Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression.

Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of BCL-XL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency. Treatment with a BCL inhibitor ABT-263 further improves HDR efficiency by 70% and KO efficiency by 40%. The increased genome editing efficiency is attributed to higher expressions of Cas9 and sgRNA in surviving cells after electroporation. HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene. Moreover, KO efficiency of 100% can be achieved in a bulk population of cells with biallelic HDR KO followed by double selection, abrogating the necessity for single cell cloning. Taken together, these simple yet highly efficient editing strategies provide useful tools for applications ranging from manipulating human iPSC genomes to creating gene-modified animal models.

Nε-(carboxymethyl)-lysine, White Matter, and Cognitive Function in Diabetes Patients.

OBJECTIVE: To study the relationship of Nε-(carboxymethyl)-lysine level (CML) with microstructure changes of white matter (WM), and cognitive impairment in patients with type 2 diabetes mellitus (T2DM) and to discuss the potential mechanism underlying T2DM-associated cognitive impairment. METHODS: The study was performed in T2DM patients (n=22) with disease course ≥5 years and age ranging from 65 to 75 years old. A control group consisted of 25 sex- and age-matched healthy volunteers. Fractional anisotropy (FA) of several WM regions was analyzed by diffusion tensor imaging scan. Plasma CML levels were measured by enzyme-linked immunosorbent assay, and cognitive function was assessed by Mini-Mental State Examination and Montreal cognitive assessment (MoCA). RESULTS: The total Mini-Mental State Examination score in the patient group (25.72±3.13) was significantly lower than the control group (28.16±2.45) (p<0.05). In addition, the total MoCA score in the patient group (22.15±3.56) was significantly lower than the control group 25.63±4.12) (p<0.01). In the patient group, FA values were significantly decreased in the corpus callosum, cingulate fasciculus, inferior fronto-occipital fasciculus, parietal WM, hippocampus, and temporal lobes relative to corresponding regions of healthy controls (p<0.05). Plasma CML level was negatively correlated with average FA values in the global brain (r=-0.58, p<0.01) and MoCA scores (r=-0.47, p<0.05). CONCLUSIONS: In T2DM, WM microstructure changes occur in older patients, and elevations in CML may play a role in the development of cognitive impairment.

A new method for evaluation of motor injury after acute brain ischemic damage.

PURPOSE: Motor impairment is an important index for assessing the extent of brain injury. The present study uses a new method, the movement capture analysis (MOCA) system, for assessing motor damage after acute ischemia. MATERIALS AND METHODS: Forty rats were divided into four groups: standard ischemia, sham-operated, Dizocilpine (MK-801), and Ginkgo biloba extract (GBE) groups. Brain ischemia was induced using the temporary right middle cerebral artery occlusion model. Longa score and MOCA were used to assess motor injury one day after ischemia. Infarct volume was delineated with 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining. The correlation of infarct volume with Longa score and MOCA data was calculated. RESULTS: Compared with the sham-operated group (0.10 ± 0.31), Longa scores of MK-801 (2.33 ± 0.73), GBE (1.80 ± 0.58), and standard (2.88 ± 0.83) groups showed a statistical difference (p < 0.05); however, it was unable to discern the difference between MK-801 and standard groups. MOCA was able to clearly discern the differences in motor disparity among the four groups, standard (1.00 ± 0.19), sham-operated group (0.17 ± 0.02), MK-801 (0.79 ± 0.08), GBE (0.38 ± 0.05) (p < 0.05). Both MK-801 (18.03 ± 0.96%) and GBE (10.82 ± 1.93%) treatment reduced infarct size compared with the standard ischemia group (25.88 ± 1.16%) (p < 0.05). The MOCA data showed a more significant correlation with infarct size than Longa score (r = 0.85:0.53). CONCLUSIONS: MOCA system proved to be more sensitive than the Longa score. It may potentially be more accurate method for behavioral evaluation in clinical trials.

[Research on the dynamics of ion debris from Sn plasma by use of dual laser pulses].

