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Nucleic acid deformations play important roles in many biological processes. The physical understanding of nucleic acid deformation by environmental stimuli is limited due to the challenge in the precise measurement of RNA and DNA deformations and the complexity of interactions in RNA and DNA. Magnetic tweezers experiments provide an excellent opportunity to precisely measure DNA and RNA twist changes induced by environmental stimuli. In this work, we applied magnetic tweezers to measure double-stranded RNA twist changes induced by salt and temperature changes. We observed RNA unwinds when lowering salt concentration, or increasing temperature. Our molecular dynamics simulations revealed the mechanism: lowering salt concentration or increasing temperature enlarges RNA major groove width, which causes twist decrease through twist-groove coupling. Combining these results with previous results, we found some universality in RNA and DNA deformations induced by three different stimuli: salt change, temperature, and stretching force. For RNA, these stimuli first modify the major groove width, which is transduced into twist change through twist-groove coupling. For DNA, these stimuli first modify diameter, which is transduced into twist change through twist-diameter coupling. Twist-groove coupling and twist-diameter coupling appear to be utilized by protein binding to reduce DNA and RNA deformation energy cost upon protein binding.

Supplementary Figure Legends 1-9, Methods from Blocking EphB1 Receptor Forward Signaling in Spinal Cord Relieves Bone Cancer Pain and Rescues Analgesic Effect of Morphine Treatment in Rodents

Supplementary Figures 1-9 from Blocking EphB1 Receptor Forward Signaling in Spinal Cord Relieves Bone Cancer Pain and Rescues Analgesic Effect of Morphine Treatment in Rodents

5-Methyl-cytosine (5mC) is one of the most important DNA modifications and plays versatile biological roles. It is well known that 5mC stabilizes DNA duplexes. However, it remains unclear how 5mC affects the kinetics of DNA melting and hybridization. Here, we studied the kinetics of unzipping and rezipping using a 502-bp DNA hairpin by single-molecule magnetic tweezers. Under constant loading rates, 5mC increases the unzipping force but counterintuitively decreases the rezipping force at various salt and temperature conditions. Under constant forces, the non-methylated DNA hops between metastable states during unzipping and rezipping, which implies low energy barriers. Surprisingly, the 5mC DNA can’t rezip after fully unzipping unless much lower forces are applied, where it rezips stochastically in a one-step manner, which implies 5mC kinetically hinders DNA hybridization and high energy barriers in DNA hybridization. All-atom molecular dynamics simulations reveal that the 5mC kinetically hinders DNA hybridization due to steric effects rather than electrostatic effects caused by the additional methyl groups of cytosines. Considering the possible high speed of DNA unzipping and zipping during replication and transcription, our findings provide new insights into the biological roles of 5mC.

Ion-mediated effective interactions are important for the structure and stability of charged particles such as colloids and nucleic acids. It has been known that the intrinsic electrostatic repulsion between like-charged particles can be modulated into effective attraction by multivalent ions. In this work, we examined the dependence of multivalent ion-mediated attraction between like-charged colloidal particles on the particle charge in a wide range by extensive Monte Carlo simulations. Our calculations show that for both divalent and trivalent salts, the effective attraction between like-charged colloidal particles becomes stronger with the increase of the particle charge, whereas it gradually becomes weakened when the particle charge exceeds a “critical” value. Correspondingly, as the particle charge is increased, the driving force for such effective attraction transits from an attractive electrostatic force to an attractive depletion force, and the attraction weakening by high particle charges is attributed to the transition of electrostatic force from attraction to repulsion. Our analyses suggest that the attractive depletion force and the repulsive electrostatic force at high particle charges result from the Coulomb depletion which suppresses the counterion condensation in the limited region between two like-charged colloidal particles. Moreover, our extensive calculations indicate that the “critical” particle charge decreases apparently for larger ions and smaller colloidal particles due to stronger Coulomb depletion and decreases slightly at higher salt concentrations due to the slightly enhanced Coulomb depletion in the intervening space between colloidal particles. Encouragingly, we derived an analytical formula for the “critical” particle charge based on the Lindemann melting law.

When stretched, both DNA and RNA duplexes change their twist angles through twist-stretch coupling. The coupling is negative for DNA but positive for RNA, which is not yet completely understood. Here, our magnetic tweezers experiments show that the coupling of RNA reverses from positive to negative by multivalent cations. Combining with the previously reported tension-induced negative-to-positive coupling reversal of DNA, we propose a unified mechanism of the couplings of both RNA and DNA based on molecular dynamics simulations. Two deformation pathways are competing when stretched: shrinking the radius causes positive couplings but widening the major groove causes negative couplings. For RNA whose major groove is clamped by multivalent cations and canonical DNA, their radii shrink when stretched, thus exhibiting positive couplings. For elongated DNA whose radius already shrinks to the minimum and canonical RNA, their major grooves are widened when stretched, thus exhibiting negative couplings.

