Fluorescent immunochromatography of vancomycin in plasma: A rapid and sensitive quantitative analysis.

Vancomycin (VAN) has a narrow therapeutic window and variable pharmacokinetic properties, which require timely monitoring of plasma concentration to adjust the dosage for better effectiveness. A sensitive and quantitative immunochromatographic method was developed to detect VAN in plasma. The linear response range of the method to detect plasma VAN was 1.25-50 µg/mL. The intra- and inter-assay coefficients were 4.68% and 8.81%, respectively, while the recovery was 89.10%-98.32%. The clinical comparative experiment results demonstrated a good correlation (r > 0.90) between the fluorescent immunochromatographic strip test and the mass spectrum. In this study, a simple, rapid, and high-sensitivity immunochromatographic method was established for detecting VAN, which helped establish the basis for further application of monitoring plasma concentration.

Acupoint catgut embedding improves senescence in a rat model of ageing by regulating mitophagy via the PINK1 pathway.

Ageing population is a tough task worldwide, and the aggravating trend of ageing population in China brings enormous pressure to healthcare system. Chinese acupuncture has shown definite anti-ageing effect as arthralgia relief, movement improvement, energy increase and immunity enhancement; however, the mechanisms underlying are far away from illumination. Increasing literature has highlighted the role of alterations in mitochondrial function as a potential central regulator in ageing biology; mitophagy plays a critical role in mitochondrial quality control. In the present study, we demonstrated that acupoint catgut embedding treatment ameliorated ageing-related alterations in appearance, muscle function and spatial memory in rats, reduced degenerated cells in hippocampus, and maintained relatively normal structures in the hippocampus tissue and neurons. These changes were proved to be associated with the regulation of mitochondrial function and autophagic activity. Furthermore, we investigated part of the molecular mechanisms and demonstrated that the PINK1 other than PINK1-Parkin signalling pathway involved in the effects of acupoint catgut embedding, and the imbalancement between mitochondrial fusion and fission and stimulation of mitochondrial biogenesis may aggravate or compensate for impaired mitochondria. The factors act downstream PINK, and the interaction between them for mitochondrial homeostasis in this process remains to be identified.

Single-molecule real-time transcript sequencing identified flowering regulatory genes in Crocus sativus.

BACKGROUND: Saffron crocus (Crocus sativus) is a valuable spice with medicinal uses in gynaecopathia and nervous system diseases. Identify flowering regulatory genes plays a vital role in increasing flower numbers, thereby resulting in high saffron yield. RESULTS: Two full length transcriptome gene sets of flowering and non-flowering saffron crocus were established separately using the single-molecule real-time (SMRT) sequencing method. A total of sixteen SMRT cells generated 22.85 GB data and 75,351 full-length saffron crocus unigenes on the PacBio RS II panel and further obtained 79,028 SSRs, 72,603 lncRNAs and 25,400 alternative splicing (AS) events. Using an Illumina RNA-seq platform, an additional fifteen corms with different flower numbers were sequenced. Many differential expression unigenes (DEGs) were screened separately between flowering and matched non-flowering top buds with cold treatment (1677), flowering top buds of 20 g corms and non-flowering top buds of 6 g corms (1086), and flowering and matched non-flowering lateral buds (267). A total of 62 putative flower-related genes that played important roles in vernalization (VRNs), gibberellins (G3OX, G2OX), photoperiod (PHYB, TEM1, PIF4), autonomous (FCA) and age (SPLs) pathways were identified and a schematic representation of the flowering gene regulatory network in saffron crocus was reported for the first time. After validation by real-time qPCR in 30 samples, two novel genes, PB.20221.2 (p = 0.004, r = 0.52) and PB.38952.1 (p = 0.023, r = 0.41), showed significantly higher expression levels in flowering plants. Tissue distribution showed specifically high expression in flower organs and time course expression analysis suggested that the transcripts increasingly accumulated during the flower development period. CONCLUSIONS: Full-length transcriptomes of flowering and non-flowering saffron crocus were obtained using a combined NGS short-read and SMRT long-read sequencing approach. This report is the first to describe the flowering gene regulatory network of saffron crocus and establishes a reference full-length transcriptome for future studies on saffron crocus and other Iridaceae plants.