Extreme ultraviolet lithography is one of the most promising technologies on the next generation of high-capacity integrated circuit manufacturing. However, techniques for ion debris mitigation have to be considered in the application of extreme ultraviolet source for lithography. In our paper the dynamics of ion debris from Sn plasma by using dual ns laser pulses were investigated. The results show that debris from plasma greatly depends on the energy of pre-pulse and the delay time between the two laser pulses. The energy of Sn ions debris was efficiently mitigated from 2. 47 to 0. 40 keV in the case of dual laser pulses, up to 6. 1 times lower than that by using single laser pulse. We also found that Sn ions debris can be mitigated at all angles by using the dual laser pulses method.

[The LIBS experiment condition optimization of alloy steel].

Laser induced plasma spectroscopy of alloy steel was produced by Nd : YAG pulsed laser at 1 064 nm, and the spectral signal was detected by high resolution and width controlled ICCD. Several Fe atomic spectral lines such as 404.581, 414.387, 427.176 and 438.355 nm were chosen for analysis, and the effects of different experimental parameters on LIBS spectral signal intensity were investigated. It is shown that the experimental parameters such as pulse energy, laser focus location and laser delay time have great influence on the LIBS signal. LIBS signals with high spectral intensity and signal-background ratio (SBR) as well as the optimum experiment conditions were obtained by optimizing these experiment parameters so as to make composition analysis of the alloy steel.

Enhanced in vitro antitumor efficacy and strong anti-cell-migration activity of a hydroxycamptothecin-encapsulated magnetic nanovehicle.

A 10-hydroxycamptothecin-encapsulated magnetic nanovehicle (HEMN) was fabricated by coencapsulating Fe(3)O(4) nanoparticles and 10-hydroxycamptothecin (HCPT) into a micelle core self-assembled from the amphiphilic copolymer methoxy-poly(ethylene glycol)-poly(d,l-lactide-co-glycolide) through a facile dialysis method. A satisfactory drug-loading content of (9.03 ± 0.67) % and a relatively high encapsulation efficiency of (53.52 ± 6.46) % were achieved. In vitro drug release was performed by membrane dialysis and a pH-dependent release behavior was observed. In comparison with free HCPT dissolved in dimethylsulfoxide, HEMNs showed a greatly improved in vitro antitumor efficacy against three different human cancer cell lines-HeLa, A549, and HepG2-and lower IC(50) values were measured. The mechanism of cell death was investigated, and it was clearly demonstrated that the apoptosis process was triggered. An in vitro wound-healing assay and a transwell assay indicated that HEMNs exerted much stronger activity in inhibiting HeLa cell migration. The cellular uptake of HEMNs in a desired area can be significantly enhanced by an external magnetic field. These results demonstrate HCPT-encapsulated magnetic nanovehicles might have important potential in clinical applications for inhibiting tumor metastasis and for targeted drug delivery.

Improved and Flexible HDR Editing by Targeting Introns in iPSCs.

Highly efficient gene knockout (KO) editing of CRISPR-Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.

Effects of Ginkgo biloba extracts on NMDA-activated currents in acutely isolated hippocampal neurons of the rat.

Ginkgo biloba extracts (GBE) have long been used as a traditional herbal medicine for treating central nervous system diseases and peripheral vascular diseases, but the underlying mechanisms have yet to be elucidated. Furthermore, traditional GBE is in the form of microsomes and only dissolves in organic solvents; its clinical applications have been greatly limited. Therefore, in the present study, nanometer GBE (nGBE) was prepared utilizing supercritical anti-solvent (SAS) upon CO(2) -supercritical fluid extraction (CO(2) -SPF). Using whole-cell patch clamp techniques, the effects of different preparations of GBE on N-methyl-D-aspartate (NMDA)-activated currents (I(NMDA) ) from acutely isolated rat hippocampal neurons were investigated and the difference in protective potency between nGBE and mGBE evaluated. The results showed that the inward current activated by NMDA could be depressed by mGBE and nGBE. The inhibitory rates were 40% ± 17% and 64% ± 15%, and the half-inhibition concentrations (IC(50) ) were 0.0210 ± 0.0055 and 0.0262 ± 0.0038 mg/mL, respectively. In comparison, the modulatory effect of nGBE (dissolved in extracellular solution) on NMDA-activated current was significantly greater than that of mGBE (dissolved in DMSO) (p < 0.05). This indicated that the modulatory effects of GBE on NMDA-activated current may contribute to the neuroprotective effects of GBE and the modulatory effect of nGBE on NMDA-activated current was greater than that of mGBE.