CpG methylation of DNA is common in mammalian cells. In sperm, the DNA has the highest level of CpG methylation and is condensed into toroidal structures. How CpG methylation affects DNA structures and interactions is important to understand its biological roles but is largely unknown. Using an RNA-DNA-RNA structure, we observed the equilibrium hopping dynamics between the condensed and extended states of DNA in the presence of polyamines or polylysine peptide as a reduced model of histone tails. Combing with the measured DNA elasticities, we report that CpG methylation of each cytosine nucleotide substantially increases DNA-DNA attraction by up to 0.2 kBT. For the DNA with 57% GC content, the relative increase caused by CpG methylation is up to 32% for the spermine-induced DNA-DNA attraction and up to 9% for the polylysine-induced DNA-DNA attraction. These findings help us to evaluate the energetic contributions of CpG methylation in sperm development and chromatin regulation.

Structural elasticity of double-strand DNAs is very important for their biological functions such as DNA-ligand binding and DNA-protein recognition. By all-atom molecular dynamics simulations, we investigated the bending elasticity of DNA with three typical sequences including poly(A)-poly(T) (AA-TT), poly(AT)-poly(TA) (AT-TA), and a generic sequence (GENE). Our calculations indicate that, AA-TT has an apparently larger bending persistence length (P ∼63 nm) than GENE (P ∼49 nm) and AT-TA (P ∼48 nm) while the persistence length of AT-TA is only very slightly smaller than that of GENE, which agrees well with those from existing works. Moreover, through extensive electrostatic calculations, we found that the sequence-dependent bending elasticity is attributed to the sequence-dependent electrostatic bending energy for AA-TT, AT-TA and GENE, which is coupled to their backbone structures. Particularly, the apparently stronger bending stiffness of AA-TT is attributed to its narrower minor groove. Interestingly, for the three DNAs, we predicted the non-electrostatic persistence length of ∼17 nm, thus electrostatic interaction makes the major contribution to DNA bending elasticity. The mechanism of electrostatic energy dominating sequence effect in DNA bending elasticity is furtherly illustrated through the electrostatic calculations for a grooved coarse-grained DNA model where minor groove width and other microscopic structural parameters can be artificially adjusted.

Background Chronic low back pain affects daily activities at home and workplaces and causes a huge economic burden. Current therapeutic options are very limited and the effects of available pharmacological agents are less than satisfactory. While NSAIDs might be effective for the short term and opioids might help with urgent pain relief and improving the life quality, their long-term use is associated with significant side effects and drug misuse or abuse. To seek alternative pharmacological agents for effective treatment, we examined the therapeutic potential of the extracts of Vaccinia variola -inoculated rabbit skin ( Analgecine, abbreviated as AGC ) in patients with chronic low back pain due to degenerative vertebral disorders. Methods In this randomized multi-center double-blind placebo-controlled phase 3 clinical trial (Chinese Clinical Trial Registry number 2009L01498), we enrolled patients (aged 26–70 years) with chronic low back pain for at least 3 months due to degenerative spinal (vertebral) disorders from 7 medical centers in China, and randomly allocated 459 participants to receive oral AGC or placebo for 28 days to study the efficacy and safety of AGC . Randomization was performed according to a centralized randomization schedule, which was blocked by study sites and generated by an unmasked statistician independent of study conduct and data analysis. Both participants and staff at each study site were masked to treatment assignment. The primary efficacy endpoint was the change of the mean pain intensity, based on an 11-point numerical rating scale, between the baseline and the last week of treatment, with the primary efficacy analysis of intention to treat. The ratio between exposed and unexposed groups was designed to be 3:1 in order to increase the likelihood of demonstrating the AGC effect upon repeated measures. Results 347 patients were assigned to receive AGC (4 units/tablet; 2 tablets twice a day) and 112 patients were to take placebo. Among them, 324 patients taking AGC and 112 receiving placebo completed the assessment. Patients receiving AGC reported significant pain relief at the end of week 2 and 3 compared to those taking placebo, with mean reduction of the pain scores as 1.7 vs. 0.9 at week 2 (p vs . 1.2 at week 3 (p AGC -treated patients reported 85 treatment emergent adverse events while 16 patients taking placebo reported 26 events, but no serious side effects were found to be related to AGC treatment. Conclusion Analgecine ( AGC , 8 units twice daily) effectively alleviates chronic low back pain due to degenerative vertebral disorders when compared to placebo and is well tolerated by tested individuals, and can be considered as a first-line treatment for chronic low pain due to degenerative vertebral diseases.

