Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.     

Rapid determination of DNA synthesis in adherent cells grown in microtiter plates

A specific, rapid, and economical method for measuring the extent of DNA synthesis in adherent rat hepatoma H4-II-E cells grown in 96-well microtiter plates is described. The adherent cells were pulsed for 1 h with [methyl-3H]thymidine, released from the substratum by trypsinization, and collected on fiberglass filters with a MASH II cell harvester. The amount of radioactivity incorporated was directly proportional to the number of cells per well. Growth curves generated by measuring [methyl-3H]thymidine incorporation and counting the number of cells per well were identical. Experiments with inhibitors of DNA, protein, and RNA synthesis demonstrated that this method selectively measured DNA synthesis. In addition, [3H]thymidine uptake showed excellent correlation with autoradiographic assessment of DNA synthesis. This specific and sensitive method for determining DNA synthesis in microtiter cultures should facilitate studies of effects of various growth-controlling agents on epithelial, fibroblastic, and other cells which grow as adherent cells in culture.     

Inhibitory role of E2F-1 in the regulation of tumor suppressor p53 during DNA damage response

Appropriate regulation of DNA damage response is pivotal for maintaining genome stability. p53 as well as E2F-1 plays a critical role during DNA damage response, however, the physiological significance of their interaction has been elusive. In the present study, we found that E2F-1 has an inhibitory effect on p53 during adriamycin (ADR)-mediated DNA damage response. Upon ADR exposure, p53 and E2F-1 were markedly induced at protein and mRNA levels in p53-procifient U2OS and HCT116 cells, and formed a stable complex as examined by co-immunoprecipitation experiments. Of note, chromatin immunoprecipitation (ChIP) experiments revealed that ADR-mediated induction coincides with the efficient recruitment of p53 and E2F-1 onto the promoters of p53-target genes, such as p21(WAF1) and BAX. Subsequent RT-PCR and luciferase reporter assays demonstrated that E2F-1 strongly attenuates p53-dependent transactivation of p53-target genes. Importantly, siRNA-mediated knockdown of E2F-1 stimulated apoptosis in response to ADR, which was associated with an accelerated response of p21(WAF1) and BAX. Collectively, our present findings suggest that E2F-1 participates in p53-mediated DNA damage response and might have a checkpoint function to limit overactive p53.     

Efficient Inhibition of wear debris-induced inflammation by locally delivered siRNA

Aseptic loosening is the most common long-term complication of total joint replacement, which is associated with the generation of wear debris. The purpose of this study was to investigate the inhibitory effect of small interfering RNA (siRNA) targeting tumor necrosis factor-α (TNF-α) on wear debris-induced inflammation. A local delivery of lentivirus-mediated TNF-α siRNA into the modified murine air pouch, which was stimulated by polymethylmethacrylate (PMMA) particles, resulted in significant blockage of TNF-α both in mRNA and protein levels for up to 4 weeks. In addition, significant down-regulation of interleukin-1 (IL-1) and interleukin-6 (IL-6) was observed in TNF-α siRNA-treated pouches. The safety profile of gene therapy was proven by Bioluminescent assay and quantitative fluorescent flux. Histological analysis revealed less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) in TNF-α siRNA-treated pouches. These findings suggest that local delivery of TNF-α siRNA might be an excellent therapeutic candidate to inhibit particle-induced inflammation.     

Enhanced immunodetection of cell-free synthesized erythropoietin under denaturing conditions

A monoclonal antibody produced by hydridoma cell line, ATCC HB8209, was used to detect and purify erythropoietin synthesized in a cell-free system. The antibody was raised against the N-terminal 20 residues of erythropoietin. It retained anti-erythropoietin activity in 6 M urea in which most of the cell-free synthesized erythropoietin became soluble and gave an enhanced activity of the antibody.     

Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Background

Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.

Methods

We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.

Results

We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2?>?apoE3?>?apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.

Conclusions

Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.