The Effect of Fatigue on Wheelchair Users' Upper Limb Muscle Coordination Patterns in Time-Frequency and Principal Component Analysis.

An assessment of shoulder muscle coordination patterns is important to gain insight into muscle fatigue during wheelchair propulsion. The objective of the present study was to quantify muscle coordination changes over time during fatiguing wheelchair propulsion, as the muscles go through distinct levels of fatigue, a) non-fatigued, b) transiting to fatigue and c) fatigued to exhaustion. We recorded surface electromyography (sEMG) signals of the anterior deltoid (AD), middle deltoid (MD), posterior deltoid (PD), infraspinatus (IS), upper trapezius (UT), sternal head of the pectoralis major (PM), biceps brachii (BB), and triceps brachii (TB) during a wheelchair incremental exercise test. Nine wheelchair users with a diagnosis of spina bifida or T6-T12 spinal cord injury volunteered for the study. Oxygen uptake and SmartWheel kinetic parameters were also recorded during the test. EMG signals were processed by wavelet and principal component analysis (PCA), allowing for an assessment of how wheelchair users modify their muscle coordination patterns over time. Analyses of covariance (ANCOVA) were conducted to identify the main effect of fatigue levels on muscle coordination patterns by controlling for the effect of increased workload as covariate. A significant effect of fatigue levels on the PC1 and PC3 loading scores was found after controlling for the effect of increasing workloads (with both cases). In addition, PC3 reflects the most dominant fatigue effect on muscle coordination patterns which are not affected by increased ergometer workload. PC3 indicates muscle imbalance when muscles are fully fatigued and muscle co-contraction when muscles are beginning to fatigue. We conclude that fatigue-related changes in neuromuscular activity during wheelchair propulsion contribute to muscle imbalance and reflect a strategy of stiffening the shoulder joint.

Assessing time-dependent association between scalp EEG and muscle activation: A functional random-effects model approach.

This study investigates time-dependent associations between source strength estimated from high-density scalp electroencephalogram (EEG) and force of voluntary handgrip contraction at different intensity levels. We first estimate source strength from raw EEG signals collected during voluntary muscle contractions at different levels and then propose a functional random-effects model approach in which both functional fixed effects and functional random-effects are considered for the data. Two estimation procedures for the functional model are discussed. The first estimation procedure is a two-step method which involves no iterations. It can flexibly use different smoothing methods and smoothing parameters. The second estimation procedure benefits from the connection between linear mixed models and regression splines and can be fitted using existing software. Functional ANOVA is then suggested to assess the experimental effects from the functional point of view. The statistical analysis shows that the time-dependent source strength function exhibits a nonlinear feature, where a bump is detected around the force onset time. However, there is the lack of significant variations in source strength on different force levels and different cortical areas. The proposed functional random-effects model procedure can be applied to other types of functional data in neuroscience.

Fractal dimension assessment of brain white matter structural complexity post stroke in relation to upper-extremity motor function.