Background: Isoflurane (IFL), one of the most widely used volatile anesthetics, has an anti-nociceptive effect at the spinal cord level. However, the reported spinal mechanisms of IFL analgesic action remain controversial. The aim of this study was to examine the effect of IFL on the excitability of spinal substantia gelatinosa (SG, lamina II) neurons and the synaptic transmission from primary nociceptive fibers to SG neurons.Methods: Parasagittal spinal slices with a dorsal root attached were cut from the Sprague-Dawley rats (4-6 weeks old). Whole-cell patch-clamp recordings were made from SG neurons at room temperature. Dorsal root stimulation was used to evoke excitatory postsynaptic currents (eEPSCs). Depolarizing current injection was used to display the firing pattern of action potentials. IFL was given as volume percent (3%) by passing the gas mixture (flow rate, 0.5 L/minute) into equilibrated artificial cerebral spinal fluid (ACSF) for at least 15 minutes at room temperature.Results: IFL significantly suppressed the peak amplitude of monosynaptic eEPSCs mediated by primary Aδ and C fibers. Regardless of neuron types, IFL significantly decreased the frequency of action potentials in all SG neurons tested.Conclusions: IFL may play analgesic role through inhibiting or weakening the excitability and synaptic transmission of spinal nociceptive pathway. Citation: Jie Liu, Gen-Lin Ji, Jian-Min Li, Tun Liu, Qun Wang, Ya-Nan Pu, et al. Isoflurane suppresses the excitability and synaptic transmission of spinal nociceptive pathway in rats. J Anesth Perioper Med 2015; 2: 61-5. doi: 10.24015/JAPM.2015.0010This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Aim of review: Diabetes is a chronic and slowly progressing disease that has a tendency todevelop rapidly deteriorating complications such as major adverse cardiovascular events (MACE), especially under the stress of surgery. While clinical strategy to prevent MACE is controversial and uncertain.Method: We conducted a comprehensive review of current clinical strategies in preventing perioperative MACE, in particularly related to diabetic patients.Results: The major findings are: 1) Current clinical studies have demonstrated that coronary artery bypass graft (CABG) is still a better therapy than percutaneous coronary intervention (PCI) on the ground of reducing repeat revascularization, myocardial infarction and death for most diabetic patients with left main-stem and multivessel coronary artery disease who require revascularization, however, it remains to be studied whether coronary revascularization before noncardiac surgery can protect diabetic patients from MACE; 2) There is lack of evidence that intensive or "tight" glycemic control perioperatively can reduce MACE, instead, a moderate or less stringent glucose management probably is safer for patients undergoing surgery;3) The recent results of clinical trials on beta-blockers appear to be disappointing in preventing MACE in surgical patients, including diabetic patients. Meanwhile, the perioperative therapy with statins, angiotensin-converting enzyme inhibitors or multifactorial interventions is promising in preventing MACE in diabetic patients.Summary: Further studies targeted at preventing MACE in diabetic patients undergoing surgery are needed in order to fight this major health problem in perioperative medicine. Citation: Jian-zhong Sun, Hai-long Dong, Li-nong Yao, Hong Liu. Preventing perioperative major adverse cardiovascular events in patients with diabetes. J Anesth Perioper Med 2015; 2: 96-106. doi: 10.24015/JAPM.2015.0014This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Treating surrounding rock as absolutely loose mass helps to determine positions of the four key reinforced points in roadway, in other words, they are juncture locations between 45 degrees angle directions of two arch haunches and two footwalls. Surrounding rock deformation can be effectively controlled with the help of reinforcement on key reinforced points, furthermore, it is possible to save lots of supporting materials, in short, it is a very effect way. New technology proposed in this paper what is about stable augmentation has deeply unearthed important effect of anchor in floor of roadway, it argues that anchor in floor has quadruplex efficacies: transferable effect of surrounding rock stress, displacement boundary effect, decreasing equivalent stress of basic angle area and the inverted wedge effect, so it can suppress floor heave effectively, and this lays a solid foundation for roadway stability. Article deeply analyzed basic mechanism about deformation of roadway under loading, exposed essentiality of roadway support, and provided important theoretical basis for floor heave control emerged more and more frequently in deep mine roadway. Key words: broken rock zone, bolt support, floor heave, equivalent stress

This paper synthetically analyzed essential features of various support methods in engineering geological soft rock, proposed a scheme that organically combines active support (like anchor bolt support) with passive support (like U-shaped steel bracket support), analyzed fundamentals of this combined support scheme, and forecasted support effect of this support scheme. It provides roadway support design with fresh ideas. U-shaped steel bracket can be translated into special anchor plate with the help of a section of simple steel channel beam, so effect of anchor bolt can be ensured; U-shaped steel bracket , this extremely long curved beam ,will be divided into several sections of short beams, so its bearing ability will be several times improved. Cost of this combined support scheme is low, its process is simple and its application prospect is wide.

