An effective technique for isolating adult activated Schwann cells

BACKGROUND： Schwann cells （SCs） are neuroglial cells of peripheral nerve and play a key role in repairing peripheral nerve injury; therefore, it provides an important evidence for transplantation of SCs which are characterized by active proliferation and adult high-purity in vitro after nerve injury in clinic, and also develops a new therapeutic way for nerve injury.OBJECTEVE： To investigate an effective technique for isolating adult activated Schwann cells,DESIGN： Controlled observational study.SETTING： Mudanjiang Medical College.MATERIALS： The experiment was completed at the Department of Medical Genetics of Harbin Medical University from March 2003 to April 2005. Health female Wistar rats, aged 2 months, weighting 150-160 g, were randomly divided into 3 groups with 5 in each group.METHODS： The right sciatic nerves from 15 Wistar rats were exposed and transected at the mid thigh under pentobarbital anesthesia （4 mg/kg, Lp）. Seven days later, the distal segments of the predegenerated nerves were removed and used to produce adult Schwann cell cultures. The distal segment of the predegenerated nerve, 20 mm in length, was resected. The nerve was cut into pieces 1 mm in length and incubated for 3 hours under CO2 at 37 ℃ with an enzyme mixture of 0.05% collagenase/dispase. Rats were divided into 3 groups： ① Group 1： The nerve fragments were explanted in poly-L-lysine and laminin-coated dishes with BS medium from the 1st to the 6th day, On the 6^th day, the fragments were removed into a new poly-L-lysine-laminin-coated dish and the BS medium was changed to BS with 10% FBS, The nerve fragments were replaced repeatedly in the same way in new dishes on the 12^th and the 18th days. ② Group 2： For the first 3 days, the nerve fragments were fed with BS with 10% FBS. This medium was changed to BS medium on the third day. The nerve fragments were removed to another dish on day 6 and BS medium was changed to BS with 25 mI.JL FBS. Hereafter the culture method was the same as for group 1. ③ Group 3： For the first 6 days, nerve fragments were incubated in a dish not coated with poly-L-lysine and laminin, in BS medium supplemented with 8×10^7 U/L of penicillin-streptomycin. On the 6th day, the nerve fragments were removed to a poly-L-lysine-laminin-coated dish and cultured in BS with 25 mI.JL FBS, On the 12th day, the nerve fragments were explanted a second dish and fed with BS containing 100 mL/L FBS. On the 18^th day, they were explanted to a third poly-L-lysine-laminin-coated dish, SCs were obtained from all 3 dishes on the 21st day, Finally, purity and density of SCs were identified and proliferation index was calculated at the same time.MAIN OUTCOME MEASURES ： Purity and density of SCs cultured with various methods in the three groups for 21 days.RESULTS ： ① Isolation and proliferation of SCs： In the group 1, they increased in number after 4 days and both purity and density of cultured SCs were significantly higher than those from group 2. In the group 2, there were few fibroblasts. In the group 3, both purity and density of cultured SCs were remarkably higher than in those from groups 1 or 2. Then optimal proliferation was soon seen and the rapid expansion of SC populations suppressed the development of contaminating fibroblasts. On the 21st day, SCs proliferated to achieve maximal density and were too crowded to be counted. With Chi-square test, the data of the purity and the density were analyzed from groups 1 to 3, the result indicated X^2=430.47, P 〈 0.05. ② Characterization and proliferation rate of SCs： Immunostaining for S100 protein was evident in the cell soma and the processes of all three groups in cultures of SCs. SCs in vitro demonstrated typical bior tri-polar morphology, had oval nuclei, and stained brightly for $100. The proliferation rate of SCs was assessed with double fluorescence staining for BrdU and S100 on the 21^st day of all three groups in cultures. About 40%-50% of the total SCs in the each group showed BrdU incorporation.CONCLUSION： The method is to use predegeneration in vivo, differential speed culture supplemented with the penicillin-streptomycin in low concentration, and changing of the concentration of FBS in the BS medium from 0 to 100 mL/L. This method allows remarkable suppression of fibroblast growth and attainment of SC proliferation and purity, in a short time, from adult nerves.