Little is known about the association between brain white matter (WM) structure and motor function in humans. This study investigated complexity of brain WM interior shape as determined by magnetic resonance imaging (MRI) and its relationship with upper-extremity (UE) motor function in patients post stroke. We hypothesized that (1) the WM complexity would decrease following stroke, and (2) higher WM complexity in non-affected cortical areas would be related to greater UE motor function. Thirty-eight stroke patients (16 with left-hemisphere lesions) underwent MRI anatomical brain scans. Fractal dimension (FD), a quantitative shape metric, was applied onto skeletonized brain WM images to evaluate WM internal structural complexity. Wolf Motor Function Test (WMFT) and Fugl-Meyer Motor Assessment (FM) scores were measured to assess motor function of the affected limb. The WM complexity was lower in the stroke-affected hemisphere. The FD was associated with better motor function in two subgroups: with left-subcortical lesions, FD values of the lesion-free areas of the left hemisphere were associated with better FM scores; with right-cortical lesions, FD values of lesion-free regions were robustly associated with better WMFT scores. These findings suggest that greater residual WM complexity is associated with less impaired UE motor function, which is more robust in patients with right-hemisphere lesions. No correlations were found between lesion volume and WMFT or FM scores. This study addressed WM complexity in stroke patients and its relationship with UE motor function. Measurement of brain WM reorganization may be a sensitive correlate of UE function in people recovering from stroke.

Association of SelS mRNA expression in omental adipose tissue with Homa-IR and serum amyloid A in patients with type 2 diabetes mellitus.

BACKGROUND: Tanis was reported as a putative receptor for serum amyloid A (SAA) involving glucose regulated protein in insulin regulated resistance. It was found to be dysregulated in diabetic rats (Psammomys obesus, Israeli sand rat) and its homologue for humans is SelS/AD-015. The present study analyzed mRNA expression of SelS in omental adipose tissue biopsies from patients with type 2 diabetes mellitus (T2DM), and age- and weight-matched nondiabetic patients, the relationship of SelS mRNA with Homa-IR and serum SAA level. METHODS: Human omental adipose tissues from ten cases of type 2 diabetic patients and twelve cases of nondiabetic individuals were analyzed for the expression level of SelS mRNA by semiquantitative polymerase chain reaction (PCR), Homa-IR estimated by standard formula and SAA level by enzyme-linked immunosorbent assay (ELISA). RESULTS: SelS mRNA expression, Homa-IR and serum SAA were higher in T2DM sufferers than in nondiabetic control group. SelS mRNA level was positively correlated with Homa-IR and SAA level in each group. CONCLUSIONS: SelS protein may be involved in insulin resistance in Chinese with T2DM by acting as the SAA receptor, thus playing an important role in the development of T2DM and atherosclerosis.

Effects of Zibu Piyin recipe on SNK-SPAR pathway in neuron injury induced by glutamate.

OBJECTIVE: To investigate the relationship between the excitotoxicity and serum-inducible kinase (SNK) and spine-associated Rap GTPase-activating protein (SPAR) pathway in primary hippocampal neuron injury induced by glutamate and furthermore, to explore the molecular mechanism of neuroprotection of Zibu Piyin Recipe (ZBPYR) and the relationship between ZBPYR and the morphological regulation of dendritic spines. METHODS: The serum containing ZBPYR was prepared by seropharmacology. Reverse transcription and polymerase chain reaction (RT-PCR) was used to detect the expression of mRNA for SNK, SPAR, postsynaptic density protein 95 (PSD-95) and N-methyl-D-aspartate (NMDA) receptor subunits (NR1, NR2A and NR2B) in primary rat hippocampal neuron cultures after pretreatment with 10 micromol/L glutamate and ZBPYR serum. RESULTS: ZBPYR serum pretreatment resulted in a significant down-regulation of glutamate-induced SNK mRNA expression (P<0.05). Significant up-regulation was seen on the mRNA expression of SPAR and PSD-95 (P<0.05). All these changes were dose-dependent. The mRNA expression of NR1, NR2A and NR2B was down-regulated to different degrees (P<0.05). CONCLUSION: The mechanism of effect of ZBPYR on glutamate-induced excitotoxicity may be related to the regulation of SNK-SPAR signal pathway. ZBPYR may play a role in protecting and maintaining the normal morphology and structure of dendritic spines, which may be achieved by inhibiting the excessive activation of NMDA receptors.

Involvement of the Snk-SPAR pathway in glutamate-induced excitotoxicity in cultured hippocampal neurons.

