MicroRNA-138 Regulates Spinal Cord Development by Activating the Shh in Fetal Rats.

INTRODUCTION: Dysregulation of spinal cord development can lead to serious neuronal damage and dysfunction, causing significant health problems in newborns. MiRNA-138 appears to be crucial for proliferation, differentiation, and apoptosis of cells. However, the regulation of miRNA-138 and downstream molecules in embryonic spinal cord development remain elusive. The aim of this experiment is to determine whether overexpression of miRNA-138 or RNA interference (RNAi) can regulate the development of spinal cord in fetal rats. METHODS: Two plasmid vectors including pLenti-III-mico-GFP (miRNA-138 open reading frame [ORF]) and pLenti-III-miR-Off (miRNA-138 short hairpin) were constructed and injected into the tail vein of rats on the 14th day of pregnancy. Hematoxylin-eosin (HE) staining was used to observe the cell morphology. QRT-PCR, Western blot, and immunostaining confirmed the regulatory relationship between miRNA-138 and downstream molecules sonic hedgehog (Shh). RESULTS: Overexpression of miRNA-138 increased neuron regeneration significantly and decreased neuronal apoptosis when compared with the control. Silencing of miRNA-138 increased neuronal apoptosis and spinal cord atrophy significantly. Furthermore, miRNA-138 ORF treatment effectively increased the expression level of miRNA-138 and also upregulated the level of Shh. Comparatively, knockdown of miRNA-138 downregulated Shh levels in myelodysplastic regions. CONCLUSION: These findings indicated that miRNA-138 overexpression could protect the spinal cord development of fetal rats, and the underlying mechanisms were associated with Shh expression. The present study provides a novel strategy to promote the molecular mechanism of embryonic spinal cord development.

Sulfation predominates the pharmacokinetics, metabolism, and excretion of forsythin in humans: major enzymes and transporters identified.

Forsythin extracted from Forsythiae Fructus is widely used to treat fever caused by the common cold or influenza in China, Japan and Korea. The present study aimed to analyze the pharmacokinetics, metabolism and excretion routes of forsythin in humans and determine the major enzymes and transporters involved in these processes. After a single oral administration, forsythin underwent extensive metabolism via hydrolysis and further sulfation. In total, 3 of the 13 metabolites were confirmed by comparison to reference substances, i.e., aglycone M1, M1 sulfate (M2), and M1 glucuronide (M7). Hydrolysis was the initial and main metabolic pathway of the parent compound, followed by extensive sulfation to form M2 and a reduced level of glucuronidation to form M7. In addition, the plasma exposure of M2 and M7 were 86- and 4.2-fold higher than that of forsythin. Within 48 h, ~75.1% of the administered dose was found in urine, with M2 accounting for 71.6%. Further phenotyping experiments revealed that sulfotransferase 1A1 and UDP-glucuronosyltransferase 1A8 were the most active hepatic enzymes involved in the formation of M2 and M7, respectively. The in vitro kinetic study provided direct evidence that M1 showed a preference for sulfation. Sulfated conjugate M2 was identified as a specific substrate of organic anion transporter 3, which could facilitate the renal excretion of M2. Altogether, our study demonstrated that sulfation dominated the metabolism and pharmacokinetics of forsythin, while the sulfate conjugate was excreted mainly in the urine.

Synthesis, biological evaluation and molecular docking of novel 5-phenyl-1H-pyrazol derivatives as potential BRAF(V600E) inhibitors.

The RAF-MEK-ERK cascade appears to be intimately involved in the regulation of cell cycle progression and apoptosis. The BRAF(V600E) mutant results in constitutive activation of the ERK pathway, which can lead to cellular growth dysregulation. A series of 5-phenyl-1H-pyrazol derivatives (3a-5e) have been designed and synthesized, and their biological activities were evaluated as potential BRAF(V600E) inhibitors. All the compounds were reported for the first time except 3e, and compound 1-(4-bromo-2-hydroxybenzyl)-3-phenyl-1-(5-phenyl-1H-pyrazol-3-yl)urea (5c) displayed the most potent inhibitory activity (BRAF(V600E) IC50 = 0.19 µM). Antiproliferative assay results indicated that compound 5c possessed high antiproliferative activity against cell lines WM266.4 and A375 in vitro, with IC50 values of 1.50 and 1.32 µM, respectively, which were comparable with the positive control vemurafenib. Docking simulations showed that compound 5c binds tightly to the BRAF(V600E) active site and acts as BRAF(V600E) inhibitor. A 3D-QSAR model was also built to provide more pharmacophore understanding towards designing new agents with more potent BRAF(V600E) inhibitory activity.