Floor grouting in soft rock roadway is one of the effective ways in floor heave control, and this method is being promoted quietly and quickly, but the applying effect is far from satisfied, the reasons are that: the depth of floor grouting is not enough and the influence of water hiding in floor hasnt been attached great importance. This paper analyzed the characteristics of floor broken rock zone in soft rock roadway and pointed out the importance of floor grouting in soft rock roadway, then proposed a practical method of floor grouting. The analysis conclusions show that: the depth of floor broken rock zone that can reach more than 4m is much greater than that of roof broken rock zone due to the double influences of surrounding rock stress and water; the important role of floor grouting in soft rock roadway is draining off free water from broken rock zone in order to strengthen roadway floor; floor grouting includes three important steps: plugging water channels, pumping out free water and grouting in deep holes. The article has revealed common blind spots of floor grouting methods existing, the method this article has noted can solve floor heave of soft rock roadway well, and it has very good application prospects. Key words: soft rock roadway, floor grouting, broken rock zone, free water in floor

Background The cholinergic arousal systems are known to critically regulate the state of consciousness. The aim of this study was to determine the effect of isoflurane on the inhibitory or excitatory neurotransmitters efflux in important nuclei within the cholinergic arousal system using in vivo intracerebral microdialysis. Methods The efflux of glutamate, gamma-aminobutyric acid (GABA), or acetylcholine in the posterior hypothalamus (PH), the basal forebrain (BF), and the somatosensory cortex (S1BF) of rats was detected using intracerebral microdialysis under an awake condition and at 0.5-2.0 minimum alveolar concentration (MAC) isoflurane anesthesia. The intrabasalis perfusion of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate on the cortical acetylcholine effluxes was also examined under both conditions. Results Isoflurane had no influence on the glutamate and GABA efflux in the PH, whereas in the BF, it dose-dependently increased glutamate efflux and decreased GABA efflux. A transient increase in glutamate efflux at 1.0 MAC and a decrease in GABA at 0.5-1.5 MAC were observed in the S1BF. Isoflurane dose-dependently decreased acetylcholine efflux in the S1BF. Perfusion of the BF with AMPA increased acetylcholine efflux in the S1BF with electroencephalographic activation during 0.75 MAC isoflurane anesthesia, suggesting an inhibitory action of isoflurane on AMPA receptors in the BF. However, N-methyl-D-aspartate had no effect on these parameters. Conclusion Isoflurane induces both excitatory and inhibitory actions in the cholinergic arousal system. The predominant inhibitory action of isoflurane over its excitatory action at the BF would result in the decrease in the acetylcholine efflux in the S1BF.

MAGE-n is a new member of MAGE gene family and has been demonstrated closely associated with hepatocellular carcinoma (HCC). In this study, MAGE-n-derived peptide-specific cytotoxic T lymphocytes (CTL) were induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with HLA-A2-restricted MAGE-n peptide-pulsed T2 cells. The induced CTLs exhibited specific lysis against T2 cells pulsed with the peptide and HLA-A2+ HCC cells expressing MAGE-n, while HLA-A2+ HCC cell lines that did not express MAGE-n could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-MHC class I monoclonal antibody. These results suggested the MAGE-n peptide could be a potential target of specific immunotherapy for HLA-A2 patients with HCC.

Foreign body aspiration is a common life-threatening event in young children. Tracheobronchial foreign body removal is usually performed by rigid tracheobronchoscopy under general anesthesia. Anesthetic and ventilation techniques vary greatly among anesthesiologists and institutions. In the present retrospective study, we report our anesthetic experience over 5 years. We describe complications and outcomes and analyze the clinical characteristics of anesthesia and ventilation.We retrospectively reviewed relevant clinical findings of 586 pediatric patients treated with rigid tracheobronchoscopy under general anesthesia. All procedures were performed under inhaled sevoflurane anesthesia combined with remifentanil infusion, with spontaneous respiration assisted by high-frequency jet ventilation (HFJV) and topical airway anesthesia.Among 586 patients, the foreign body was successfully removed by rigid tracheobronchoscopy in 558 patients, and no foreign body was found in 28 patients. Laryngospasm was observed during the procedure in five patients. Hypoxemia was observed in 15 patients (2.6%). No severe complications or deaths occurred. The mean operation time was 22 min and the average hospital stay was 2 days.Inhaled sevoflurane anesthesia combined with remifentanil infusion, with spontaneous respiration assisted by HFJV and topical airway anesthesia, is safe and effective for tracheobronchial foreign body removal.