The serum-induced kinase (Snk)-spine-associated Rap GTPase-activating protein (SPAR) signaling pathway is reported as a new molecular mechanism in activity-dependent remodeling of synapses. However, the relationship between Snk-SPAR pathway and glutamate-induced excitotoxicity is not well understood. We report here that in cultured hippocampal neurons, glutamate stimulation induces the activation of Snk-SPAR pathway, and leads to a loss of mature dendritic spines. The time-dependent changes in Snk and SPAR expression after glutamate exposure are also elucidated. Furthermore, the activation of Snk-SPAR pathway induced by glutamate treatment can be blocked by an NMDA receptor antagonist, MK801. These results demonstrate that Snk-SPAR pathway may play a pivotal role in glutamate-induced excitotoxic damage in CNS through regulating the stability of synapse.

[Effects of Ginkgo biloba extract against excitotoxicity induced by NMDA receptors and mechanism thereof].

OBJECTIVE: To observe the effects of Ginkgo biloba extract (GBE) on N-methyl-D-aspartate (NMDA) excitotoxicity and focal cerebral ischemia, and further explore the neuroprotective mechanisms of GBE. METHODS: Neonatal SD rat hippocampus was taken out to make into cell suspension. immunohistochemistry with neuron nucleoprotein monoclonal antibody (NeuN) was used to calculate the percentage of NeuN positive cells. Twelve days after incubation the suspension of neurons were randomly divided into 4 groups: normal control group (exposed to normal saline for 15 min and then to DMEM without NMDA and glycine for 24 h), NMDA group (exposed to culture fluid with NMDA of the terminal concentration of 100 micromol/L and glycine of the terminal concentration of 10 micromol/L for 15 min and then to DMEM without NMDA and glycine for 24 h), MK-801 group (exposed to MK-801, an NMDAR antagonist, for 2 min, to culture fluid with NMDA for 15 min, and then to DMEM without NMDA and glycine for 24 h), and GBE pretreatment group (exposed to GBE of the terminal concentration of 150 microg/ml for 3 d, culture fluid with NMDS for 15 min, and then to DMEM without NMDA and glycine foe 24 h). Trypan blue staining was used to calculate the survival rate of the neurons. The lactic dehydrogenase (LDH) level in the supernatant of cultured cell suspension was detected. Whole-cell patch clamp recording was carried out to evaluate the modulatory effects of GBE on NMDA-activated currents in the rat hippocampal neurons. 108 SD rats were randomly divided into 5 groups: sham operation group (n = 12), standard middle cerebral artery occlusion (MCAO) group (n = 24, undergoing MCAO and then reperfusion), MK-801 acute administration group (n = 24, undergoing MCAO and immediate peritoneal administration of MK-801 1 mg/kg), GBE acute administration group (n = 24, undergoing peritoneal injection of GBE 100 mg/kg immediately after the MCAO), and GBE pretreatment group (n = 24, undergoing peritoneal administration of GBE every day for 7 days before the MCAO). The 4 groups were re-divided into 4 subgroups with 3 approximately 4 rats: 0.5 h ischemia, and 3 h, 1 d, and 7 d ischemia-reperfusion (IR) subgroups. The neurological symptoms were evaluated by Longa's scoring after the rats became conscious. The rats were killed at different time-points, their brains were taken out to undergo 2, 3, 5-triphenyl-tetrazolium chloride staining, the areas of cerebral infarction were calculated, and immunohistochemistry was used to evaluate the contents of NeuN and microtubule-associated protein (MAP-2). RESULTS: The cell viability of the GBE group was 85% +/- 5%, significantly higher than that of the NMDA group (39.8% +/- 2.1%, P < 0.01), and significantly lower than that of the MK-801 group (93.8% +/- 2.7%, P < 0.05). The LDH efflux of the GBE group was 87 U/L +/- 8 U/L, significantly lower than that of the NMDA group (138 U/L +/- 12 U/L, P < 0.01) and significantly higher than that of the MK-801 group (47 U/L +/- 7 U/L, P < 0.05). The inward current (I(NMDA)) of the NMDA group was significantly activated, The inhibitory rate of the NMDA-activated I(NMDA) of the GBE group was 40% +/- 17%, significantly lower than that of the MK-801 group (78% +/- 18%, P < 0.05); After washing out with standard extracellular solution, the I(NMDA) could recover to 91% +/- 8% in the GBE group, but not in the MK-801 group (P < 0.05), which indicated that GBE had lower affinity to NMDA receptor than MK-801. The Longa's scores of the 3 h and 24 h IR subgroups of the GBS pretreatment group were all significantly lower than those of the corresponding subgroups of the standard MCAO and GBE acute administration groups. The symptoms of the MK-801 were the most severe. Cerebral infarction began to appear in the 1-day subgroups. The cerebral infarction areas of the 1 d subgroups of the GBF pretreatment and MK-801 groups were 11.5% +/- 1.3% and 6.5% +/- 0.9% respectively, both significantly smaller than those of the MCAO and GBE acute administration groups (24.5% +/- % and 22.9% +/- 1.3% respectively, both P < 0.01), however, there was no significant difference in the cerebral infarction area between the GBE acute administration and MCAO group. It was true too for the cerebral infarction areas of the 7 d subgroups. Except in the control group, loss of NeuN positive neuron was seen in all groups, especially the MCAO and GBE acute administration groups. Except in the control group, the MAP-2 positive neurons were decreased in all groups, especially the MCAO and GBE acute administration groups, and 1 day and 7 days after the IR MAP-2 positive neurons were almost unseen in the MCAO and GBE acute administration groups, however, could be seen in small amounts in the GBE and MK-801 groups (all P < 0.01). CONCLUSION: GBE pretreatment protects the neurons from excitotoxicity induced by over-activated NMDA receptor and focal cerebral ischemia, which can be explained by the mild blocking effect of GBE on NMDA receptor with low affinity, comparing with MK-801, and GBE is expected to interfere in excitotoxicity in clinic without neurotoxic behaviors.

