Establishment of duplex TaqMan RT-PCR method for detection of Entamoeba histolytica and Giardia lamblia in fecal sample / 中国热带医学

@#Abstract: Objective To establish the duplex TaqMan RT-PCR method for detection of Entamoeba histolytica and Giardia lamblia in fecal samples. Methods Primer pairs and probes for Entamoeba histolytica and Giardia lamblia were designed and duplex TaqMan RT-PCR amplification system was constructed. PCR products were inserted into the pUC57 plasmid, and the lower limit of detection of the method was determined. Clinical stool samples were tested in order to evaluated the efficacy of the method. Results The detection limits of duplex TaqMan RT-PCR were 31.6 copies/μL for Entamoeba histolytica and 32 copies/μL for Giardia lamblia, respectively. Of the total of 212 clinical stool samples tested, all 3 samples with E. histolytica-positive patients by microscopy were positive by PCR, while 1 from 209 samples with E. histolytica-negative patients by microscopy were positive by PCR, and the remaining samples were negative. For Giardia lamblia, all 8 samples positive by microscopy were positive by PCR, and 1 from 204 sample with a microscopy-negative patient was positive by PCR, and the remaining samples were negative. The amplification product sequencing and blast analysis were used to confirm that the amplified sequence in the specimen of a patient with negative microscopy but positive PCR belongs to the targeted pathogen, supported by clinical symptoms and laboratory test results. PCR results for other diarrhea-causing pathogens were negative, indicating no cross-reactivity. Conclusions The dual TaqMan RT-PCR method developed in this study can not only detect microscopy-positive samples of Entamoeba histolytica and Giardia lamblia but also can detect samples that were missed by microscopy, with higher sensitivity than the microscopy method. Further, this detection method does not cross-react with other diarrhea-causing pathogens, including cross-react with other diarrhea-causing pathogens including Iodamoeba butschlii, Blastocystis hominis, Plesiomonas, Aeromonas, Salmonella, Shigella, Sphaerozoum fuscum, and Entamoeba hartmani, thus has a good specificity.

Effect of fatigue load on internal mechanical properties of the intervertebral disc / Efecto de la carga de fatiga en las propiedades mecánicas internas del disco intervertebral

SUMMARY: Lumbar disc herniation is considered to be the main pathological factor for the common clinical disease of low back pain. Biomechanical factor is an important cause of lumbar disc herniation, so it is urgent to analyze the stress/strain behavior of intervertebral disc under different loading condition. Slow repetitive loading is considered to be an important factor of spine and disc injuries, and the effect of fatigue load on internal displacement in the intervertebral disc was investigated by applying the optimized digital image correlation technique in this study. The first finding was that fatigue load had a significant effect on the displacement distribution in the intervertebral disc under compression. Superficial AF exhibited the largest axial displacements before fatigue load, while it exhibited the smallest axial displacements after fatigue load. Inner AF exhibited slightly smaller radial displacements than outer AF before fatigue load, while it exhibited significantly greater radial displacements than outer AF displacements after fatigue load. The second finding was that fatigue load had a certain effect on the internal displacement distribution in the flexed intervertebral disc under compression. Middle AF exhibited the smallest axial displacements before fatigue load, while deep AF exhibited the smallest axial displacements after fatigue load. The radial displacement distribution did not change before and after fatigue load, as the radial displacement in outer AF was the smallest, while the radial displacement in inner AF was the largest. The third finding was that with the increase in fatigue time and amplitude, the Young's modulus of the intervertebral disc increased significantly. This study can provide the basis for clinical intervertebral disc disease prevention and treatment? and is important for mechanical function evaluation of artificial intervertebral disc as well.