Synthesis, biological evaluation and 3D-QSAR studies of novel 4,5-dihydro-1H-pyrazole niacinamide derivatives as BRAF inhibitors.

A series of novel 4,5-dihydropyrazole derivatives containing niacinamide moiety as potential V600E mutant BRAF kinase (BRAF(V600E)) inhibitors were designed and synthesized. Results of the bioassays against BRAF(V600E) and WM266.4 human melanoma cell line showed several compounds to be endowed potent activities with IC(50) and GI(50) value in low micromolar range, among which compound 27e, (5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)6-methylpyridin-3-yl methanone (IC(50)=0.20 µM, GI(50)=0.89 µM) was bearing the best bioactivity comparable with the positive control Sorafenib. Docking simulation was performed to determine the probable binding model and 3D-QSAR model was built to provide more pharmacophore understanding that could use to design new agents with more potent BRAF(V600E) inhibitory activity.

Dual functional amphiphilic sugar-coated AIE-active fluorescent organic nanoparticles for the monitoring and inhibition of insulin amyloid fibrillation based on carbohydrate-protein interactions.

Amyloid-related diseases, such as Alzheimer's disease, are all considered to be related to the deposition of amyloid fibrils in the body. Insulin is a protein hormone that easily undergoes aggregation and fibrillation to form more toxic amyloid-like fibrils. So far, it is still challenging to develop a new protocol to study the ex situ detection and in situ inhibition of amyloid fibrillation. Here, we reported a modular synthetic strategy to construct nine amphiphilic sugar-coated AIE-active fluorescent organic nanoparticles (FONs, TPE2/3/4X, X = G, M or S) with glucosamine (G), mannose (M) or sialic acid (S) as a hydrophilic moiety and tetraphenylethylene (TPE) as a hydrophobic AIE core. The carbohydrate-protein interactions between insulin and TPE2/3/4X were investigated by fluorescence spectroscopy, circular dichroism spectroscopy and transmission electron microscopy. Among the nine FON AIEgens, TPE2G was screened out as the best dual functional FON for the ex situ detection and in situ inhibition of the insulin fibrillation process, indicating that the glycosyl moiety exhibited a crucial effect on the detection/inhibition of insulin fibrillation. The molecular dynamics simulation results showed that the binding mechanism between TPE2G and native insulin was through weak interactions dominated by van der Waals interactions and supplemented by hydrogen bonding interactions to stabilize an α-helix of the insulin A chain, thereby inhibiting the insulin fibrillation process. This work provides a powerful protocol for the further research of amyloid-related diseases based on carbohydrate-protein interactions.

3,5-Dibromo-4-oxo-2,2,6,6-tetra-methyl-piperidin-1-yl oxide.

In the title compound, C(9)H(14)Br(2)NO(2), the substituted ring exhibits a chair conformation. A crystallographic mirror plane, passing through the N atom, the O atoms and the C atom in the 4-position, bis-ects the mol-ecule.

Simultaneous determination of forsythin and its major metabolites in human plasma via liquid chromatography-tandem mass spectrometry.

Forsythiae suspensa is widely used in China as a traditional Chinese medicine. Forsythin is extracted from Forsythiae Fructus and has undergone phase II clinical trials as an antipyretic drug in China. The main metabolites of forsythin in human plasma are aglycone sulfate (KD-2-SO3H) and aglycone glucuronide (KD-2-Glc). In the present study, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the simultaneous analysis of forsythin, KD-2-Glc, and KD-2-SO3H, in human plasma. After precipitating proteins with methanol, these three analytes were separated on a Gemini-C18 column along with teniposide as an internal standard. Mass spectrometry detection, under multiple reaction monitoring, was then carried out in negative mode using the Triple Quad™ 6500+ LC-MS/MS system coupled with an electrospray ionization (ESI) ion source. The transitions of m/z 371.1→356.1 for forsythin, m/z 547.2→356.0 for KD-2-Glc and m/z 451.2→356.2 for KD-2-SO3H were chosen to effectively maintain the balance between selectivity and sensitivity. The developed method was linear over the following concentrations in human plasma samples: 1.00-1000 ng/mL for forsythin, 2.50-2500 ng/mL for KD-2-Glc, and 5.00-5000 ng/mL for KD-2-SO3H. Assays were validated and satisfied the acceptance criteria recommended by the CFDA guidance. Furthermore, this LC-MS/MS method was successfully implemented in a Phase I, first-in-human, dose-escalation pharmacokinetic study among Chinese healthy participants after single oral administration of forsythin tablets.