     

Ontogeny and phylogeny of Metaurostylopsis cheni sp. n. (Protozoa,Ciliophora), with estimating the systematic position of Metaurostylopsis

Chen, X., Huang, J. & Song, W. (2010). Ontogeny and phylogeny of Metaurostylopsis cheni sp. n. (Protozoa, Ciliophora), with estimating the systematic position of Metaurostylopsis. —Zoologica Scripta, 40, 99–111. The ciliate genus Metaurostylopsis seems to be a highly divergent marine‐habiting group, of which neither systematic position nor the variation of their ontogeny has been critically checked. In the present work, the morphology and morphogenesis during asexual division of a new form, Metaurostylopsis cheni sp. n., isolated from the Yellow Sea, China, were investigated and comparison among known congeners was performed. The new species has two types of cortical granules, the larger ones of which are flattened and oval or circular in outline with a longitudinal groove, yellow–green in colour, and arranged along the cirral rows and dorsal kineties, whereas the smaller ones are colourless or grayish and sparsely distributed. The main morphogenetic features are: (i) the entire parental ciliature, including the old oral apparatus, is renewed, (ii) the oral primordium of the proter originates de novo and beneath the surface of the buccal cavity, that is, sub‐apokinetally, (iii) the anlagen of the marginal rows and of the dorsal kineties are formed intrakinetally and (iv) fusion of the macronuclear nodules results in an irregular mass with only few branches. The small subunit ribosomal RNA (SSU rRNA) gene of M. cheni was sequenced. Phylogenetic analysis based on SSU rRNA gene sequence data shows that M. cheni clusters with all other Metaurostylopsis spp. sequenced to date indicating that the genus is monophyletic and is probably closely related to the Apokeronopsis–Thigmokeronopsis‐group, within the order Urostylida.     

Establishment of Transfer Functions of the Pollen-Climatic Factors in Northern China and the Quantitative Climatic Reconstruction at DJ Core

The 2.4 meter-long core was extracted from the Diaojiao lake (41º18′N, 112º21′E) at the foot of the northern part of Daqingshan Mts. Pollen analysis from collections subsampled in the laboratory at 2 cm intervals, revealed plentiful pollen and spores from over 10 arboreal genera, including Pinus, Betula, Picea, Abies, Carpinus, Quercus, Ulmus and more than 20 non-arboreal genera, mainly of Artemisia, Labiatae, Nitraria, Polygonaceae, Ranunculus, Thalictrum, Umbel- liferae, Caryophyllaceae and Cyperaceae. Fern spores, aquatic pollen and algae were also observed in some parts of the core. The transfer functions were established by the stepwise regression analysis using the climatic factors and 13 pollen taxa. The different Fl and F2 value were used as the thresh- old value of F test (i. e. used for selecting and deleting factors). Each regressed equation was obtained from 70 times of calculations with a step-wise increase of 0.1 for Fl and F2 and those having the smallest regression deviation and the largest multiple correlation coefficient were the final four transfer functions. Substituting the pre-factor obtained from the stratigraphic sampled into the regression equations, the estimates of temperature and precipitation in January and in July, and annual mean temperature values could be calculated. Some climatic stages were inferred from total pollen influx and pollen percentage from the core using a transfer function: humid-cool (from 10 000 to 7 800 a BP), arid-cold (9 200 to 7 900 a BP), arid-warm (7 900 to 7 100 a BP), humid-warm (7 100 to 4 400 a BP), arid-warm (4 400 to 3 000 a BP), arid-cold (3 000 to 2 100 a BP). The highest annual mean temperature during Holocene was ca. 4 ℃ higher and the lowest was ca. 2 ℃ lower than the present temperature. Annual precipitation was 250 mm higher and 300 mm lower than the present.     

Statin Use and the Risk of Parkinson's Disease: An Updated Meta-Analysis

Introduction

In response to the ongoing debate over the relationship between the use of statins and the risk of Parkinson''s disease (PD), we performed a systematic review and meta-analysis of observational studies to examine their association.

Methods

We conducted a review of the literature using electronic databases supplemented by a manual search to identify potentially relevant case-control or cohort studies. Summary relative risk (RRs) and 95% confidence intervals (CIs) were calculated using a random-effects model. Sensitivity and subgroup analyses were also conducted.

Results

Eleven studies (five case-control and six cohort) with a total of 3,513,209 participants and 21,011 PD cases were included. Statin use was associated with a lower risk of PD, with a summary RR of 0.81 (95% CI 0.71–0.92). Sensitivity analysis demonstrated the robustness of results. Subgroup analyses showed that neither study design nor study region significantly influenced the effect estimates. However, subgroup studies adjusted for age or sex had a greater inverse association than did unadjusted analyses (age-adjusted RR 0.75, 95% CI 0.60–0.95; age-unadjusted RR 0.86, 95% CI 0.75–0.99 and sex-adjusted RR 0.76, 95% CI 0.59–0.98; sex-unadjusted RR 0.85, 95% CI 0.79–0.92).

Conclusions

Results of this systematic review suggest that statin use is associated with a reduced PD risk. However, randomized controlled trials and more observational studies should be performed before strong conclusions are drawn.