease; Aβ40; iminopyrimidinone; X-ray crystallography
ZNF24 is a member of the SCAN domain family of Krüppel-like zinc finger (ZF) transcription factors, which plays a critical role in cell proliferation and differentiation. However, how ZNF24 enters the nucleus in order to exert its function remains unclear since its nuclear localization signal(s) (NLS) has not been identified. Here, we generated a series of GFP-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to ZF1-2. Deletion of ZF1-2 caused cytoplasmic accumulation of ZNF24. Fusion of the ZF1-2 to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the ZF1-2 is both necessary and sufficient for nuclear localization. ZNF24 containing histidine to leucine mutations that disrupt the structure of ZF1 or/and ZF2 retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. K286A and R290A mutation led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that ZNF24 interacted with importin-β and this interaction required the ZF motifs. The β-Catenin (CTNNB1) luciferase assays showed that the ZNF24 mutants defective in nuclear localization could not promote CTNNB1promoter activation as the wild-type ZNF24 did. Taken together, these results suggest that consecutive ZF1-2 is critical for the regulation of ZNF24 nuclear localization and its transactivation function.
Mycobacterium tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a dominant target antigen for cell-mediated immunity in the early phase of tuberculosis. The fms-like tyrosine kinase 3 ligand (FL) that induces potent immune response has been used as an adjuvant in vaccine development. In this study, a new recombinant plasmid (pIRES-epitope-peptides-FL) encoding three T cell epitopes of ESAT-6 and FL was constructed, and the immunogenicity of the DNA vaccine was assessed in C57BL/6 mice immunized with the plasmid DNA vaccine. Additionally, a strategy of intramuscular injection with the DNA vaccine (prime) and intranasal administration of the epitope peptides (boost) was employed to induce higher immune reaction of the mice. The results showed that mice vaccinated with the recombinant plasmid DNA vaccine and boosted with the peptides not only increased the levels of Th1 cytokines (IFN-γ and IL-12), the number of IFN-γ+ T cells and activities of cytotoxic T lymphocytes as well as IgG, but also enhanced protection against Mycobacterium tuberculosis challenge. In conclusion, these data indicate that the novel recombinant pIRES-epitope-peptides-FL plasmid is a useful DNA vaccine for preventing Mycobacterium tuberculosis infection.
early secretory antigenic target-6 (ESAT-6); fms-like tyrosine kinase 3 ligand (FL); Mycobacterium tuberculosis; recombinant plasmid; T cell epitopes
The expression of vascular endothelial growth factor (VEGF) is regulated by microenvironmental factors within the tumors, such as hypoxia, free radicals, pH imbalance and nutrient deficiency. The purpose of this study was to observe VEGF activity in tumor cells under different stress conditions. A plasmid was generated, consisting of green fluorescent protein (GFP) fused to a 1,217-bp sequence, which was located downstream and upstream of the transcriptional start site of VEGF, respectively. The plasmid was stably transfected into 4T1 mouse breast carcinoma cells. Cells were cultured in a medium with nitric oxide (NO) donor sodium nitroprusside (SNP), hypoxia-mimetic agent deferoxamine mesylate (DFX), H2O2, absence of serum and lowered or elevated pH, or were heat-shocked, followed by measurement of VEGF activity by reverse transcription polymerase chain reaction (RT-PCR) and ELISA. Hypoxia, SNP and H2O2 led to increments of VEGF mRNA and protein expression, as well as of GFP expression. The pH alterations, serum deprivation and heat shock reduced VEGF mRNA expression, but had little effect on GFP expression. The results demonstrated that VEGF expression may be influenced by a number of microenvironmental factors and these factors may play important roles in regulating VEGF expression during tumorigenesis.
vascular endothelial growth factor promoter; tumor cells; microenvironment; free radical
Our objective was to dynamically observe changes in peripheral blood Th17, Treg cells, and interleukin (IL)-17 levels in HIV-1/AIDS patients before and after highly active antiretroviral therapy (HAART). The study design consisted of a randomized case-controlled study. A total of 33 HIV-1/AIDS patients were chosen to receive a HAART regimen and 30 healthy volunteers were assigned as controls. Peripheral blood Th17 and Treg cells were measured by flow cytometry before or 6 and 12 months after HAART therapy. The plasma IL-17 level was determined by ELISA. The percentage of Th17 cells to total CD4+ cells was 1.2±0.37% in HIV/AIDS patients before treatment, which was significantly lower than that in uninfected controls (4.7±1.43%). After HAART therapy for 6 or 12 months, the Th17 percentage increased to 2.5±1.03% and 3.7±1.56%, respectively. The percentage of Treg cells to CD4+ cells is 9.16±3.33% in HIV/AIDS patients, which was significantly elevated compared to controls (4.43±0.97%). HAART therapy for 6 and 12 months significantly decreased Treg cell percentage (7.19±2.91% and 5.53±1.88%, respectively). Interestingly, the ratio of Th17/Treg cells was significantly decreased in HIV/AIDS patients before treatment, while HAART treatment partially normalized the Th17/Treg ratio. IL-17 levels were 5.3±2.5 and 17.7±6.60 pg/ml in HIV/AIDS patients and controls, respectively; the HAART regimen increased the IL-17 level to 7.7±2.4 and 10.4±3.1 pg/ml at 6 and 12 months, respectively. The percentage of Th17 cells correlated with IL-17 level, but both negatively correlated with viral load before treatment, whereas the percentage of Treg cells positively correlated with viral load before HAART therapy. The imbalance of peripheral blood Th17 and Treg cells may play a crucial role in the pathogenesis of AIDS. HAART can restore the balance of Th17 and Treg cells as well as the IL-17 level, which may gradually rebuild the immune equilibrium in HIV/AIDS patients.
Historically, the incidence of gentamicin resistance in Campylobacter has been very low, but recent studies reported a high prevalence of gentamicin-resistant Campylobacter isolated from food-producing animals in China. The reason for the high prevalence was unknown and was addressed in this study. PCR screening identified aminoglycoside resistance genes aphA-3 and aphA-7 and the aadE–sat4–aphA-3 cluster among 41 Campylobacter isolates from broiler chickens. Importantly, a novel genomic island carrying multiple aminoglycoside resistance genes was identified in 26 aminoglycoside resistant Campylobacter coli strains. Sequence analysis revealed that the genomic island was inserted between cadF and COO1582 on the C. coli chromosome and consists of 14 open reading frames (ORFs), including 6 genes (the aadE–sat4–aphA-3 cluster, aacA-aphD, aac, and aadE) encoding aminoglycoside-modifying enzymes. Analysis by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing indicated that the C. coli isolates carrying this unique genomic island were clonal, and the clone of PFGE subtype III and sequence type (ST) 1625 was particularly predominant among the C. coli isolates examined, suggesting that clonal expansion may be involved in dissemination of this resistance island. Additionally, we were able to transfer this genomic island from C. coli to a Campylobacter jejuni strain using natural transformation under laboratory conditions, and the transfer resulted in a drastic increase in aminoglycoside resistance in the recipient strain. These findings identify a previously undescribed genomic island that confers resistance to multiple aminoglycoside antibiotics. Since aminoglycoside antibiotics are used for treating occasional systemic infections caused by Campylobacter, the emergence and spread of this antibiotic resistance genomic island represent a potential concern for public health.