Glucocorticoid Receptor/HCN4 Channels Interaction in Spinal Dorsal Horn Participates in the Regulation of Neuropathic Pain after Peripheral Nerve Injury in Rats.

We studied the interaction between glucocorticoid receptor (GR) and HCN4 channels in the rat model of spared nerve injury (SNI) in Sprague-Dawley rats (n=124). The animals were randomly divided into 6 groups: sham-operated (SO; n=24), SNI (reference group; n=20), and 4 experimental SNI groups intrathecally treated with dexamethasone (DEX; GR agonist; n=20), RU38486 (GR antagonist; n=20), ZD7288 (HCN channels blocker; n=20), and ZD7288+DEX (n=20). The paw mechanical withdrawal threshold (PWT) was measured one day before surgery (SO group) and on days 1, 3, 7, 14, and 21 after surgery. Behavioral results showed that mechanical hyperalgesia appeared on day 1 after SNI, while PWT decreased gradually with time. The expression of GR and HCN4 channels in L4-L6 dorsal horn of the spinal cord was detected by Western blotting and immunohistochemistry. In the reference group, SNI significantly increased GR expression up to day 14 after surgery in comparison with the SO group. The expression of GR showed a tendency to increase in the DEX group (with the maximum expression on days 14 and 21), significantly increased in the RU38486 group (maximum on day 7). In the ZD7288 group, GR expression was lower than in the SNI group and did not change throughout the experiment, suggesting that ZD7288 could block the expression of GR. In the DEX group, the expression of HCN4 channels was significantly higher on day 1 after SNI, but there were no differences in this parameter between the RU38486 and ZD7288 groups. In the ZD7288+DEX group, the expression of HCN4 channels significantly increased on days 14 and 21 after SNI. Thus, GR and HCN4 have the same linkage in the formation of central sensitization after SNI, but antagonists have no significant effect on the improvement of pain behavior.

COX-2-derived prostaglandins as mediators of the deleterious effects of nicotine in chronic kidney disease.

Tobacco smoking has been identified as a risk factor in the progression of chronic kidney disease (CKD). In previous studies, we showed that nicotine induces cyclooxygenase (COX)-2 expression in vivo and in vitro and that the administration of nicotine in vivo worsens the severity of renal injury in a model of subtotal renal ablation. In the present study, we tested the role of COX-2-derived prostaglandins on the deleterious effects of nicotine in CKD. Sham and 5/6 nephrectomy (5/6Nx) rats received tap water or nicotine (100 µg/mL) in the drinking water for 12 wk. Additional groups also systemically received the COX-2 inhibitor NS-398 (1.5 mg·kg-1·day-1 via osmotic minipump). The administration of nicotine worsened renal injury and proteinuria in 5/6Nx rats and increased proteinuria in sham rats. 5/6Nx rats had increased cortical production of the prostaglandins PGE2, PGI2, PGD2, and PGF2α and of thromboxane A2. In these rats, nicotine reduced the production of all prostaglandins examined except thromboxane A2. Treatment with the COX-2 inhibitor NS-398 resulted in complete inhibition of all prostaglandins studied and ameliorated renal injury and proteinuria in 5/6Nx rats on nicotine but not in 5/6 Nx rats on tap water. Nicotine also reduced the expression of megalin in all groups examined, and this was partially prevented by COX-2 inhibition. In the present study, we showed that in CKD, nicotine worsens renal injury at least in part by producing an imbalance in the production of prostaglandins. This imbalance in the production of prostaglandins likely plays a role in the deleterious effects of smoking on the progression of CKD.

JMJD3 enhances invasiveness and migratory capacity of non-small cell lung cancer cell via activating EMT signaling pathway.