Progresses and Strategies of Gene Mapping and Isolating of Photo(thermo) sensitive Genic Male Sterile Rice

Considering the research on classical genetics of photoperiod(therm) sensitive genic male sterile rice, it is important to select the sterile lines and their segregating population controlled by one pair of gene in mapping and isolating sterile genes. It is discussed the advantages, disadvantages and the reasons leading to various mapping results of chromosome location of sterile genes through gene marker, isozyme marker and DNA marker techniques. In comparison to isolation of photo(thermo) sensitive sterile genes via various plant gene cloning techniques, it was concluded that map based cloning was acceptable, but it is still difficult to locate the loci of sterile genes within 1cM. On the other hand, “sensitivity to environment”, an important characteristic of sterile lines can be fully utilized by DD PCR and (or) RDA techniques. Therefore, these two techniques were considered as the effective ways to isolate sterile genes.

A NEW METHOD OF ISOLATING KUPFFER CELLS FROM BIOPSY TISSUE OF RAT LIVER

The isolation of a high yield and purity of Kupffer cells has been reported in detail.1 This paper reports into the research about isolation Kupffer cells from biopsy tissue of liver. This method includes 5 important steps: (1) take fresh liver tissue, and mince with scissors. (2) spin at low speed to wash off red blood cells. (3) digest in collagenase for suitable time. (4) isolate Kupffer cells on a percoll density gradient. (5) cell charaterization was observed by N.S.E stain and peroxidatic activity with lumino-meter measurement and phagocytosis with latex beads.2.3

Isolating cDNA clones from rice induced by Magnaporthe grisea using PCR based differential screening method

The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that

A rapid approach for isolating a single fungal spore from rice blast diseased leaves

Single spore isolation is a fundamental approach in plant pathology and mycology to isolate and identify plant fungal pathogens from diseased samples.However,routine single spore isolation procedure is time-consuming and has a high risk of contamination by other microorganisms.In this study,we developed a rapid approach for isolating a single spore of the fungal pathogen,Pyricularia oryzae,from rice blast diseased leaves in the paddy field with low potential of contamination.First,rice blast leaves with single lesions were selected in the paddy field,and a single lesion was cut out and pressed and dragged gently across the surface of water agar.Next,a germinated single spore with a barely visible piece of agar was cut out of water agar with a dissecting needle under a stereomicroscope at approximately 120-fold magnification.Last,the germinated single spore with water agar was transferred onto oatmeal tomato agar for growth and preservation.Based on our experience,a skilled technician or student can successfully isolate single spore from over 150 independent diseased samples with nearly no contaminations in a single working day.This approach is also suitable for isolating single spore from other fungal disease samples that produce abundant aerial conidia.

Isolated Pelvic Hyperthermochemotherapeutic Perfusion-An Experimental Study on Isolating Efficacy

Hyperthermochemotherapeutic perfusion model through isolated pelvic vessels was developed to evaluate the leakage of hyperthermia and drugs (such as adriamycin) from the isolated pelvic circulation to systemic circulation and its associated side/toxic effects. The isolated pelvic circulation was perfused through a femoral artery catheter with hyperthermic (48 ℃ to 55 ℃) adriamycin solution (50 μg/ml) for 30 min. The efflux was drained through a femoral vein catheter. And the pelvic temperature was kept at the level of 43±0.5 ℃. The temperature of pelvic circulation was kept at 4 ℃ to 5 ℃ greater than the systemic/core temperature. The adriamycin concentration of pelvic efflux was 12 to 46 folds of that of systemic serum. The difference between them was very significant ( P <0.001). As the perfusion pressure was increased, which kept lower than the mean systemic artery pressure, the leakage of the adriamycin from the isolated pelvic circulation to systemic circulation was increased, but there was no significant difference between them ( P >0.05). During isolated perfusion, the systemic blood dynamics remained stable and there were no organic injuries on the important organs. It was suggested that the isolating efficacy of the modality of isolated pelvic hyperthermochemotherapeutic perfusion through vessels was rather high. The hyperthermia and drugs could be effectively limited in the isolated pelvic region with minor side effects on the systemic circulation and important organs.