RESUMEN: La hernia de disco lumbar se considera el principal factor patológico para la enfermedad clínica común del dolor lumbar. El factor biomecánico es una causa importante de hernia de disco lumbar, por lo que es urgente analizar el comportamiento de esfuerzo / tensión del disco intervertebral bajo diferentes condiciones de carga. La carga repetitiva lenta se considera un factor importante de lesiones de columna y disco, y en este estudio el efecto de la carga de fatiga sobre el desplazamiento interno en el disco intervertebral se investigó mediante la aplicación de la técnica de correlación de imagen digital optimizada. El primer hallazgo fue que la carga de fatiga tuvo un efecto significativo en la distribución del desplazamiento en el disco intervertebral bajo compresión. El AF superficial exhibió los desplazamientos axiales más grandes antes de la carga de fatiga, mientras que exhibió los desplazamientos axiales más pequeños después de la carga de fatiga. El AF interno exhibió desplazamientos radiales ligeramente más pequeños que el AF externo antes de la carga de fatiga, mientras que exhibió desplazamientos radiales significativamente mayores que los desplazamientos AF externos después de la carga de fatiga. El segundo hallazgo fue que la carga de fatiga tenía un cierto efecto sobre la distribución del desplazamiento interno en el disco intervertebral flexionado bajo compresión. El AF medio exhibió los desplazamientos axiales más pequeños antes de la carga de fatiga, mientras que el AF profundo exhibió los desplazamientos axiales más pequeños después de la carga de fatiga. La distribución del desplazamiento radial no cambió antes ni después de la carga de fatiga, ya que el desplazamiento radial en la FA externa fue el más pequeño, mientras que el desplazamiento radial en la FA interna fue el más grande. El tercer hallazgo fue que con el aumento del tiempo de fatiga y la amplitud, el módulo de Young del disco intervertebral aumentó significativamente. Este estudio puede proporcionar la base para la prevención y el tratamiento clínico de la enfermedad del disco intervertebral, y también es importante para la evaluación de la función mecánica del disco intervertebral artificial.

Automatic Detection and Classification of Rib Fractures on Thoracic CT Using Convolutional Neural Network: Accuracy and Feasibility

Objective@#To evaluate the performance of a convolutional neural network (CNN) model that can automatically detect and classify rib fractures, and output structured reports from computed tomography (CT) images. @*Materials and Methods@#This study included 1079 patients (median age, 55 years; men, 718) from three hospitals, between January 2011 and January 2019, who were divided into a monocentric training set (n = 876; median age, 55 years; men, 582), five multicenter/multiparameter validation sets (n = 173; median age, 59 years; men, 118) with different slice thicknesses and image pixels, and a normal control set (n = 30; median age, 53 years; men, 18). Three classifications (fresh, healing, and old fracture) combined with fracture location (corresponding CT layers) were detected automatically and delivered in a structured report. Precision, recall, and F1-score were selected as metrics to measure the optimum CNN model. Detection/diagnosis time, precision, and sensitivity were employed to compare the diagnostic efficiency of the structured report and that of experienced radiologists. @*Results@#A total of 25054 annotations (fresh fracture, 10089; healing fracture, 10922; old fracture, 4043) were labelled for training (18584) and validation (6470). The detection efficiency was higher for fresh fractures and healing fractures than for old fractures (F1-scores, 0.849, 0.856, 0.770, respectively, p = 0.023 for each), and the robustness of the model was good in the five multicenter/multiparameter validation sets (all mean F1-scores > 0.8 except validation set 5 [512 x 512 pixels; F1-score = 0.757]). The precision of the five radiologists improved from 80.3% to 91.1%, and the sensitivity increased from 62.4% to 86.3% with artificial intelligence-assisted diagnosis. On average, the diagnosis time of the radiologists was reduced by 73.9 seconds. @*Conclusion@#Our CNN model for automatic rib fracture detection could assist radiologists in improving diagnostic efficiency, reducing diagnosis time and radiologists’ workload.

Screening and activity verification of monoclonal antibody against PcrV protein of pseudomonas aeruginosa / 中国免疫学杂志

Objective:To obtain a high specificity and high affinity anti-PcrV protein monoclonal antibody which can be used for the treatment of Pseudomonas aeruginosa infected.Methods: The PcrV gene was amplified by PCR using P.aeruginosa PAO1 genome DNA as the template.The expression vector(pET-28a-PcrV) was constructed and transformed into E.coli BL21(DE3).The re-combinant PcrV protein was expressed by IPTG induction and purified by Ni2+affinity chromatography.The specific binders of PcrV were screened by phage display.The genes encoding VH and VL were amplified respectively by PCR using the plasmid of positive clone as the template.Then the recombinant expression vectors were constructed and transfected into 293E cells.Monoclonal antibody were purified by the Protein A affinity resin from the culture supernatants.The affinity of antibody was detected by ELISA and the function of YG5 was verified in murine pneumonia model caused by P.aeruginosa.Results: Recombinant PcrV protein was expressed and purified.A full human monoclonal antibody(named as YG5) against PcrV was obtained by phage display.The results of ELISA showed that YG5 had a high affinity with EC50=61 ng/ml.Furthermore,it was found that YG5 could protect mice from infection caused by P.aeruginosa.Conclusion:Our findings present a novel human monoclonal antibody YG5 against PcrV,which inhibits the infection casued by P.aeruginosa and may be a potential drug for treatment of P.aeruginosa infection.