Treating bone cancer pain continues to be a clinical challenge and underlying mechanisms of bone cancer pain remain elusive. Here, we report that EphB1 receptor forward signaling in the spinal cord is critical to the development of bone cancer pain and morphine tolerance in treating bone cancer pain. Tibia bone cavity tumor cell implantation (TCI) produces bone cancer–related thermal hyperalgesia, mechanical allodynia, spontaneous and movement-evoked pain behaviors, and bone destruction. Production and persistence of these pain behaviors are well correlated with TCI-induced upregulation of EphB1 receptor and its ligand ephrinB2 in the dorsal horn and primary sensory neurons. Spinal administration of an EphB1 receptor blocking reagent EphB2-Fc prevents and reverses bone cancer pain behaviors and the associated induction of c-Fos and activation of astrocytes and microglial cells, NR1 and NR2B receptors, Src within the N-methyl-D-aspartate receptor complex, and the subsequent Ca2+-dependent signals. The exogenous ligand ephrinB2-Fc upregulates level of phosphorylation of NR1 and NR2B receptors depending on the activation of EphB1 receptor. Spinal administration of EphB2-Fc and ephrinB2-Fc induces downregulation of EphB1 and ephrinB2, respectively, accompanied with increased activity of matrix metalloproteinase (MMP)-2/9. Blocking MMP-2 or MMP-9 reverses EphB1-Fc treatment–induced downregulation of EphB1 receptor. In addition, spinal blocking or targeted mutation of EphB1 receptor reverses morphine tolerance in treating bone cancer pain in rats and defensive pain in mice. These findings show a critical mechanism underlying the pathogenesis of bone cancer pain and suggest a potential target for treating bone cancer pain and improving analgesic effect of morphine clinically. Cancer Res; 71(13); 4392–402. ©2011 AACR.

It remains to be established whether spinal cord ischemic tolerance can be induced by limb remote ischemic preconditioning (RIPC), and the mechanisms underlying the neuroprotective effects of RIPC on the spinal cord need to be clarified.Spinal cord ischemia was studied in New Zealand White rabbits. In experiment 1, all rabbits were subjected to 20-min spinal cord ischemia by aortic occlusion. Thirty minutes before ischemia, rabbits were subjected to sham intervention or RIPC achieved by bilateral femoral artery occlusion (10 min ischemia/10 min reperfusion, two cycles). Dimethylthiourea (500 mg/kg, intravenously), a hydroxyl radical scavenger, or vehicle was given 1 h before RIPC. Antioxidant enzyme activity was measured along with spinal cord histology and neurologic function. In experiment 2, rabbits were subjected to spinal cord ischemia, with or without RIPC. In addition, rabbits were pretreated with various doses of hexamethonium.RIPC improved neurologic function and reduced histologic damage. This was associated with increased endogenous antioxidant activity. Dimethylthiourea inhibited the protective effects of RIPC. In contrast, there was no effect of hexamethonium on the protective effect of RIPC.An initial oxidative stress acts as a trigger to upregulate antioxidant enzyme activity, rather than the neural pathway, and plays an important role in the formation of the tolerance against spinal cord ischemia by limb RIPC.

The neuroprotective effect of the cyclooxygenase (COX) inhibitor has been demonstrated in acute and chronic neurodegenerative processes. But its function under cerebral ischemic conditions is unclear. This study was designed to evaluate the neuroprotective efficacy of emulsified flurbiprofen axetil (FA, COX inhibitor) and its therapeutic time window in a model of transient middle cerebral artery occlusion (MCAO) in rats.Forty-eight male SD rats were randomly assigned into six groups (n = 8 in each group); three FA groups, vehicle, sham and ischemia/reperfusion (I/R) groups. Three doses of FA (5, 10 or 20 mg/kg, intravenous infusion) were administered just after cerebral ischemia/reperfusion (I/R). The degree of neurological outcome was measured by the neurologic deficit score (NDS) at 24, 48 and 72 hours after I/R. Mean brain infarct volume percentage (MBIVP) was determined with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining at 72 hours after I/R. In three other groups (n = 8 in each group), the selected dosage of 10 mg/kg was administrated intravenously at 6, 12 and 24 hours after I/R.The three different doses of FA improved NDS at 24, 48 and 72 hours after I/R and significantly reduced MBIVP. However, the degree of MBIVP in the FA 20 mg/kg group differed from that in FA 10 mg/kg group. Of interest is the finding that the neuroprotective effect conferred by 10 mg/kg of FA was also observed when treatment was delayed until 12 - 24 hours after ischemia reperfusion.COX inhibitor FA is a promising therapeutic strategy for cerebral ischemia and its therapeutic time window could last for 12 - 24 hours after cerebral ischemia reperfusion, which would help in lessening the initial ischemic brain damage.