Association of long interspersed nucleotide element-1 and interferon regulatory factor 6 methylation changes with nonsyndromic cleft lip with or without cleft palate.

OBJECTIVE: To examine the possible associations between methylation changes in the promoter regions of long interspersed nucleotide element-1 (LINE-1) and interferon regulatory factor 6 gene (IRF6) and nonsyndromic cleft lip with or without cleft palate (NSCL/P). METHODS: A case-control investigation was performed to compare 37 infants affected by NSCL/Ps with 60 babies without cleft malformations. Their genomic DNA samples were obtained, and the LINE-1 and IRF6 methylation levels were measured by using Sequenom MassArray. Unconditional logistic regression was adopted to estimate the odds ratio. RESULTS: Infants with NSCL/Ps had a higher methylation level at LINE-1 and IRF6 promoter regions than controls. High levels of LINE-1 (≥64.07%) and IRF6 (≥6.46%) methylation were associated with an increased risk of NSCL/P (LINE-1, OR = 2.63, 95% CI: 1.07-6.57; IRF6, OR = 4.73, 95% CI: 2.10-13.07), and the associations remained to be significant after adjusting for potential confounders. Similar associations were also found for cleft lip only, cleft lip, and palate. CONCLUSION: Our study suggested that aberrant methylation of LINE-1 and IRF6 might contribute to the development of NSCL/Ps. Further studies are needed to replicate the findings.

Association of SNP rs1867277 in FOXE1 Gene and Cleft Lip with or without Cleft Palate in a Han Chinese Population.

BACKGROUND: The genetic factors causing cleft lip with or without cleft palate (CL ± P) are still unclear. The SNPs in FOXE1 gene were associated with CL ± P. However, the results have been inconsistent. OBJECTIVE: We explored the associations of four SNPs in FOXE1 gene and CL ± P by a family based study. MATERIALS AND METHODS: 128 children with CL ± P and their parents were recruited. rs3758249 and rs1867277 were genotyped by high-resolution melting curve (HRM) method, whereas rs1443434 and rs907577 were genotyped by Sequenom MassARRAY® method. The software PLINK, FBAT and FAMHAP were used for analyzing data. RESULTS: rs1867277 was associated with CL ± P (Pm = 0.0395). The patients were divided into two subgroups, individuals with cleft lip only and persons with cleft lip and palate. There were no associations in subgroup analyses. CONCLUSION: We confirmed the association of FOXE1 gene and CL ± P by a family based study. For the first time, rs1867277 was significantly associated with CL ± P.

Lilium liangiae, a new species in the genus Lilium (Liliaceae) that reveals parallel evolution within morphology.

The former genus Nomocharis, which has been merged as a clade within the genus Lilium (Liliaceae), represents one of the most complicated and unclear groups included in the latter. Research on members of the Nomocharis clade has been quite limited due to the sampling difficulties caused by its selective environmental preferences. In this study, we propose a new species within this clade, Lilium liangiae, as a further bridge connecting the former genus Nomocharis with other members of the genus Lilium. We conducted morphological clustering, phylogenetic, and comparative genomics analyses of nuclear internal spacers and the newly generated complete chloroplast genome, in conjunction with previously published sequences, and performed ancestral state reconstruction to clarify the evolutionary pattern of important traits in Lilium. The clustering results of 38 morphological traits indicated that the new species is allied to Nomocharis, further increasing the morphological polymorphism in the latter. The phylogenetic results and morphological clustering both supported L. liangiae belonging to the subclade Ecristata in Nomocharis, its closest affinity being Lilium gongshanense. Inconsistencies in phylogenetic relationships were detected between nuclear and plastid datasets, possibly due to ancient hybridization and ongoing introgression. Comparative genomics revealed the conservation and similarity of their chloroplast genomes, with variations observed in the expansion and contraction of the IR regions. A/T and palindromic repeat sequences were the most abundant. Seven highly variable regions (Pi≥0.015) were identified as potential molecular markers based on the chloroplast genomes of 47 species within Lilium. Both nuclear and plastid genes exhibited very low variability within the Nomocharis clade, contrasting with their highly variable morphological appearance. The ancestral state reconstruction analysis suggests that the campanulate flower form, as in L. liangiae, arose at least three times within the genus Lilium, revealing parallel evolution in the latter. Overall, this study adds important genetic and morphological evidence for understanding the phylogenetic relationships and parallel evolution patterns of species within the genus Lilium.