Water-soluble Glucosamine-coated AIE-Active Fluorescent Organic Nanoparticles: Design, Synthesis and Assembly for Specific Detection of Heparin Based on Carbohydrate-Carbohydrate Interactions.

Two water-soluble carbohydrate-coated AIE-activate fluorescent organic nanoparticles TPE3G and TPE4G were designed and synthesized for the detection of heparin. Different from the reported strategy, we not only utilized the general detection mechanism of electrostatic interactions, but also introduced the concept of carbohydrate-carbohydrate interactions (CCIs) to enrich the detection mechanism of heparin. TPE3G can serve as an efficient "turn-on" probe with higher selectivity towards heparin than TPE4G. TEM studies revealed that the micro-aggregated TPE3G was encapsulated with the heparin chain to form a complex self-assemblied composite and emits strong fluorescence. It is believed that the results illustrated in this study provide a novel strategy based on CCls to design water-soluble and more efficient bio-probes for various biological and clinical applications.

Aggregation-Induced-Emission Materials with Different Electric Charges as an Artificial Tongue: Design, Construction, and Assembly with Various Pathogenic Bacteria for Effective Bacterial Imaging and Discrimination.

Imaging-based total bacterial count and type identification of bacteria play crucial roles in clinical diagnostics, public health, biological and medical science, and environmental protection. Herein, we designed and synthesized a series of tetraphenylethenes (TPEs) functionalized with one or two aldehyde, carboxylic acid, and quaternary ammonium groups, which were successfully used as fluorescent materials for rapid and efficient staining of eight kinds of representative bacterial species, including pathogenic bacteria Vibrio cholera, Klebsiella pneumoniae, and Listeria monocytogenes and potential bioterrorism agent Yersinia pestis. By comparing the fluorescence intensity changes of the aggregation-induced-emission (AIE) materials before and after bacteria incubation, the sensing mechanisms (electrostatic versus hydrophobic interactions) were simply discussed. Moreover, the designed AIE materials were successfully used as an efficient artificial tongue for bacteria discrimination, and all of the bacteria tested were identified via linear discriminant analysis. Our current work provided a general method for simultaneous broad-spectrum bacterial imaging and species discrimination, which is helpful for bacteria surveillance in many fields.

Upregulation of miR-98 Inhibits Apoptosis in Cartilage Cells in Osteoarthritis.

OBJECTIVE: We aimed to investigate the effects of microRNA-98 (miR-98) on apoptosis in cartilage cells of osteoarthritis (OA) patients. METHODS: Knee cartilage tissue samples were collected from 31 OA patients, 21 autopsies, and 26 amputation patients due to trauma. The clinicopathological data were recorded. Quantitative real-time polymerase chain reaction was performed to compare the miR-98 expression levels from cartilage cells obtained from the OA and non-OA patients. Clinicopathological characteristics of the patients were also analyzed. Primary chondrocytes were separated from cartilage tissues and transfected with plasmids or siRNA to overexpress or inhibit miR-98. Annexin V-PI double staining and TUNEL assays were used to examine apoptosis in the primary chondrocytes after transfection. Finally, a rat OA model was used to confirm the effects of miR-98 on apoptosis in cartilage cells in vivo. RESULTS: Compared with the normal cartilage tissues, miR-98 expression was reduced in the OA cartilage tissues (p < 0.01). The miR-98 expression levels were also significantly correlated with the OA stage (p < 0.05). In vitro, transfection with the miR-98 inhibitor increased apoptosis in the cartilage cells (p < 0.05), and transfection with a miR-98 mimic inhibited apoptosis in cartilage cells (p < 0.05). In the OA rat model, exogenous injection of the miR-98 mimic inhibited apoptosis in the rat cartilage cells thus alleviating OA. CONCLUSION: MiR-98 expression is reduced in the cartilage cells of OA patients and the overexpression of miR-98 inhibits cartilage cell apoptosis, while inhibition of microRNA-98 leads to cartilage cell apoptosis. These findings provide a theoretical basis for the development of novel targeted therapies for OA.