Efficiency of Plating Media and Enrichment Broths for Isolating <i>Salmonella</i>Species from Human Stool Samples: A Comparison Study

A comparative study was performed to evaluate best practice culture media and enrichment broths for recovering Salmonella species from human stool samples. A total of 1297 human stools were collected and processed in this study. Evaluation of agar media was carried out by direct plating (DP), 1096 stool samples were inoculated on Modified Semisolid Rappaport-Vassiliadis (MSRV), Xylose-Lysine-Deoxycolate (XLD), MacConkey (MAC), and Hektoen Enteric (HE) agars. Evaluation of enrichment broths were carried out by enrichment all 1297 stool samples in Selenite broth (SB), Rappaport-Vassiliadis (RV) broth, and Buffered Peptone Water (BPW), followed by plating on MSRV, MAC, and HE agars. A total of 102 Salmonella-positive stools by DP, 85.3% (87/102) were recovered utilizing MSRV while recovery from XLD, MAC, and HE agars were 34.3% (35/102), 34.3% (35/102), and 29.4% (30/102) respectively. A total 299/1297 stools samples were Salmonella-positive on at least one plating medium after enrichment procedure were 77.3% (177/299) for SB, 86.0% (197/299) and 78.6% (180/299) for RV and BPW respectively. All Salmonella isolated in this study was nontyphi Salmonella. Presently, the data suggest that the use of MSRV over MAC, HE, and XLD agars for isolation nontyphi Salmonella species from human stools is more efficacious. Additionally, use of MSRV in combination with MAC and HE agars following enrichment in RV broth enhances recovery of nontyphi Salmonella species. However, RV broth is inhibitory to typhi Salmonella, thus use of MSRV medium in combination with MAC, HE or XLD agars in direct plating following enrichment in non-selective BPW is an alternate method for recovery of both typhi and nontyphi Salmonella species contaminated in human stool samples.

Reproductive Isolating Mechanisms in the Bangladesh Coastal Bullfrog Hoplobatrachus litoralis and Its Congeneric Species Revealed by Crossing Experiments and Examination on Spermatogenesis of the Hybrids

To elucidate reproductive isolating mechanisms in the Bangladesh coastal bullfrog Hoplobatrachus litoralis and its congeneric species, we performed crossing experiments using three species： H. litoralis, H. tigerinus and H. rugulosus. In addition, we conducted histological observations on spermatogenesis of the hybrids. The reciprocal hybrids between H. litoralis and H. tigerinus developed normally with somewhat lower viability at the metamorphosis stage compared with the controls. Most of the metamorphosed frogs became mature. On the other hand, almost all hybrids between female H. rugulosus and male H. litoralis or H. tigerinus died of underdevelopment at the tadpole stage, and only a few hybrids metamorphosed normally and survived to maturity. The inner structures of the testes of the control H. litoralis and H. tigerinus were completely normal, with seminiferous tubules filled with compact bundles of normal spermatozoa. Those of the reciprocal hybrids between H. litoralis and H. tigerinus were almost normal or slightly abnormal, with seminiferous tubules that contained pycnotic nuclei in addition to normal bundles of normal spermatozoa, which demonstrates slight abnormalities in spermatogenesis. In contrast, the hybrids between female H. rugulosus and male H. litoralis or H. tigerinus had no bundles of spermatozoa nor spermatids in the seminiferous tubules, which indicates entirely abnormal spermatogenesis. Meiotic chromosome figures in the reciprocal hybrids between H. litoralis and H. tigerinus showed slight abnormalities, with the occurrence of univalents and increase of rod-shaped bivalents. These results indicate that H. litoralis and H. tigerinus are not isolated from each other by hybrid inviability nor by hybrid sterility, although the hybrids showed somewhat abnormal spermatogenesis in hybrids and that H. rugulosus is isolated from both H. litoralis and H. tigerinus by incomplete hybrid inviability and complete hybrid sterility.