Novel indoline-2,3-dione derivatives as inhibitors of aminopeptidase N (APN).

Aminopeptidase N (APN/CD13), as a zinc-containing ectoenzyme, plays a critical role in the process of tumor angiogenesis, invasion and metastasis. Through the docking-based virtual screening of chemical databases and the further activity assay, we discovered that compound 10c exhibits potent and selective inhibitory ability towards APN. In addition, a series of indoline-2,3-dione derivates have been designed and synthesized as APN inhibitors. The results of preliminary activity evaluation showed that compound 12a (IC(50) = 0.074 ± 0.0026 µM) exhibited the best inhibitory activity against APN, which could be used for further anticancer agent research.

Design, synthesis and biological evaluation of novel L-isoserine tripeptide derivatives as aminopeptidase N inhibitors.

Aminopeptidase N (APN/CD13) is one of the essential proteins for tumour invasion, angiogenesis and metastasis as it is over-expressed on the surface of different tumour cells. Based on our previous work that L-isoserine dipeptide derivatives were potent APN inhibitors, we designed and synthesized L-isoserine tripeptide derivatives as APN inhibitors. Among these compounds, one compound 16l (IC50 = 2.51 ± 0.2 µM) showed similar inhibitory effect compared with control compound Bestatin (IC50 = 6.25 ± 0.4 µM) and it could be used as novel lead compound for the APN inhibitors development as anticancer agents in the future.

Design, synthesis and preliminary activity evaluation of novel 3-amino-2-hydroxyl-3-phenylpropanoic acid derivatives as aminopeptidase N/CD13 inhibitors.

Aminopeptidase N (APN/CD13) over expressed on tumour cells, plays a critical role in tumour invasion, metastasis and tumour angiogenesis. In this article, we described the design, synthesis and preliminary activity studies of novel 3-amino-2-hydroxyl-3-phenylpropanoic acid derivatives as APN inhibitors. The in vitro enzymatic inhibitions on APN from porcine kidney showed that compound 7e had the most potent inhibitory activity against APN with the IC(50) value to 1.26 ± 0.01 µM, which is better than that of bestatin (IC(50) = 2.55 ± 0.11 µM). In addition, compound 7e also showed better inhibitory activity against APN on human ovary clear cell carcinoma cell ES-2 than bestatin with the IC(50) value to 30.19 ± 1.02 µM versus 60.61 ± 0.1 µM. Compound 7e could be used as the lead compound in the future for anti-cancer agent research.

[Epidemiological analysis of selected congenital limb malformations in Hengyang].

OBJECTIVE: To describe the epidemiological characteristics of selected congenital limb malformations (CLM) in newborns of Hengyang. METHODS: During the period of 2008-2010, cluster sampling survey was adopted to investigate the congenital limb malformations of neonates born to women resident in Hengyang, including Nanyue District, Zhuhui District, Changning City and Hengshan County. Each newborn was examined for the screening of CLM after birth. Limb malformations were grouped into the isolated (ILM) and the syndromic (SLM) form, depending on associated malformations of the affected. Prevalence rates, CLM spectrum and clinical manifestations were analyzed. RESULTS: A total of 170 CLM cases were identified among 52,307 newborns during the study period, resulting overall rate of 32.50/10(4). The rates for isolated and syndromic CLM were 28.29 and 4.21 per 10 000 births respectively. The rates for polydactyly, congenital talipes equinovarus, syndactyly and limb reduction defects were 13.00/10(4), 9.56/10(4), 5.16/10(4) and 3.63/10(4), respectively. No significant difference in rates of overall CLM or specified CLM was observed across urban-rural, gender and maternal age groups. Of the cases affected by polydactyly, syndactyly and limb reduction defects, malformation involved upper limbs, lower limbs and the both accounted for 68.14%, 14.16% and 17.70%. Preterm birth, low birth-weight, still birth and neonatal death were observed more frequently in syndromic cases than in isolated patients. CONCLUSION: The high CLM prevalence rate and fatality rate in Hengyang suggest that effective measures should be taken to prevent malformations and to improve survival of the affected.