A dialdehyde-diboronate-functionalized AIE luminogen: design, synthesis and application in the detection of hydrogen peroxide.

A dialdehyde-diboronate-functionalized tetraphenylethene (TPE-DABF) was reported as a H2O2-specific AIE luminogen. TPE-DABF, bearing multiple reductive units (aldehyde, boronate and fructose) in one molecule, afforded an excellent H2O2 selectivity over other ROS in biological buffer, and can be used for sensitive detection of glucose under neutral conditions.

A mass vaccination campaign targeting adults and children to prevent typhoid fever in Hechi; expanding the use of Vi polysaccharide vaccine in southeast China: a cluster-randomized trial.

BACKGROUND: One of the goals of this study was to learn the coverage, safety and logistics of a mass vaccination campaign against typhoid fever in children and adults using locally produced typhoid Vi polysaccharide (PS) and group A meningococcal PS vaccines in southern China. METHODS: The vaccination campaign targeted 118,588 persons in Hechi, Guangxi Province, aged between 5 to 60 years, in 2003. The study area was divided into 107 geographic clusters, which were randomly allocated to receive one of the single-dose parenteral vaccines. All aspects regarding vaccination logistics, feasibility and safety were documented and systematically recorded. Results of the logistics, feasibility and safety are reported. RESULTS: The campaign lasted 5 weeks and the overall vaccination coverage was 78%. On average, the 30 vaccine teams gave immunizations on 23 days. Vaccine rates were higher in those aged < or = 15 years (90%) than in adolescents and young adults (70%). Planned mop-up activities increased the coverage by 17%. The overall vaccine wastage was 11%. The cold chain was maintained and documented. 66 individuals reported of adverse events out of all vaccinees, where fever (21%), malaise (19%) and local redness (19%) were the major symptoms; no life-threatening event occurred. Three needle-sharp events were reported. CONCLUSION: The mass immunization proved feasible and safe, and vaccine coverage was high. Emphasis should be placed on: injection safety measures, community involvement and incorporation of mop-up strategies into any vaccination campaign. School-based and all-age Vi mass immunizations programs are potentially important public health strategies for prevention of typhoid fever in high-risk populations in southern China.

Design, synthesis and biological evaluation of novel (E)-α-benzylsulfonyl chalcone derivatives as potential BRAF inhibitors.

Activating mutations in the BRAF serine/threonine kinase are found in more than 70% of human melanomas, >90% of which are BRAF(V600E). It provides new therapeutic opportunities in malignant melanoma. In silico and in vitro screening of our compound collection has identified Hit 2 as BRAF(V600E) inhibitor. Based on its structure, a series of novel (E)-α-benzylsulfonyl chalcone derivatives (13-40) were designed and synthesized. Compound 38 exhibited the most potent inhibitory activity with an IC(50) value of 0.17 µM for BRAF(V600E) and GI(50) value of 0.52 µM for mutant BRAF-dependent cells. The results of cell based pERK activity and cellular selectivity suggested that those compounds could selectively inhibit proliferation of mutant BRAF-dependent melanoma cell line through inhibition of oncogenic BRAF.

Genistein and its synthetic analogs as anticancer agents.

Genistein, one of the predominant isoflavones derived from soybeans, has been shown beneficial effects in cancer prevention and treatment. There is an accumulating body of experimental evidences suggesting that genistein affects cancer progression by increasing apoptosis, inducing cell cycle arrest,modulating intracellular signaling pathways, and inhibiting invasion and metastasis of cancer cells. During last decade, many researchers have conducted extensive studies by synthesizing amounts of structurally-modified derivatives based on the isoflavone skeleton of genistein to enhance its anticancer activity, some analogs of which possess more potent activities of the prevention and/or treatment of various cancers. In this review, we summarized the current knowledge regarding anticancer effects,structure-activity relationships and action mechanisms of genistein and its synthesized analogs, which would be beneficial to the rational design of new genistein derivatives as anticancer drugs.

[Effectiveness of an immunization campaign with group A and C meningococcal polysaccharide vaccine in controlling an outbreak of group C meningococcal disease].