OBJECTIVE: This study aimed to explore the expression of JMJD3 in non-small cell lung cancer (NSCLC) and to further study its association with clinical features and prognosis of patients. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the level of JMJD3 in 46 pairs of NSCLC tissues and para-cancerous specimens. The relationship between JMJD3 level and clinical features of NSCLC and patients' prognosis was analyzed. And JMJD3 expression in NSCLC cells was further verified by qRT-PCR. In addition, JMJD3 knockdown model was constructed using siRNA in cell lines including A549 and SPC-A1, and the effect of JMJD3 on the biological function of NSCLC cells was analyzed by cell counting kit-8 (CCK-8) and transwell migration and invasive-ness assays. Lastly, Western blot was performed to explore the potential mechanism. RESULTS: In this investigation, qRT-PCR results indicated that JMJD3 expression in above-mentioned tumor tissues was conspicuously higher than that in normal tissues. In addition, compared with patients with low level of JMJD3, patients with high level of JMJD3 had a higher incidence of lymphatic metastasis and distant metastasis and a lower overall survival rate. Meanwhile, the proliferation, invasiveness and migratory capacity of cells in the sh-JMJD3 group was conspicuously decreased when compared with the cells in negative control group. Western Blot results indicated that the levels of key proteins in EMT signaling pathway such as E-cadherin, N-cadherin, Vimentin, TGF-ß, and MMP-9 were notably decreased in sh-JMJD3 group. Besides, the addition of TGF-ß cytokines synergistically promoted the malignant progression of NSCLC induced by JMJD3. CONCLUSIONS: JMJD3 expression was found conspicuously increased in NSCLC, which might be close relevant to NSCLC lymphatic or distant metastasis as well as patients' poor prognosis. Therefore, we speculated that JMJD3 could promote invasiveness and migratory capacity of non-small cell lung cancer cells by activating EMT process.

Development and validation of a novel 13-plex PCR system for commonly used short tandem repeats in horses (Equus caballus).

BACKGROUND: Due to the thriving development of the modern horse industry and the occurrence of horse related crimes, the demand for methods of individual horse identification, parentage tests and other genetic analyses is increasing. Previous methods had disadvantages that decreased the accuracy of the results, lacked the inclusion of all commonly used short tandem repeats (STR) or increased the experimental cost and time. OBJECTIVES: We aimed to develop a novel 13-plex STR typing system to resolve the above issues. STUDY DESIGN: Experimental study. METHODS: Twelve autosomal and most commonly used di-nucleotide STRs (AHT4, AHT5, ASB2, ASB17, ASB23, HMS2, HMS3, HMS6, HMS7, HTG4, HTG10 and VHL20), and a Y-chromosomal STR (YJ10) were included. We redesigned the primers of eight STRs to establish a novel multiplex PCR system and tested this system for species specificity, sensitivity and repeatability. RESULTS: Full profiles were easily generated in one fast PCR reaction using a low-cost polymerase, as little as 1 ng of horse DNA template and 13 pairs of primers labelled with fluorescent dyes. No full profile was generated from DNA templates of humans or other commonly encountered animals. We also established an allelic ladder that contained 110 alleles based on 200 horses from 12 breeds and calculated standard population genetic parameters based on 150 Thoroughbreds. Stutter analysis showed that the averages of the stutter ratios were distinctly lower than those of lower allele ratios and the combined probability of paternity exclusion for this system were 0.994659935 (CPEduo ) and 0.999854032 (CPEtrio ). MAIN LIMITATIONS: A nonspecific and relatively low peak at 316 bp was frequently observed in locus HMS2, which is a nonexistent allele in all horses and should be ignored. CONCLUSIONS: Our results indicate that this 13-plex STR genotyping system is sensitive, species-specific, cost-effective and robust for applications in the horse industry and forensic investigation.

Are older drivers' driving patterns during an on-road driving task representative of their real-world driving patterns?