Study on the Correlation between Pain and Cytokine Expression in the Peripheral Blood of Patients with Bone Metastasis of Malignant Cancer Treated Using External Radiation Therapy.

The incidence of cancer is increasing worldwide on a yearly basis, with the number of patients with bone metastases also increasing annually. Events associated with bone metastases can seriously affect patient quality of life, through pain, hypercalcemia, bone marrow regeneration disorders, and spinal cord compression. In this nonrandomized controlled clinical trial study, we focused on the relationship between bone metastasis, pain, and cytokines before and after radiotherapy. We hypothesized that radiotherapy alters the cytokine profile of the local bone environment. Combined with the analgesic effects of radiotherapy, certain cytokines may be very sensitive to radiation. External radiation therapy is commonly used to treat cancer patients with bone metastases and can effectively relieve metastasis-related pain, although its underlying mechanisms have not been fully elucidated. For this case-control study, we recruited 30 cancer patients with bone metastasis and 30 healthy individuals. Peripheral venous blood from healthy individuals was collected. The clinical characteristics and peripheral venous blood were collected from patients one week before and one week after radiotherapy. The preradiotherapy and postradiotherapy pain scores, quality of life (QOL), and blood cytokine profiles of the patients to that of the controls were collected to identify pain-related cytokines. Finally, the pain score and the quality of life score improved significantly after radiotherapy. Moreover, the preradiotherapy and postradiotherapy blood cytokine profiles of the patients showed significant differences, indicating that the analgesic effect of radiotherapy against bone metastases is mediated via altered cytokine production. Furthermore, some cytokines were more sensitive to radiotherapy. The levels of MIP-1δ, MCP-2, TIMP-1, RANTES, IGFBP3, and TNF-α showed significant differences in the pairwise comparative analysis and may therefore mediate pain associated with bone metastasis.

Design, synthesis and biological evaluation of novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives as aminopeptidase N/CD13 inhibitors.

A series of novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were designed, synthesized and assayed for their activities against aminopeptidase N (APN/CD13) and MMP-2. The results showed that most compounds exhibited higher inhibitory activities against APN than that of MMP-2. Within this series, compound 12h (IC(50)=6.28 ± 0.11 µM) showed similar inhibitory activities compared with Bestatin (IC(50)=5.55 ± 0.01 µM), and it could be used as novel lead compound for the future APN inhibitors development as anticancer agents.

Novel aminopeptidase N (APN/CD13) inhibitors derived from 3-phenylalanyl-N'-substituted-2,6-piperidinedione.

Aminopeptidase N (APN/CD13) over expressed on tumor cells, plays a critical role in tumor invasion, metastasis, and tumor angiogenesis. Here we described the design, synthesis and preliminary activity studies of novel APN inhibitors with 3-phenylalanyl-N'-substituted-2,6-piperidinedione scaffold. The results showed that compound 7c had the most potent inhibitory activity against APN with the IC(50) value to 5.00 +/-3.17 microM, which could be used as the lead compound in the future for anticancer agent research.

[FGFR2 gene mutation in a Chinese patient with Apert syndrome].

OBJECTIVE: To determine the disease-causing mutation in a Chinese patient with Apert syndrome (AS). METHODS: Genomic DNA was extracted from peripheral blood samples of the AS patient and his parents. Polymerase chain reaction (PCR) was used to amplify the exons 7 and 9 of fibroblast growth factor receptor 2 (FGFR2) gene. Then PCR products were sequenced bi-directionally. RESULTS: A heterozygous 934C to G transversion in exon 7 of the FGFR2 gene was detected in the patient, which resulted in the substitution of tryptophan residue for serine at position 252 of FGFR2 protein (S252W). This mutation has been reported in AS patients previously. CONCLUSION: This Chinese AS results from the 934 C to G mutation in exon 7 of FGFR2 gene.

Oxygen-glucose deprivation activates 5-lipoxygenase mediated by oxidative stress through the p38 mitogen-activated protein kinase pathway in PC12 cells.