OBJECTIVE: To assess the safety, immunogenicity and efficacy of group A and C meningococcal polysaccharide vaccine (A/C MPV) in response to an outbreak of group C meningococcal disease. METHODS: A vaccination campaign with A/C MPV was prompted 6 weeks after the use of group A MPV in Laibin city, Guangxi, where an outbreak of group C meningococcal meningitis occurred in 2002. Vaccinees were observed for local and systemic reactions after the vaccination and followed up for the meningococcal disease for 5 years. Blood samples were collected from 71 people in the epidemic and 43 in the non-epidemic areas before and 1 month after the vaccination and examined by ELISA to detect IgG antibodies to group A and C polysaccharides. RESULTS: The vaccination coverage was 97%. No significant adverse reactions were observed. The positive rates of group C antibodies after vaccination was between 97.67% and 100% among the populations in the epidemic and non-epidemic areas, as well as among those negative and positive for group C antibodies prior to the vaccination. The geometric mean anti-C concentrations ranged 30.81 microg/ml to 37.44 microg/ml, showing no significant difference between groups. The incidence rate of meningococcal disease in students with timely immunization (218.58/100,000) dropped by 69.02%, when compared to that in those with delayed immunization (705.72/100,000). No clinical cases were identified during the follow-up period of 15,760 person-years. CONCLUSION: The vaccination campaign with the Chinese group A/C MPV seemed successful in controlling the group C meningococcal outbreak. The vaccine was shown to be safe even administered after the group A vaccine only 6 weeks apart. It could induce high levels of antibodies in vulnerable population and significantly increase antibody levels in seropositive individuals, thus providing a protection of at least 5 years.

[Application of cluster randomization method on typhoid Vi vaccine trails].

OBJECTIVE: To describe the design and application of cluster randomized controlled method on typhoid Vi vaccine trial, and to assess the effect of implementation. METHODS: Simple size calculation of cluster-randomized trial was used to determine the sample size of the two groups and a vaccination campaign was conducted. The study group was given typhoid Vi vaccine and the control group was given meningococcal A vaccine. RESULTS: According to sample size calculation, a total sample of 96,121 participants was required and the study areas were divided into 108 clusters. In practice, 53 study clusters with 44,054 participants and 54 control clusters with 48,422 participants were stratified and matched according to size, location (urban or rural), characteristics (school, department, factory, demography) were randomized respectively. Confounding factors of two groups including age, sex, resident area, income, level of education were compared. It was found that the ratio of all confounding factors between the two groups were comparable and balanced. CONCLUSION: Confounding factors can be better controlled between study group and the control group by applying cluster-randomized method on vaccine trail which enabled the intervention to be more scientifically evaluated; The implementation of cluster randomization trial was simple and easy to be accepted.

[Epidemiologic surveillance for bacterial meningitis in 140 000 children under 5 years of age in Nanning district, Guangxi province].

OBJECTIVE: To characterize the incidence, epidemiologic features, etiologic agents and sequelae of bacterial meningitis in children under 5 years of age in Nanning, Guangxi. METHODS: A population-based surveillance was conducted to evaluate children with signs and symptoms of meningitis. All hospitals, township health centers and village clinics in the surveillance area were structured to participate in the case referral and evaluation. Cerebrospinal fluid (CSF) and blood specimens were obtained and processed using standardized microbiologic methods. RESULTS: During the 26-month surveillance period, among the children under 5 years old, a total of 1272 cases who met the screening criteria of meningitis were studied. 265 of 1272 cases were identified as clinically diagnosed meningitis, with an incidence rate of 86.36 per 100 000 population. The annual incidence rate under the 38 cases of confirmed bacterial meningitis appeared to be 12.38/100 000. Staphylococcus species accounted for the largest proportion of laboratory-confirmed bacterial meningitis, followed by E. coli and S. pneumoniae. The highest attack rate occurred in neonates < 1 month, followed by children aged 1 - 12 months in the confirmed patients. Meningitis caused by Sp and Hi mainly occurred in children aged 1 - 12 months. All cases of meningitis due to Hi and Sp were children aged 1 - 24 months. 13.16% and 0.00% of the cases survived with complications and sequelae, and the case-fatality rate was 18.42%. 40 bacterial isolates were identified from 1193 blood cultures and 23 from 1211 cerebrospinal fluid samples, but no Neisseria meningitidis was found. CONCLUSION: Meningitis due to Hi was first confirmed in Guangxi with the incidence of 0.98 per 100 000 population. The annual incidence rate of confirmed bacterial meningitis was 12.38 per 100 000, which was considered an important public health problem in children. Staphylococci was the predominant pathogen in confirmed bacterial meningitis.