OBJECTIVE: The current study investigated whether older drivers' driving patterns during a customized on-road driving task were representative of their real-world driving patterns. METHODS: Two hundred and eight participants (male: 68.80%; mean age = 81.52 years, SD = 3.37 years, range = 76.00-96.00 years) completed a customized on-road driving task that commenced from their home and was conducted in their own vehicle. Participants' real-world driving patterns for the preceding 4-month period were also collected via an in-car recording device (ICRD) that was installed in each participant's vehicle. RESULTS: During the 4-month period prior to completing the on-road driving task, participants' median real-world driving trip distance was 2.66 km (interquartile range [IQR] = 1.14-5.79 km) and their median on-road driving task trip distance was 4.41 km (IQR = 2.83-6.35 km). Most participants' on-road driving task trip distances were classified as representative of their real-world driving trip distances (95.2%, n = 198). CONCLUSIONS: These findings suggest that most older drivers were able to devise a driving route that was representative of their real-world driving trip distance. Future research will examine whether additional aspects of the on-road driving task (e.g., average speed, proportion of trips in different speed zones) are representative of participants' real-world driving patterns.

Deficiency of brain structural sub-network underlying post-ischaemic stroke apathy.

BACKGROUND AND PURPOSE: This study aimed to reveal the structural basis of post-ischaemic stroke apathy, especially in relation to disruptions in structural connectivity. METHODS: Eighty-eight participants were included. The Apathy Evaluation Scale, clinician version, was used to characterize the severity of apathy. Diffusion tensor imaging tractography was used to examine white matter integrity and to reconstruct white matter networks using 90 nodes based on the automated anatomical labeling atlas. The degree for each node was extracted to determine the relationship to the severity of apathy. RESULTS: Apathy was not significantly associated with damage to any single brain region. The degrees of 24 nodes (limbic system, three nodes; frontal lobe, six; basal ganglia, two; temporal lobe, three; parietal lobe, three; insula, two; occipital lobe, five) were significantly correlated to the Apathy Evaluation Scale scores. These 24 nodes constituted an apathy-related sub-network and its global and local efficiencies were negatively correlated with apathy levels (global, r = -0.54, P < 0.01; local, r = -0.64, P < 0.01). Multivariate logistic regression indicated that decreased global efficiency of this sub-network was an independent risk factor for apathy (odds ratio 0.03, 95% confidence interval 0.01-0.04, P = 0.007). Efficiencies of the non-apathy-related sub-network (the remaining 66 nodes) did not correlate or predict the presence of apathy. CONCLUSIONS: Post-stroke apathy is not due to the dysfunction of a single region or circuit. Rather, it results from disconnection of a complex sub-network of brain regions. This provides new insights into the neuroanatomical basis of post-stroke apathy.

The study of the tumor stem cell properties of CD133+CD44+ cells in the human lung adenocarcinoma cell line A549.

We studied the tumor stem cell properties of the CD133+CD44+ subpopulation in the human lung adenocarcinoma cell line A549. A549 cells were classified into subpopulations based on differential expression patterns for CD133 and CD44. Cells from different subpopulations were cultured and subcutaneously injected into 32 nude mice. Our results as following, (1) The majority of A549 cells died, whereas only about 4.11% of cells divided and proliferated to form cell clones. (2) The expression of CD133 and CD44 in proliferative cancer cells was statistically significantly different from that in normal A549 cells (p < 0.001). (3) Cell proliferation in group A (CD133+CD44+) was the fastest among all groups. Cell proliferation in A549 cells was slower than in group A but faster than in groups B (CD133-CD44-), C (CD133-CD44+), and D (CD133+CD44-). (4) The tumorigenic capacity in cells from group A was significantly higher than that in cells from groups B (p<0.001), C (p<0.001) and D (p<0.04). In conclusion, CD133+CD44+ cells in the adenocarcinoma cell line A549 have expressive significant cancer stem cell properties with continuous proliferative capacity and differentiation potential.

Non-equilibrium thermodynamic analysis of transport properties in the nanofiltration of ionic liquid-water solutions.