Preparation of minor ginsenosides C-Mc, C-Y, F2, and C-K from American ginseng PPD-ginsenoside using special ginsenosidase type-I from Aspergillus niger g.848.

BACKGROUND: Minor ginsenosides, those having low content in ginseng, have higher pharmacological activities. To obtain minor ginsenosides, the biotransformation of American ginseng protopanaxadiol (PPD)-ginsenoside was studied using special ginsenosidase type-I from Aspergillus niger g.848. METHODS: DEAE (diethylaminoethyl)-cellulose and polyacrylamide gel electrophoresis were used in enzyme purification, thin-layer chromatography and high performance liquid chromatography (HPLC) were used in enzyme hydrolysis and kinetics; crude enzyme was used in minor ginsenoside preparation from PPD-ginsenoside; the products were separated with silica-gel-column, and recognized by HPLC and NMR (Nuclear Magnetic Resonance). RESULTS: The enzyme molecular weight was 75 kDa; the enzyme firstly hydrolyzed the C-20 position 20-O-ß-D-Glc of ginsenoside Rb1, then the C-3 position 3-O-ß-D-Glc with the pathway Rb1→Rd→F2→C-K. However, the enzyme firstly hydrolyzed C-3 position 3-O-ß-D-Glc of ginsenoside Rb2 and Rc, finally hydrolyzed 20-O-L-Ara with the pathway Rb2→C-O→C-Y→C-K, and Rc→C-Mc1→C-Mc→C-K. According to enzyme kinetics, K m and V max of Michaelis-Menten equation, the enzyme reaction velocities on ginsenosides were Rb1 > Rb2 > Rc > Rd. However, the pure enzyme yield was only 3.1%, so crude enzyme was used for minor ginsenoside preparation. When the crude enzyme was reacted in 3% American ginseng PPD-ginsenoside (containing Rb1, Rb2, Rc, and Rd) at 45°C and pH 5.0 for 18 h, the main products were minor ginsenosides C-Mc, C-Y, F2, and C-K; average molar yields were 43.7% for C-Mc from Rc, 42.4% for C-Y from Rb2, and 69.5% for F2 and C-K from Rb1 and Rd. CONCLUSION: Four monomer minor ginsenosides were successfully produced (at low-cost) from the PPD-ginsenosides using crude enzyme.