A Simple Method for Isolating Chloroplast DNA and Mitochondria DNA from the Same Rapeseed Green Leaf Tissue

In the study, we present a fast, simple and inexpensive protocol for isolating chloroplast and mitochondrial DNA from one rapeseed leaf tissue sample. The chloroplast and mitochondria were separated from the same green leaf tissue by differential centrifugations. The protocol is the first report that isolates plant chloroplast DNA （cpDNA） and mitochondrial DNA （mtDNA） from the same sample homogenate. The organelle DNA yield is 2-10 micrograms per gram of tissue; the DNA was fully restrictable and was successfully used for sequencing.

A method for isolating rat cardiomyocytes by acute enzymatic hydrolysis

Objective: This r esea rch showed a method of isolate rat cardio myocytes though acute enzymatic hydrolysis. Method: Our method was divided into five steps: preparation, heart ext raction, perfusion, mechanical dissociation and pu rifica tion and r ecalcifica ti on. Fir stly, make a preparation, including solutions, equipment and so on. Secondly, after anesthesia satisfactorily, open the chest of rat and take the heart out. Thirdly, transect the aorta and hang the aorta onto the Langendroff perfusion system such that the tip is just distal to the aortic valve. For th, perfuse the hea rt with diges tion solution for 17-20 min afte r Ca2+ free tyrode solution for 5 min. Finally, remove the heart from the system quickly and dissociate mechanically in the KB solution and recalcification after filter. Result: Though the microscope, good-state cardiomyocytes (clear stripes, strong sense of three-dimensional, stick well) can be observed. Conclusion: Those cells can be used to undergo medical electrophysiological experiment, including the study of iron channel.

Method for Isolating Mitochondrial DNA from Etiolated Tissue of Cabbage

Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for separation and purification of mitochondria and extraction of mitochondrial DNA（mtDNA） from etiolated tissues of cabbage was developed. We took a method combined mannitol density gradient with differential centrifugation, selected appropriate rotational speed, extended DNase I treating time and changed mitochondria cracking condition. The results showed that the extracted mitochondria in this protocol had complete structure, appeared to ellipsoid and had not been contaminated with other impurities under the Jannus Green B staining. The isolated mitochondrial DNA had high purity and yield through detecting the optical density, nuclear specific primer PCR and agarose gel electrophoresis. The results indicated that mitochondrial DNA extracted by this protocol had high quality and enabled to be used in futher genetic studies.

Dynamic response of concrete pavement structure with asphalt isolating layer under moving loads

A three-dimensional finite element model （3D FEM） is built using ABAQUS to analyze the dynamic response of a concrete pavement structure with an asphalt isolating layer under moving loads. The 3D model is prepared and validated in the state of no asphalt isolating layer. Stress and deflection at the critical load position are calculated by changing thickness, modulus of isolating layer and the combination between the isolating layer and concrete slab. Analysis result shows that the stress and deflection of the concrete slab increase with the increase of thickness. The stress and deflection of the concrete slab decrease with the increase of combination between the isolating layer and concrete slab. The influence of changing the isolating layer modulus to the stress and deflection of the concrete slab is not significant. From the results, asphalt isolating layer design is suggested in concrete pavement.

Rapid and efficient method for isolating plant high molecular weight(HMW)DNA with high purity

PREPARATION of HMW DNA (Megabase-size) is the basis for construction of genomic library with large DNA inserts such as bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC), and for long-range physical mapping. It can also be used for the macro-study of repeat sequences. Since HMW DNA during preparation is inclined to be sheared physically and digested by internal nucleases, it is very difficult to prepare the HMW DNA. Initially, plant HMW DNA was prepared by embedding protoplasts in the low melting-point (LMP) agarose; however, it had several disadvantages: (ⅰ) Culture of protoplasts was time-consuming, costly and tedious. ( ⅱ ) It was only used successfully for limited