5-Lipoxygenase (5-LOX) is a key enzyme catalyzing arachidonic acid to form leukotrienes. We have reported that ischemic-like injury activates 5-LOX in PC12 cells; however, the mechanisms are unknown. To determine whether ischemic-like injury activates 5-LOX mediated by oxidative stress through the p38 MAPK pathway, we transfected GFP-5-LOX into PC12 cells and induced ischemic-like injury by oxygen-glucose deprivation (OGD). We found that the transfected GFP-5-LOX was localized primarily in the nuclei and translocated to the nuclear envelope after OGD/recovery reaching a maximum 2 hr after a 2-hr exposure to OGD. The nonselective 5-LOX inhibitor caffeic acid, 5-LOX-activating protein inhibitor MK886, and selective 5-LOX inhibitor zileuton attenuated the cell injury and reduced the production of 5-LOX products, cysteinyl leukotrienes, after OGD/recovery. However, only caffeic acid inhibited OGD/recovery-induced 5-LOX translocation. OGD/recovery also increased reactive oxygen species (ROS), which was inhibited by caffeic acid only. Hydrogen peroxide, an exogenous ROS, evoked similar cell injury and 5-LOX translocation, and the inhibitors had effects on the changes after H(2)O(2) similar to those after OGD/recovery. Both OGD/recovery and H(2)O(2) increased the phosphorylated p38 MAPK level, which was inhibited by caffeic acid and the ROS scavenger edaravone, but not by MK886 or zileuton. Moreover, SB203580 (a p38 MAPK inhibitor) and edaravone inhibited the cell injury and 5-LOX translocation induced by OGD/recovery and H(2)O(2). Thus, we conclude that OGD/recovery-induced ischemic-like injury induces 5-LOX activation, which is mediated by oxidative stress through activating the p38 MAPK pathway.

Pranlukast attenuates ischemia-like injury in endothelial cells via inhibiting reactive oxygen species production and nuclear factor-kappaB activation.

The anti-inflammatory effects of pranlukast, an antagonist of cysteinyl leukotriene receptor 1, may be rendered not only by antileukotriene activity but also by other pharmacological activities. Previous studies indicate that pranlukast reduces ischemic tissue injury partially through decreasing vascular permeability, but its effect on ischemic injury in endothelial cells is not known. Thus, in this study, we investigated the effect of pranlukast on ischemia-like injury induced by oxygen-glucose deprivation (OGD) in EA.hy926 cells, a human endothelial cell line, and the possible mechanisms. We found that cell viability was reduced, lactate dehydrogenase release was increased 4-8 hours after OGD, and necrosis was induced 8 hours after OGD. Production of reactive oxygen species (ROS) increased by 211%, 176%, and 128%, respectively, 0.5, 1, and 2 hours after OGD. Nuclear factor-kappaB (NF-kappaB) was translocated to the nuclei 4-8 hours after OGD. Pranlukast ameliorated the reduced viability, the increased lactate dehydrogenase release, and necrosis after OGD. It also reduced ROS production and inhibited NF-kappaB nuclear translocation after OGD. The ROS scavenger, edaravone, inhibited OGD-induced nuclear translocation of NF-kappaB as well. Edaravone and pyrrolidine dithiocarbamate (a specific NF-kappaB inhibitor) protected endothelial cells from the OGD-induced injury. However, zileuton, a 5-lipoxygenase inhibitor, did not affect the cell injury, ROS production, and NF-kappaB nuclear translocation after OGD. The exogenous leukotriene D4 did not induce cell injury, ROS production, and NF-kappaB translocation. Thus, we conclude that pranlukast protects endothelial cells from ischemia-like injury via decreasing ROS production and inhibiting NF-kappaB activation, which is leukotriene independent.

Design, synthesis, and preliminary studies of the activity of novel derivatives of N-cinnamoyl-L-aspartic acid as inhibitors of aminopeptidase N/CD13.

A series of novel derivatives of N-cinnamoyl-l-aspartic acid were designed, synthesized, and assayed for their inhibitory activities against aminopeptidase N. The preliminary biological assay showed that compound 8c has the most potent inhibitory activity against APN with an IC(50) of 11.1+/-0.9 microM, this could be used as the lead compound in future research on anticancer agents.

SiNiSan Ameliorates the Depression-Like Behavior of Rats That Experienced Maternal Separation Through 5-HT1A Receptor/CREB/BDNF Pathway.