Thenanofiltration of aqueous solutions of the ionic liquids (ILs) 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF(4)), and 1-butyl-3-methylimidazolium bromide ([Bmim]Br) with a polyamide nanofiltration membrane was investigated. The practical transport coefficients, including hydrodynamic permeability (L(p)), reflection (sigma) and solute permeability (omega) were calculated in terms of a non-equilibrium thermodynamics approach. It was found that L(p) and sigma diminished as the concentration of the IL solutions increased. These characteristics are similar to those observed in inorganic electrolyte-water systems. In addition, it was shown that the rejection and volume flux for both ionic liquid solutions rose with feed pressure, while it decreased with feed concentration. The maximum rejection efficiencies for [Bmim]Br and [Bmim]BF(4) are 67 % and 60 %, respectively, on our experimental scale. All the data suggests that a highly efficient process for IL separation could be developed when the operating conditions are optimized further.

Characterization of microsatellite loci in the lesser dawn bat (Eonycteris spelaea).

We constructed a genomic DNA library enriched for CA repeat motifs in Eonycteris spelaea. Nine microsatellite loci were isolated and tested on a population of 39 samples from Yunnan Province, China. These nine loci had three to 22 alleles per locus. Observed and expected heterozygosity values ranged from 0.079 to 0.963 and from 0.078 to 0.959. Two loci revealed significant departure from Hardy-Weinberg equilibrium and no linkage disequilibrium was found between loci pairs. These microsatellites can be a powerful molecular tool for population-level studies of E. spelaea.

Isolation and characterization of microsatellite loci in the long-fingered bat Miniopterus fuliginosus.

We isolated and characterized 10 microsatellite loci in the long-fingered bat Miniopterus fuliginosus. These loci were tested on 48 individuals from Anhui Province of China, and all loci were highly polymorphic. The mean number of observed alleles per locus was 13.6 (range from six to 27). Observed and expected heterozygosity values ranged from 0.364 to 0.957, and from 0.676 to 0.951, respectively. After Bonferroni correction, four loci deviated significantly from Hardy-Weinberg equilibrium. No pairs of loci were in linkage disequilibrium. These polymorphic markers will be used to examine population structure and genetic diversity in this species.

Isolation and characterization of microsatellite loci in the western long-fingered bat, Miniopterus magnater.

We isolated and characterized 10 microsatellite loci in the western long-fingered bat, Miniopterus magnater. These loci were tested on 48 individuals from Anhui Province of China, and all loci were highly polymorphic. The mean number of observed alleles per locus was 13.6 (range from six to 27). Observed and expected heterozygosity values ranged from 0.364 to 0.957, and from 0.676 to 0.951, respectively. After Bonferroni correction, four loci deviated significantly from Hardy-Weinberg equilibrium. No pairs of loci were in linkage disequilibrium. These polymorphic markers will be used to examine population structure and genetic diversity in this species.

Characterization of microsatellite loci in fulvous fruit bat Rousettus leschenaulti.

Rousettus leschenaulti is an abundant species in many countries of South-East Asia, including south China. We isolated seven microsatellite loci in R. leschenaulti from genomic DNA enriched for CA repeats with the enriched library method. A total of 56 samples from a population in the Guangxi Province of China were tested with these microsatellite markers. The polymorphism ranged from seven to 16 alleles, and the observed heterozygosity was 84-94%. It is the first time microsatellite markers were characterized from R. leschenaulti, and these markers can be an important tool for analysing population structure and genotypic diversity.

Characterization of the main effects, epistatic effects and their environmental interactions of QTLs on the genetic basis of yield traits in rice.

Main effects, epistatic effects and their environmental interactions of QTLs are all important genetic components of quantitative traits. In this study, we analyzed the main effects, epistatic effects of the QTLs, and QTL by environment interactions (QEs) underlying four yield traits, using a population of 240 recombinant inbred lines from a cross between two rice varieties tested in replicated field trials. A genetic linkage map with 220 DNA marker loci was constructed. A mixed linear model approach was used to detect QTLs with main effects, QTLs involved in digenic interactions and QEs. In total, 29 QTLs of main effects, and 35 digenic interactions involving 58 loci were detected for the four traits. Thirteen QTLs with main effects showed QEs; no QE was detected for the QTLs involved in epistatic interactions. The amount of variations explained by the QTLs of main effect were larger than the QTLs involved in epistatic interactions, which in turn were larger than QEs for all four traits. This study illustrates the ability of the analysis to assess the genetic components underlying the quantitative traits, and demonstrates the relative importance of the various components as the genetic basis of yield traits in this population.