Study on a conical bearing for acceleration-sensitive equipment

Seismic isolation effectively reduces seismic demands on building structures by isolating the superstructure from ground vibrations during earthquakes.However,isolation strategies give less attention to acceleration-sensitive systems or equipment.Meanwhile,as the isolation layer’s displacement grows,the stiffness and frequency of traditional rolling and sliding isolation bearings increases,potentially causing self-centering and resonance concerns.As a result,a new conical pendulum bearing has been selected for acceleration-sensitive equipment to increase self-centering capacity,and additional viscous dampers are incorporated to enhance system damping.Moreover,the theoretical formula for conical pendulum bearings is supplied to analyze the device’s dynamic parameters,and shake table experiments are used to determine the proposed device’s isolation efficiency under various conditions.According to the test results,the newly proposed devices have remarkable isolation performance in terms of minimizing both acceleration and displacement responses.Finally,a numerical model of the isolation system is provided for further research,and the accuracy is demonstrated by the aforementioned experiments.

Dynamic analysis of a novel multilink-spring mechanism for vibration isolation and energy harvesting

Due to technical limitations,existing vibration isolation and energy harvesting(VIEH)devices have poor performance at low frequency.This paper proposes a new multilink-spring mechanism(MLSM)that can be used to solve this problem.The VIEH performance of the MLSM under harmonic excitation and Gaussian white noise was analyzed.It was found that the MLSM has good vibration isolation performance for low-frequency isolation and the frequency band can be widened by adjusting parameters to achieve a higher energy harvesting power.By comparison with two special cases,the results show that the MLSM is basically the same as the other two oscillators in terms of vibration isolation but has better energy harvesting performance under multistable characteristics.The MLSM is expected to reduce the impact of vibration on high-precision sensitive equipment in some special sites such as subways and mines,and at the same time supply power to structural health monitoring devices.

Seismic control of multi-degrees-of-freedom structures by vertical mass isolation method using MR dampers

Vertical mass isolation(VMI)is one of the novel methods for the seismic control of structures.In this method,the entire structure is assumed to consist of two mass and stiffness subsystems,and an isolated layer is located among them.In this study,the magnetorheological damper in three modes:passive-off,passive-on,and semi-active mode with variable voltage between zero and 9 volts was used as an isolated layer between two subsystems.Multi-degrees-of-freedom structures with 5,10,and 15 floors in two dimensions were examined under 11 pairs of near field earthquakes.On each level,the displacement of MR dampers was taken into account.The responses of maximum displacement,maximum inter-story drift,and maximum base shear in controlled and uncontrolled buildings were compared to assess the suggested approach for seismic control of the structures.According to the results,the semi-active control method can reduce the response by more than 12%compared to the uncontrolled mode in terms of maximum displacement of the mass subsystem of the structures.This method can reduce more than 16%and 20%of the responses compared to the uncontrolled mode in terms of maximum inter-story drift and base shear of the structure,respectively.

Emulsifying property,antioxidant activity,and bitterness of soybean protein isolate hydrolysate obtained by Corolase PP under high hydrostatic pressure

Enzymatic hydrolysis of proteins can enhance their emulsifying properties and antioxidant activities.However,the problem related to the hydrolysis of proteins was the generation of the bitter taste.Recently,high hydrostatic pressure(HHP)treatment has attracted much interest and has been used in several studies on protein modification.Hence,the study aimed to investigate the effects of enzymatic hydrolysis by Corolase PP under different pressure treatments(0.1,100,200,and 300 MPa for 1-5 h at 50℃)on the emulsifying property,antioxidant activity,and bitterness of soybean protein isolate hydrolysate(SPIH).As observed,the hydrolysate obtained at 200 MPa for 4 h had the highest emulsifying activity index(47.49 m^(2)/g)and emulsifying stability index(92.98%),and it had higher antioxidant activities(44.77%DPPH free radical scavenging activity,31.12%superoxide anion radical scavenging activity,and 61.50%copper ion chelating activity).At the same time,the enhancement of emulsion stability was related to the increase of zeta potential and the decrease of mean particle size.In addition,the hydrolysate obtained at 200 MPa for 4 h had a lower bitterness value and showed better palatability.This study has a broad application prospect in developing food ingredients and healthy foods.