The effects of chitosan oligosaccharides on OPG and RANKL expression in a rat osteoarthritis model

Abstract Purpose: To investigate the effect of chitosan oligosaccharides (COS) against osteoarthritis (OA) and preliminarily discuss the osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL) and RANK expression in a rat OA model. Methods: Thirty-six 6-week-old Male SD rats were randomly divided into three groups: sham-operated group(CON), OA-induction group(OA), COS intervention group(n=12/group). At 4 weeks after the operation, COS (50 ul) intervention weekily for consecutive 5 weeks. The OA and CON groups received an injection of 50 ul physiological saline. At death, 11 weeks following surgery, cartilage was harvested and total RNA and protein were extracted. Both the morphological changes of the cartilage were observed and harvested the total RNA and protein. Meanwhile, the expression of OPG, RANKL and RANK in cartilage were determined. Results: The expression of OPG and RANKL were both enhanced in the cartilage of the OA model. Compared with the OA group, COS treatment improved the cartilage damage (both extent and grade). Furthermore, the COS group showed highly OPG and lower RANKL. Simultaneously, COS treatment upregulated the ratio of OPG/RANKL and downregulated the RANKL/RANK. Conclusion: Chitosan oligosaccharides may be used as a unique biological agent to prevent and treat osteoarthritis, and this effect is associated with modulation of the expression of osteoprotegerin and receptor activator of NF-κB ligand.

Swimming plus medication reduces the expressions of cytokines in rats with chronic abacterial prostatitis / 中华男科学杂志

Objective@#To observe the effects of swimming plus medication on the expressions of cytokines in rats with chronic abacterial prostatitis (CAP).@*METHODS@#Forty healthy adult male SD rats were randomly divided into five groups of equal number, normal control, CAP model control, medication, exercise therapy, and exercise + medication. The CAP model was made by Xiaozhiling injection, and at 7 days after modeling, the rats in the medication and exercise + medication groups were treated intragastrically with Qianlie Shutong Capsules (0.016 g/ml) at 20 ml per kg of the body weight qd, those in the exercise therapy and exercise + medication groups were made swim at a regular time once a day, 35 minutes on the first day and 5 minutes more on the second until 50 minutes once, for 4 successive weeks, and those in the normal control, model control and exercise therapy groups received normal saline only. After 14 and 28 days of treatment, all the rats were killed and their prostates harvested for observation of histopathological changes and determination of the expressions of TNF- α, IL-1β and IL-6 in the prostatic tissue homogenate by ELISA.@*RESULTS@#After 14 days of treatment, the expression levels of TNF-α, IL-1β and IL-6 were significantly elevated in the groups of CAP model control (［183.08±8.07］ pg/ml, ［57.55±3.53］ pg/ml and ［256.15±13.95］ ng/L), medication (［118.49±8.06］ pg/ml, ［42.64±4.64 ］ pg/ml and ［200.74±9.33］ ng/L), exercise therapy (［169.63±10.64］ pg/ml, ［50.45±5.71］ pg/ml and ［245.23±6.49］ ng/L), and exercise + medication (［107.82±7.81］ pg/ml, ［40.35±6.93］ pg/ml and ［187.04±10.85］ ng/L) as compared with those in the normal control (［20.36±1.82］ pg/ml, ［14.64±1.91］ pg/ml and ［70.58±2.09］ ng/L) (P<0.05). At 28 days, the levels of TNF- α, IL-1β, IL-6 were remarkably lower in the exercise + medication group (［29.30±3.78］ pg/ml, ［16.91±1.24］ pg/ml and ［ 88.65±6.74］ ng/L) than in the medication group (［39.67±3.19］ pg/ml, ［26.27±3.49］ pg/ml and ［110.26±6.33］ ng/L) (P<0.05) and close to those of the normal control group (［19.34±1.76］ pg/ml, ［13.68±1.06］ pg/ml and ［71.34±2.50］ ng/L). During the treatment, no obvious pathological changes were found in the prostate tissue of the normal control rats, while significant chronic prostatic inflammation was observed in the CAP models, and the inflammation was relieved in different degrees after intervention, most significantly in the exercise + medication group.@*CONCLUSIONS@#Swimming can relieve prostatic inflammation and swimming plus medication can effectively reduce the expressions of cytokines and alleviate histological damage in the prostatic tissue of CAP rats.