[Growth and metabolism of human skin fibroblasts cultured on microcarriers].

Human foreskin fibroblasts were cultured in vitro on microcarriers in spinners and traditional static flasks. The cultured cells obtained with these approaches were compared in cell shape, cell growth, cell production, and the metabolisms of glucose, lactate, and ammonium. The cells from microcarrier cultures through medium exchange were 8 times more than those from traditional static flasks. It was found that the specific growth rate and specific glucose consumption rate in microcarrier spinner cultures were 0.64/d-1 and 5.56 mmol/10(9) cell/d, respectively, higher than those in static flask cultures. However, the average lactate yield on glucose consumption in spinner cultures was only 0.955 mmol/mmol, lower than that in static flask cultures, 1. 125 mmol/mmol. This indicated that the energy metabolism in spinner cultures was significantly more efficient than that in static flasks. The experimental results from this work suggest that the microcarrier culture system is a suitable way to expand the seeding cells for tissue engineering, due to its ideal cultivation environment provided.

Canine ehrlichiosis caused simultaneously by Ehrlichia canis and Ehrlichia platys.

To identify the causative agent of canine ehrlichiosis that has occurred in the suburbs of Guangzhou, China, since 1998, the 16S rRNA gene was amplified and sequenced. Two sequences of 1,482 and 1,483 base pairs were obtained and named as Gzh981 and Gzh982, respectively. The level of similarity of these two was 91.50%, and Gzh981 closely resembled the 16S rRNA gene of Ehrlichia canis, whereas Gzh982 resembled Ehrlichia platys. We therefore conclude that E. canis and E. platys together caused recent outbreaks of canine ehrlichiosis in China.

[On-line measurement of oxygen uptake rate in the cultivation of Vero cells using the dynamic method].

The oxygen uptake rate(OUR) during the cultivation of Vero cells in 1.5 L CelliGen bioreactor was on-line determined using the dynamic method. The results showed that the cell growth and metabolic state during the exponential growth phase was lineally related to the OUR. This implies that the on-line measurement of OUR can be used to promptly monitor the physiological state of cultured cells and to efficiently avoid contamination because of frequent sampling in the large-scale cultivation of mammalian cells.

Aerosol delivery of robust polyethyleneimine-DNA complexes for gene therapy and genetic immunization.

Aerosol delivery of plasmid DNA to the lungs offers the possibility of direct application of gene preparations to pulmonary surfaces as a means of treating a variety of genetic pulmonary disorders. However, the process of jet nebulization rapidly degrades naked DNA, viral vectors, and many lipid-based formulations. While complexing DNA with cationic lipids has been shown to significantly stabilize plasmid DNA, losses of biological activity often occur during nebulization, severely limiting the efficiency of aerosol delivery of many such complexes. In conjunction with the design of aerosol delivery systems appropriate for DNA delivery, we have developed formulations using polyethyleneimine (PEI, a polycationic polymer) and DNA that result in a high level of pulmonary transfection (10- to 100-fold greater than many cationic lipids) and are stable during nebulization. In addition, these PEI-based formulations exhibit a high degree of specificity for the lungs. The properties of PEI-based formulations that make them resistant to nebulization and efficient as DNA delivery vectors for pulmonary sites have been investigated. Potential applications of this technology, including the use of aerosolized PEI-DNA for genetic immunization, are discussed.

Genetic immunization with lung-targeting macroaggregated polyethyleneimine-albumin conjugates elicits combined systemic and mucosal immune responses.