Prediction of Damping Capacity Demand in Seismic Base Isolators via Machine Learning

Base isolators used in buildings provide both a good acceleration reduction and structural vibration control structures.The base isolators may lose their damping capacity over time due to environmental or dynamic effects.This deterioration of them requires the determination of the maintenance and repair needs and is important for the long-termisolator life.In this study,an artificial intelligence prediction model has been developed to determine the damage and maintenance-repair requirements of isolators as a result of environmental effects and dynamic factors over time.With the developed model,the required damping capacity of the isolator structure was estimated and compared with the previously placed isolator capacity,and the decrease in the damping property was tried to be determined.For this purpose,a data set was created by collecting the behavior of structures with single degrees of freedom(SDOF),different stiffness,damping ratio and natural period isolated from the foundation under far fault earthquakes.The data is divided into 5 different damping classes varying between 10%and 50%.Machine learning model was trained in damping classes with the data on the structure’s response to random seismic vibrations.As a result of the isolator behavior under randomly selected earthquakes,the recorded motion and structural acceleration of the structure against any seismic vibration were examined,and the decrease in the damping capacity was estimated on a class basis.The performance loss of the isolators,which are separated according to their damping properties,has been tried to be determined,and the reductions in the amounts to be taken into account have been determined by class.In the developed prediction model,using various supervised machine learning classification algorithms,the classification algorithm providing the highest precision for the model has been decided.When the results are examined,it has been determined that the damping of the isolator structure with the machine learning method is predicted successfully at a level exceeding 96%,and it is an effective method in deciding whether there is a decrease in the damping capacity.

Potential role of hippocampal neurogenesis in spinal cord injury induced post-trauma depression

It has been reported both in clinic and rodent models that beyond spinal cord injury directly induced symptoms, such as paralysis, neuropathic pain, bladder/bowel dysfunction, and loss of sexual function, there are a variety of secondary complications, including memory loss, cognitive decline, depression, and Alzheimer's disease. The largescale longitudinal population-based studies indicate that post-trauma depression is highly prevalent in spinal cord injury patients. Yet, few basic studies have been conducted to address the potential molecular mechanisms. One of possible factors underlying the depression is the reduction of adult hippocampal neurogenesis which may come from less physical activity, social isolation, chronic pain, and elevated neuroinflammation after spinal cord injury. However, there is no clear consensus yet. In this review, we will first summarize the alteration of hippocampal neurogenesis post-spinal cord injury. Then, we will discuss possible mechanisms underlie this important spinal cord injury consequence. Finally, we will outline the potential therapeutic options aimed at enhancing hippocampal neurogenesis to ameliorate depression.

Impact of surface-reflected seismic waves on the seismic isolation performance of circular tunnel isolation layers

Seismic isolation is an effective strategy to mitigate the risk of seismic damage in tunnels.However,the impact of surface-reflected seismic waves on the effectiveness of tunnel isolation layers remains under explored.In this study,we employ the wave function expansion method to provide analytical solutions for the dynamic responses of linings in an elastic half-space and an infinite elastic space.By comparing the results of the two models,we investigate the seismic isolation effect of tunnel isolation layers induced by reflected seismic waves.Our findings reveal significant differences in the dynamic responses of the lining in the elastic half-space and the infinitely elastic space.Specifically,the dynamic stress concentration factor(DSCF)of the lining in the elastic half-space exhibits periodic fluctuations,influenced by the incident wave frequency and tunnel depth,while the DSCF in the infinitely elastic space remain stable.Overall,the seismic isolation application of the tunnel isolation layer is found to be less affected by surface-reflected seismic waves.Our results provide valuable insights for the design and assessment of the seismic isolation effect of tunnel isolation layers.