Research of design and manufacturing technology of titanium personalized orthodontic bracket / 口腔疾病防治

Objective @# A titanium personalized orthodontic bracket is designed and manufactured using the technology of computed tomography (CT), computer aided design (CAD), finite element analysis (FEA), and selective laser melting (SLM) to well match the patient tooth so as to realize mold-free manufacturing. @*Methods@#A model of a titanium personalized orthodontic bracket which is built by CT scanning machine and Pro/E software, is imported into Ansys software to carry out finite element simulation analysis with nonlinear contact method. Then, the titanium personalized orthodontic bracket is proposed after the model data above is imported to SLM molding equipment via titanium powder melted with layer by layer using a high-speed scanning galvanometer. @*Results @#The maximum equivalent stress of the titanium personalized orthodontic bracket is distributed uniformly and reasonably, the titanium personalized orthodontic bracket formed by SLM molding equipment can provide high accuracy and there is a high similarity between the bottom of the orthodontic bracket and the tooth surface shape.@*Conclusion @#The combination of CT, CAD, FEA, SLM technology can fulfill model-free manufacturing of the personalized orthodontic bracket and thus shorten the manufacturing cycle.

Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector

Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 x 10(4),1 x 10(5),and 1x10(6) of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 x 10(4),1 x 10(5) and 1 x 10(6), respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.

Application of 360 degree circular decompression and transpedicle screw fixation in the treatment of ossification of thoracic posterior longitudinal ligament by posterior approach / 中国骨伤

<p><b>OBJECTIVE</b>To investigate the clinical effects of 360 degree circular decompression and transpedicle screw fixation to treat the ossification of thoracic posterior longitudinal ligament by posterior approach.</p><p><b>METHODS</b>From December 2009 to November 2013, 18 patients with ossification of thoracic posterior longitudinal ligament ossification were treated with 360 degree circle decompression and transpedicle screw fixation by posterior approach. There were 8 males and 10 females,ranging in age from 32 to 67 years old, with an average of 51 years old. Four patients were accompanied with ligamentum flavum ossification. Longitudinal ossifications in 5 cases were located in the upper thoracic vertebra and 13 cases in the middle and lower thoracic vertebra. Five cases were typical type, 4 cases were segmental type, 6 cases were continuous type and 3 cases were mixed type. All the patients had the posterior spinal canal decompression combined with longitudinal ligament resection, interbody fusion with bone graft and internal fixation surgeries. The operation time,blood loss and complications were recorded. JOA score were applied to evaluate the neurological function recovery pre-surgery, 2 days post-surgery and the latest follow-up. The surgery effects were evaluated by Epstein-Schwall method.</p><p><b>RESULTS</b>The operation time ranged from 3 to 6 hours (mean, 4.2 hours). The blood loss ranged from 800 to 4 000 ml (mean, 1 800 ml). All the patients were followed up, and the duration ranged from 6 months to 3 years, with a mean of 1.8 years. The JOA score increased from preoperative 4.30 ± 2.60 to 7.60 ± 2.40 2 days after surgery, and 7.80 ± 1.90 at the latest follow-up (t = 4.61, P < 0.001). The JOA scores between 2 days after surgery and the latest follow-up had no significant differences (t = 0.28,P = 0.78). The neurological recovery rate was 74% 2 days after surgery and 71% at the latest follow-up. There were 4 cases got an excellent result,10 good,3 fair and 1 poor according to Epstein-Schwall evaluation method. Four patients had cerebrospinal fluid leakage, 3 patients had intercostal nerve paralysis or pain, and 1 patient had superficial incision infection. The neurological function in 3 patients became worse in the second day posteratively , and among them, 2 patients were recovered at the latest follow-up and 1 patient had no changes. All the patients got fusion of bone graft and no internal fixation loosening and fractures occurred.</p><p><b>CONCLUSION</b>360 degree circular decompression and transpedicle screw fixation can resect different types of thoracic longitudinal ligament ossification, and can achieve a good clinical effect.</p>