Background: Early adverse life stress is an important dangerous factor in the development of psychiatric disorders, particularly depression. Available clinical antidepressant agents, such as fluoxetine, [a selective serotonin reuptake inhibitor (SSRI)], are unsatisfactory because of their side effects. SiNiSan (SNS) is a classic Chinese medicine prescription regarded to disperse stagnated liver qi to relieve qi stagnation. Therefore, this study was designed to detect the effects and molecular mechanism of SNS treatment in rats subjected to maternal separation (MS). Method: Male neonatal Wistar rats were divided into six groups including control + ddH2O, MS + ddH2O, MS + fluoxetine (5 g/kg), MS + SNS -low dose (2.5 g/kg), MS + SNS -medium dose (5 g/kg), MS + SNS -high dose (10 g/kg). The volume of drugs and ddH2O in each group are according to the weight of rats every day (10 mL/kg). Each group comprised 16 pups with 8 young and 8 adult pups. Except for the control group, all MS groups were separated from their mothers for 4 h/day from 9:00 to 13:00 during postnatal days (PNDs) 1 to 21. After MS, the six groups were intragastrically administered with ddH2O, fluoxetine, and different doses of SNS until PND 28 (for young pups) and PND 56 (for adult pups). The pups were weighed every day, and depression-like behavior was assessed by sucrose preference test, open field test, and forced swimming test. Serotonin 1A (5-HT1A) receptor, phosphorylated protein kinase A (p-PKA) substrate, cAMP response element-binding protein (CREB), p-CREB and brain-derived neurotrophic factor (BDNF) in the hippocampus were examined by Western blot, and in situ 5-HT1A receptor expression was measured by IHC. Results: Young and adult MS rats exhibited depression-like behavior. However, the depression-like behavior was ameliorated by SNS in both age groups. The levels of 5-HT1A receptor, p-CREB, and BDNF in the hippocampus were reduced in young and adult MS rats. SNS treatment significantly up-regulated the expression of 5-HT1A receptor, p-CREB, and BDNF in the hippocampus of adult MS rats. However, few significant effects on the protein expression were observed in the young MS rats. Conclusion: MS in infancy could develop depression-like behavior in young and adult. SNS treatment may perform antidepressant effects on young and adult MS rats through the BDNF/PKA/CREB pathway.

Novel 3-phenylpropane-1,2-diamine derivates as inhibitors of aminopeptidase N (APN).

Aminopeptidase N (APN) is an essential peptidase involved in the process of tumor invasion and metastasis. Here we describe a novel class of inhibitor with 3-phenylpropane-1,2-diamine as scaffold to APN. Preliminary activity evaluation with enzyme inhibition studies showed that compound 12i exhibited potent and selective inhibitory activity towards APN with the IC(50) value 15.5+/-1.2microM.

[Transfection with 5-lipoxygenase/green fluorescence protein for evaluating injury-induced 5-lipoxygenase translocation to the nuclear membrane in PC12 cells].

OBJECTIVE: To evaluate the translocation of 5-lipoxygenase (5-LOX)) after injuries by transfection with green fluorescence protein (GFP)/5-LOX in PC12 cells. METHODS: PC12 cells were stably transfected with pEGFP-C2/5-LOX (GFP/5-LOX) or pEGFP-C2 vectors (control). After treatment with oxygen-glucose deprivation (OGD), H(2)O(2) or NMDA, GFP/5-LOX localization in the cells was observed under a fluorescence microscope. Wild-type 5-LOX was determined by immunostaining after the treatment. RESULT: In the GFP/5-LOX-transfected cells, GFP/5-LOX was primarily localized in the nucleus; while in the GFP-transfected cells, GFP was localized in both the cytoplasm and nucleus. After OGD and H(2)O(2) treatments, GFP/5-LOX was translocated to the nuclear membrane in 50.6 % and 57.7% cells respectively. However, after NMDA treatment or in GFP-transfected cells, no translocation was observed. Wild-type 5-LOX was distributed in the nuclei and cytoplasm, and all the 3 treatments induced 5-LOX translocation to the nuclear membrane. CONCLUSION: In the PC12 cells stably transfected with GFP/5-LOX, GFP/5-LOX is primarily distributed in the nuclei; the OGD-, H(2)O(2)- and NMDA-induced 5-LOX translocation exhibits different properties.