To test the hypothesis that spinal cord protection induced by ischemic postconditioning is mediated by an increase of endogenous antioxidant enzyme activities during reperfusion phase in spinal cord.Seventy-eight male New Zealand rabbits were randomly divided into 3 groups: Sham group (n = 18) undergoing sham operation without aortic occlusion; ischemia/reperfusion (I/R) group (n = 30) undergoing occlusion of the infrarenal abdominal aorta for 20 min, followed by reperfusion; and postconditioning (PostC) group (n = 30) undergoing occlusion of the infrarenal abdominal aorta for 20 min followed by 3 cycles of 30 s reperfusion/30 s ischemia just at the onset of reperfusion. 30 min, and 1, 3, 6, 24, and 48 h after reperfusion 5 rabbits from each group (and 3 from the Sham group) were killed with their spinal cords taken out, and spectrophotometric method was used to determine the antioxidant enzyme activity and malondialdehyde (MDA) content 6, 24, and 48 h after reperfusion motor function scoring of the hind limbs was conducted.(1) The motor function scores of the PostC group were significantly higher than those of the I/R group 6, 24, and 48 h after reperfusion (all P0.05). (2) The activities of superoxide dismutase (SOD) and catalase (CAT) in the spinal cord tissue of the PostC group 30 min-6 h after reperfusion were all significantly higher than those of the I/R group (all P0.05). There were no significant differences in the activity of glutathione peroxidase (GSH-px) at different time points between the PostC and I/R groups. (3) The MDA levels 24 h and 48 h after reperfusion of the PostC group were both significantly lower than that of the I/R group (both P0.01).Ischemic postconditioning shows effect against spinal cord ischemic-reperfusion injury mediated, at least partially, by up-regulating the activities of SOD and CAT in spinal cord during early reperfusion phase.

To explore the effects of remote ischemic preconditioning (RIPC) on cerebral ischemia/reperfusion (I/R) injury.Seventy male SD rats were randomly divided into seven groups (n=10 for each): (1) CONTROL GROUP: animals were subjected to I/R. (2)RIPC groups: RIPC was performed by 3 cycles of occlusion/reperfusion of bilateral femoral arteries for 5 minutes/5 minutes. According to the different intervals between RIPC and middle cerebral artery occlusion (MCAO), 6 RIPC subgroups were established: RIPC 30 minutes, 1, 2, 12, 24 and 48 hours subgroups, with intervals between RIPC and cerebral I/R of 30 minutes, 1, 2, 12, 24 and 48 hours respectively. The duration of MCAO was 120 minutes and reperfusion for 24 hours. The neurological dysfunction score (NDS) was evaluated at 24 hours after reperfusion. The infarction volume was then assessed with tetrazolium chloride (TTC) staining.The NDSs of RIPC 1, 2 and 24 hours groups were significantly lower than that of control group (all P0.05), while with no significant differences of RIPC 30 minutes, 12 and 48 hours groups (all P0.05). The area percentage of infarction in RIPC 1 hour [(17.9+/-7.5)%, P=0.016], RIPC 2 hours [(18.3+/-11.2)%, P=0.019] and RIPC 24 hours [(20.2+/-11.9)%, P=0.047] groups was significantly smaller than that in control group [(30.5+/-9.8)%], while no significant differences were observed in RIPC 30 minutes, 12 and 48 hours groups (all P0.05).The RIPC the brain against focal cerebral I/R injury in rats, and the protection window is 1 to 2 hours after pretreatment and resumes after 24 hours.

Background Cholinergic arousal system plays an important role in the maintenance of consciousness. The authors investigated whether the intrabasalis injection of orexin-A or orexin-B and the electrically stimulated pedunculopontine tegmentum nuclei (PPTg: the origin of cholinergic ascending pathways) may alter acetylcholine efflux and electroencephalographic activity in the somatosensory cortex in relation to the orexinergic system in isoflurane-anesthetized rats. Methods Either orexin-A (10, 30, or 100 pmol) or orexin-B (10, 30, or 100 pmol) (n = 6 each) was injected into the basal forebrain while the electroencephalogram was measured during 1.0 minimum alveolar concentration (1.2%) isoflurane anesthesia. Injection of Ringer's solution was used as a control. The PPTg was electrically stimulated twice with the following conditions: 1-s stimulus train (0.2 ms, 100 Hz, 400 microA) per min for 20 min. Twenty minutes before the second PPTg stimulation, Ringer's solution or 20 microg SB334867, an orexin-1 receptor antagonist (n = 5 each) was injected into the basal forebrain. Results Injection of orexin-A (30 and 100 pmol) and orexin-B (100 pmol) significantly increased the acetylcholine efflux in the somatosensory cortex (P < 0.05). Injection of orexin-A (10, 30, 100 pmol) and orexin-B (30, 100 pmol) changed the burst and suppression patterns to arousal electroencephalogram. Compared with orexin-B, injection of a lower dose of orexin-A induced increase in the acetylcholine efflux and arousal electroencephalogram. SB334867 significantly attenuated the increases in the acetylcholine efflux and electroencephalographic activation evoked by PPTg stimulation. Conclusion The authors demonstrated that orexin-A was more potent than orexin-B in producing alteration of cholinergic basal forebrain neuronal activity and that the cortical activation induced by the PPTg stimulation against isoflurane anesthesia may be mediated through the orexin-1 receptors in the basal forebrain.