Efficiency of improved RAPD and ISSR markers in assessing genetic diversity and relationships in Angelica sinensis (Oliv. ) Diels varieties of China

Background Angelica sinensis is a well-known traditional Chinese medicinal plant. We aimed to assess the genetic diversity and relationships in A. sinensis cultivars collected from different locations of China and also some other Angelica species. Results We employed an improved random amplified polymorphic DNA (RAPD) technique for the amplification of DNA materials from ten Angelica cultivars, and the results were verified by inter-simple sequence repeat (ISSR) analysis. Twenty six RAPD primers were used for RAPD, and the amplified bands were found highly polymorphic (96%). Each primer amplified 8-14 bands with an average of 10.25. The cluster dendrogram showed that the similarity coefficients ranged from 0.41 to 0.92. The similarity coefficients were higher among different cultivars of A. sinensis, and lower among different species. Twenty ISSR primers were used for the amplification, and each primer generated 6-10 bands with an average of 7.2 bands per primer. The cluster dendrogram showed that the similarity coefficients ranged from 0.35 to 0.89. Conclusions This study genetically characterized the Angelica species, which might have a significant contribution to the genetic and ecological conservation of this important medicinal plant. Also, this study indicates that the improved RAPD and ISSR analyses are important and potent molecular tools for the study of genetic diversity and authentication of organisms.

Evaluation of macular function in open angle and angle-closure glaucomatous eyes by microperimetry / 中华实验眼科杂志

Background The biomeasurement and imageology of retinal nerve fiber layer(RNFL) thickness showed the damage of retinal structure in the early and middle stage of glaucomatous eye,however,the subtle functional damage of glaucoma can not be timely reflected only with automatic static perimeter.Microperimetry is a method of quantitatively assessing mean sensitivity (MS) of macular zone.Objective This study was to evaluate and compare the macular functional change in early and middle stage of primary open angle glaucoma (POAG) and chronic angle-closure glaucoma (CACG) with MP-1 microperimeter.Metbods This trail protocol was approved by Ethic Committee of Peiking University Third Hospital,and written informed consent was obtained from each subject prior to entering the study group.A cross-sectional and case-controlled study was designed.A total of 126 eyes from 126 subjects were enrolled in the trail,including 53 eyes of 53 normal subjects,50 eyes of POAG patients and 23 eyes of CACG patients.A macular 10° program was set with MP-1 microperimetry to record the MS of various subareas.The macula was zoned into central 2°,6°,and 10° visual fields and 4 quadrants in each ring.The MS of different rings and subareas was detected and compared among POAG patients,CACG patients and normal controls.Results The MS values of central 2°,6°,10° and whole macular area were (15.09 ± 3.03),(15.72 ± 3.22),(13.99 ± 3.63) and (14.91±3.07)dB in the POAG group,which were significantly lower than those of (17.29±1.59),(18.05±1.24),(16.76±1.89) and (17.37±1.46)dB in the normal control group (all at P=0.000).The MS values of central 2°,6°,10° and whole macular area was (16.00±2.39),(15.83±2.63),(14.45±3.15) and (15.42±2.54) dB in the CACG group,and the reduced MSs were seen at the 6°,10° rings and whole macular area in the CACG group compared with the normal control group (P =0.004,0.013,0.011).Within the 6° ring,the MS values in the inferotemporal quadrant were declined in the POAG group and CACG group compared with the normal control group (P =0.000,0.022),but the difference was not statistically significant between the POAG group and the CACG group (P =0.311).In addition,the MS value in the inferonasal quadrant was significantly lower than that of the normal control group (P =0.005); while that in the CACG group was not significantly different in comparison with the normal control group (P=0.119).In the POAG group,the MS value of the inferotemporal quadrant was significantly lower than that of the superonasal or superotemopral quadrant (P =0.043,0.016),but no significant differences were found among the 4 quadrants in the CACG group (all at P＞0.05).Conclusions The mild damage of retinal function appears in the early and middle stage of POAG and CACG.More serious MS reducing occurs in the inferotemporal and inferonasal quadrants of POAG.