Genetic immunization is a novel form of vaccination in which transgenes are delivered into the host to produce the foreign protein within host cells. Although systemic immune responses have been relatively easy to induce by genetic immunization, the induction of regional and mucosal immunity has often been more challenging. To address the problem of eliciting mucosal immunity in the lung, we utilized macroaggregated albumin to target plasmid DNA to the lung. Macroaggregated albumin is trapped in the lung after i. v. injection, and it is routinely used in radiolabeled form as an imaging modality to evaluate pulmonary blood flow. To couple DNA to this targeting agent, polyethyleneimine (a polycation that binds DNA and enhances transfection) was conjugated to serum albumin, and the conjugate was aggregated by heating to produce particles of 25-100 microm. The resulting particles bound plasmid DNA avidly, and when injected i.v. in mice, the particles distributed in the peripheral lung tissue in the alveolar interstitium. Particle-bound luciferase plasmid transfected a variety of cell lines in vitro, and after i.v. injection, gene expression was detected exclusively in the lung. Using human growth hormone as the encoded foreign Ag for immunization, i.v. injection of the particle-bound plasmid elicited both pulmonary mucosal and systemic immune responses, whereas naked DNA injected either i.v. or i.m. elicited only systemic responses. Thus, particle-bound plasmid DNA may have utility for genetic immunization by intravascular delivery to the lung and potentially to other organs and tissues.

Attachment kinetics of Vero cells onto CT-3 microcarriers.

The effects of cell seeding density, microcarrier concentration, agitation speed and age of seeding cells on the rate of cell attachment to the surface of CT-3 microcarriers were investigated. It was shown that the attachment followed first-order kinetics. When either the cell seeding density or the microcarrier concentration was increased, the kinetic constant increased due to an increase in the probability of collision between cells and microcarriers. However, at higher microcarrier concentrations, the increase in the kinetic constant with increasing microcarrier concentration was not significant. Cell attachment was decelerated upon increasing the agitation speed because of the shorter cell-microcarrier contact time. In addition, it was also demonstrated that cell attachment occurred more efficiently when seeding cells from the middle or early exponential growth phase were used. The process of cell attachment onto CT-3 microcarriers was investigated, and our results showed that the attachment stage was the rate-limiting step. These results will facilitate the optimization of the Vero cell culture process.

Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes.

OBJECTIVE: Increased nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity in chondrocytes is associated with cartilage matrix inorganic pyrophosphate (PPi) supersaturation in chondrocalcinosis. This study compared the roles of the transforming growth factor beta (TGFbeta)-inducible plasma cell membrane glycoprotein-1 (PC-1) and the closely related B10 NTPPPH activities in chondrocyte PPi metabolism. METHODS: NTPPPH expression was studied using reverse transcriptase-polymerase chain reaction and Western blotting. Transmembrane PC-1 (tmPC-1), water-soluble secretory PC-1 (secPC-1), and transmembrane B10 were expressed by adenoviral gene transfer or plasmid transfection, and expression of PPi was assessed in cultured articular chondrocytes and immortalized NTPPPH-deficient costal chondrocytes (TC28 cells). RESULTS: PC-1 and B10 messenger RNA were demonstrated in articular cartilages in situ, in untreated cultured normal articular chondrocytes, and in TC28 cells. Expression of tmPC-1 and secPC-1, but not B10, rendered the NTPPPH-deficient TC28 cells able to increase expression of extracellular PPi, with or without addition of TGFbeta (10 ng/ml) to the media. More plasma membrane NTPPPH activity was detected in cells transfected with tmPC-1 than in cells transfected with B10. Furthermore, confocal microscopy with immunofluorescent staining of articular chondrocytes confirmed preferential plasma membrane localization of PC-1, relative to B10. Finally, both PC-1 and B10 increased the levels of intracellular PPi, but PC-1 and B10 appeared to act principally in different intracellular compartments (Golgi and post-Golgi versus pre-Golgi, respectively). CONCLUSION: PC-1 and B10 NTPPPH activities were not redundant in chondrocytes. Although increased PC-1 and B10 expression caused elevations in intracellular PPi, the major effects of PC-1 and B10 were exerted in distinct subcellular compartments. Moreover, PC-1 (transmembrane and secreted), but not B10, increased the levels of extracellular PPi. Differential expression of PC-1 and B10 could modulate cartilage mineralization in degenerative joint diseases.