Neurological deficits after spinal cord or spine surgery pose a serious problem. There have been many attempts to monitor spinal cord function during spine or spinal cord surgery by using somatosensory evoked potentials (SEP) recorded from the scalp (Grundy and Villani, 1988; Levy et al., 1984). Unfortunately these potentials are susceptible to the effects of anesthetic agents and vary during the course of anesthesia and surgery (McPherson et al., 1985; McPherson and Ducker, 1988; Clark and Rosner, 1973; Samra et al., 1987; McPherson and Levitt, 1991).

To predict the HLA-A2-restricted CTL epitopes of tumor antigens associated with hepatocellular carcinoma (HCC).MAGE-1, MAGE-3, MAGE-8, P53 and AFP were selected as objective antigens in this study for the close association with HCC. The HLA-A*0201 restricted CTL epitopes of objective tumor antigens were predicted by SYFPEITHI prediction method combined with the polynomial quantitative motifs method. The threshold of polynomial scores was set to -24.The SYFPEITHI prediction values of all possible nonamers of a given protein sequence were added together and the ten high-scoring peptides of each protein were chosen for further analysis in primary prediction. Thirty-five candidates of CTL epitopes (nonamers) derived from the primary prediction results were selected by analyzing with the polynomial method and compared with reported CTL epitopes.The combination of SYFPEITHI prediction method and polynomial method can improve the prediction efficiency and accuracy. These nonamers may be useful in the design of therapeutic peptide vaccine for HCC and as immunotherapeutic strategies against HCC after identified by immunology experiment.

Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1.A HLA-A2.1-restricted CTL epitope was identified by using an improved "reverse immunology" strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1.Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro.Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.

To investigate the potency and antitumor effect of specific cytotoxic T lymphocytes induced by hepatocarcinoma antigen peptide EPVTKAEML-loaded dendritic cells (DCs).The healthy donors of HLA-B7 genotype were selected by PCR-SSP. Isolated and cultured DCs from HLA-B7 donors' spleen tissues were loaded with the peptide EPVTKAEML from hepatocarcinoma cell line HHCC. The specific CTL reaction and inhibitory effect of anti-HLA-I molecule mAb on CTL cytotoxicity were detected in-vitro by (51)Cr-release assay.4 donors with HLA-B7 genotype were found. Their spleen T lymphocytes could be induced to proliferate and differentiate into specific CTLs by DCs loaded with the peptide. Antibody-blocking test demonstrated that the cytotoxity of CTLs was strictly limited by MHC-I molecule.EPVTKAEML-loaded DCs can induce efficient and specific antitumor immune reaction in-vitro.

Since transfection of established tumors with immunostimulatory genes can elicit antitumor immunity, we treat mouse HCC with in vivo transfection of superantigen SEA and/or costimulatory molecule CD80 and evaluated the safety and efficacy.Mice with HCC were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin A and/or CD80. Then the mice were evaluated for tumor regression, systemic immunologic responses, survival times and treatment-associated toxicity.Of all treated mice, the overall response rates (complete or partial remission) for SEA, CD80 and SEA/CD80 treated mice in this study were 65%, 60% and 75% separately, and were significantly higher than that of untreated mice. Most of the treat mice completed the therapy without any significant reaction. CTL activity increased with time of treatment and correlated temporally with an objective tumor response. Also our results indicated that local intratumoral expression of SEA did not lead to detectable deletion or anergy of SEA-reactive spleen T cells. Survival times for hepatoma mice in this study treated by intratumoral injection of SEA, CD80 and SEA/CD80 were prolonged significantly (P0.01) compared with the control mice.