[Inhibitory effect of caffeine on GABA-activated current in acutely isolated rat dorsal root ganglion neurons].

By means of whole-cell patch clamp technique, the modulatory effect of caffeine on GABA-activated currents (I(GABA)) was investigated in acutely isolated rat dorsal root ganglion (DRG) neurons. The majority of the neurons examined (113/116) were sensitive to GABA (1~1000 micromol/L). GABA activated a concentration-dependent inward current, which manifested obvious desensitization. In 58 out of 108 neurons, caffeine induced a small inward current, while in others no detectable current was observed. After the neurons were treated with caffeine (0.1~100 micromol/L) prior to the application of GABA (100 micromol/L) for 30 s, GABA-activated inward currents were obviously inhibited. Caffeine shifted the GABA dose-response curve downward and decreased the maximum response to 57% without changing K(d) value. These results indicate that the inhibitory effect is non-competitive. The pretreatment with caffeine (10 micromol/L) inhibited I(GABA) which was potentiated by diazepam (1 micromol/L). Intracellular application of H-8 almost completely abolished the inhibitory effect of caffeine on I(GABA). Because GABA can induce primary afferent depolarization (PAD), our results suggest that caffeine may be able to antagonize the effect of presynaptic inhibition of GABA in primary afferent.

[Glutamate-neuroexcitotoxicity protective study of monoclonal antibody against human NMDA receptor key subunit].

OBJECTIVE: To study if monoclonal antibody against human NMDA receptor key subunit (NR1) may protect neurons from excitotoxicity. METHODS: We cultured primary hippocampal neurons from 10 newborn rats and made the glutamate excitotoxicity model, examined the ratio of surviving neuron (Trypan blue dye staining) and LDH assay to study the protective effects of mAbN1, There were 2 plates in parallel per group, the experiments were repeated 4 times. RESULTS: 0.3 micro mol/L mAbN1 could protect cultured hippocampal neurons from neuroexcitotoxicity induced by 500 micro mol/L glutamate with increased survival rate 35%. Epitope peptide could block the protection of mAbN1, which suggests that the protection of mAbN1 is specific. CONCLUSION: mAbN1 is a novel NMDAR blocker with in vitro neuroprotective activity which may provide a basis for antibody therapy of neuroexcitotoxicity.

Amelioration effects of berberine on diabetic microendothelial injury model by the combination of high glucose and advanced glycation end products in vitro.

Microvascular complications are much earlier and common in diabetes. Advanced glycation end products (AGEs), together with high glucose, play a key role in the endothelial dysfunction of diabetic vascular complications. So it is of more significance to expedite the therapies to block the formation and/or the effects of AGEs. Berberine has been showed to have anti-diabetic effects, however the effects on diabetic complications were less explored, especially the effects on the microvascular complications and the formation and pathways of AGEs which have not been reported. Therefore, the present study established an in vitro model of diabetic microendothelial (microEC) injury by the combination of high glucose and AGEs to mimic the clinical situations and examine the effects and mechanisms of berberine on high glucose-AGEs-induced microEC injuries and on the formation of AGEs. We prepared AGEs, established the high glucose-AGEs injured microEC models by MTT assay, which was further supported by significantly decreased nitric oxide (NO) release, NO synthase (NOS) and thrombomodulin production with ELISA, western blot and RT-PCR analysis. Berberine treatments showed significant improvements as indicated by significantly increased NO release, NOS and thrombomodulin production. Moreover, we also observed significant inhibition effects of berberine on AGEs formation. We concluded that the in vitro model of diabetic microEC injury could be established by the combination treatments of high glucose and AGEs, while berberine could improve the diabetic microvascular injury in vitro and inhibit the formation of AGEs, suggesting the potential clinical therapies with berberine for diabetes and its vascular complications.