To predict, synthesize, and identify HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes of the tumor the novel antigen MAGE-n.Long-distance prediction system SYFPEITHI combined with polynomial method was used to predict the HLA-A2-restricted CTL epitopes of the tumor antigen MAGE-n. The candidate epitopes were synthesized with solid phase strategies, purified with reverse phase high-performance liquid chromatography and identified by mass spectrometry, The binding affinity and biding stability of the synthesized peptides were examined by cellular competition-based HLA-A2 peptide binding assay, T2 peptide stabilization assay, and peptide-major histocompatibility complex dissociation assay.Five HLA-A2-restricted CTL epitopes of MAGE-n were selected: The epitopes QLVFGIEVV (159-167), IMPKTGGLI (195-203), and FLIIVLMI (201-209) with high HLA-A2 binding affinity (LC(50)15 micro mol/L) and binding stability (DT(50)6 h) were selected as candidate epitopes for further study in immunotherapy for tumor.Epitope prediction combined with epitope reconstruction improves the study of HLA-A2-restricted CTL epitopes of the tumor antigen MAGE-n. The selected epitopes of MAGE-n may be used in the design of therapeutic peptide vaccine for hepatocellular carcinoma.

To investigate the targeted killing effect to SKBr3 cells due to the expression of a secreted fusion protein consisting of anti-erbB2 antibody and reversed caspase-3.A recombinant plasmid pCMV-e23scFv-PEII-revcasp 3 was constructed by subcloning reversed caspase-3 gene to the downstream of anti-erbB2 antibody and transfected into Jurkat cells. The cell lines which secreted expressing fusion protein stably were selected. The fusion protein in media was detected by ELISA and the media was used to culture human breast cancer SKBr3 cells. The recombinant plasmids with liposomes was administrated to BALB/C nude mouses bearing SKBr3 tumor by intramuscular injection. The targetting effect of the recombinant fusion protein caspase-3 was detected by indirect immunofluorescence staining.Fusion protein can be expressed and secreted by Jurkat cells stably and kill SKBr3 cells. Significant prolonged survival time (prolonged by 72%) and inhibition of tumor growth in vivo (within inhibition ratio of 77%) were seen in the group administered with recombinant plasmids. Indirect immunofluorescence staining showed that the recombinant fusion protein caspase-3 has targetting effect.Secreted expression of the fusion protein consisting of anti-erbB2 antibody and reversed caspase-3 can targetedly induce SKBr3 cells to death.

To establish a strain of human cervical carcinoma cell line and to provide a cervical carcinoma animal model.The cervical carcinoma specimens incised aseptically were cultured in vitro by tissue culture methods, giving a tumor cell growth curve. Morphology of the cells was observed, with cell cycling analysis and chromosome analysis performed. The tumor markers (ER, PR, Keratine, PCNA) expressions of the cell line were detected by immuno-cytochemical technique.A human cervical carcinoma cell line HCC-0214 (H) has been obtained by in vitro tissue culture methods. The cells have been maintained for 16 months through 131 passages, showing a stable growth with a population doubling time of 35.48 h and a tendency to pile up without contact inhibition. The ultrastructure showed typical desmosomes and numerous tonofilaments. Chromosome analysis revealed the number of chromosomes per cell varied from 35-156 with a stem-line number of 58-80 (64.8%). The morphology of chromosomes showed human tumor cell structure. The tumor markers (ER, PR, Keratine, PCNA) of the cells showed a high expression. The DNA index was 1.931 by flow cytometry (FCM). The histopathology of the transplanted tumors in nude mice was the same as the original tumor, though with none successful by serum culture.A human cervical carcinoma cell line HCC-0214 established by tissue culture is identical to the primary cancer cell in biological characters. After the cells have been passaged for more than 16 months continually, their characteristics are still retained. Therefore, HCC-0214 may be used as a stable cell line.

To construct an eukaryotic superantigen gene expression vector containing the recombinant gene of SEA and CD80 molecule transmembrane region (CD80TM), and to express staphylococcus enterotoxin A (SEA) on the membrane of hepatocellular carcinoma (HCC) cell to form a superantigen gene modified tumor vaccine for HCC.SEA and linker-CD80TM gene were amplified through PCR from plasmid containing cDNA of SEA and CD80. Gene fragments were then subcloned into the multiple cloning sites of retroviral vector pLXSN. Recombinant plasmid was transferred into HepG2 cells mediated with lipofectamine, positive clones were selected in culture medium containing G418. RT-PCR and indirect immunofluorescence studies confirmed that SEA was expressed specifically on HCC cell membrane. INFgamma-ELISPOT study demonstrated that SEA protein was expressed on the membrane of HCC cells. Cytotoxicity of HepG2-SEA primed CTLs (SEA-T) was analyzed by (51)Cr release assay. T cells cultured with rhIL-2 (IL-2-T) were used as control.Restriction digestion and sequence analyses confirmed the correctness of length, position and orientation of inserted fusion genes. SEA was expressed on the surface of HepG2 cells, HepG2-SEA had strong stimulating effect on production of HepG2 specific CTL (P0.001). SEA-T had enhanced cytotoxicity to HepG2 cells (P0.05).Tumor cell membrane expressed superantigen can be used to reinforce the immune effect of tumor cell vaccine for HCC, which provides a new method of the enhanced active immunotherapy for HCC.