Enzyme kinetics and molecular modeling studies of G6PDMahidol associated with acute hemolytic anemia.

G6PDMahidol enzyme is the most common variant in the Achang Chinese ethnic group and clinically manifests as class II. In this study, G6PDMahidol enzyme was characterized by molecular modeling to understand its kinetics. G6PDMahidol, G6PDG487A and G6PDWT proteins were heterologously expressed in the G6PD-deficient DF213 E. coli strain, purified and their steady-state kinetic parameters were determined. Compared with G6PDWT, the Km and Vmax of NADP+ with G6PDG487A were about 28-fold and 12-fold lower, respectively. The Ki values of dehydroepiandrosterone (DHEA), NADPH and ATP with G6PDG487A showed 29.5-fold, 2.36-fold reduction and 1.83-fold increase, respectively. A molecular modeling of G6PDG487A was performed based on the X-ray structure of human G6PD (PDB: 2BH9). It is suggested that Ser-163 might affect the stability of G6PDG487A -helix d and -strand E, besides the conformation of -strand D. In conclusion, the biochemical and structural properties of G6PDG487A and G6PDWT enzymes are significantly different, which may be responsible for clinical diversity of G6PD deficiencies.

Eight-year experience in esophageal cancer surgery.

Aim: Esophageal cancer remains a major and lethal health problem. In Nepal, not much has been explored about its management. The aim of this study was to conduct a retrospective review of esophageal cancer patients undergoing surgery or combined modality treatment at a cancer hospital in Nepal. Materials and Methods: Resectable cases were treated primarily with surgery. Locally advanced cases with doubtful or obviously unresectability underwent preoperative chemo/radiation or chemoradiation followed by surgery. Results: Among 900 patients, 103 were treated with curative intent. Mean age of patients was 54 years, and 100% of the patients presented with complaint of dysphagia. Surgery as a single modality of treatment was done in 57% of cases, and the remaining underwent combined modality treatment. Transthoracic and transhiatal approaches were used in 95% and 5% of cases, respectively. Nodal sampling, two-field (2-FD), and three-field lymphadenectomy (3-FD) were done in 18%, 59%, and 20% of cases, respectively. A majority of patients had pathological stage III disease (46.6%). In-hospitality mortality was 5%, and anastomotic leakage rate was 14%. In 87% of patients, R0 resection was achieved. Overall, 4-year survival was 20%. A R0 resection, early-stage disease and 3-FD favored the survival advantage (P < 0.05). Conclusion: The mortality, complication, and survival results were in the acceptable range. R0 resection and radical nodal dissection should be standard practice.

Protective Effects of Eplerenone on Podocyte Injury in Adriamycin Nephropathy Rats / 华中科技大学学报（医学）（英德文版）

To investigate the protective effects of eplerenone on adriamycin-induced renal injury and the possible mechanisms involved,36 male Sprague-Dawley rats were randomly divided into control group,adriamycin nephropathy (AN) group and eplerenone-treated group (100 mg·kg-1·d-1 eplerenone).Blood pressure,24-h urinary protein,serum potassium,sodium and creatinine were measured 28 days after adriamycin injection (a single tail intravenous injection of 6.5 mg/kg adriamycin).The morphological changes of renal tissues were observed by light and electron microscopy.lmmunohistochemistry and Western blotting were performed to examine the expression of TGF-β1 and desmin in renal cortex.The results showed that 28 days after adriamycin injection,there were no significant changes in the level of serum potassium,sodium,creatinine concentrations and blood pressure values in the rats of the three groups.Meanwhile,the 24-h proteinuria excretion in the AN group was significantly higher than that in the control group (P＜0.01),but that in the eplerenone-treated group was substantially reduced when compared with that in the AN group (P＜0.05).Mild mesangial cell proliferation and matrix expansion,diffuse deformation and confluence of foot processes in podocytes were found in the AN group.By contrast,rats in the eplerenone-treated group exhibited obvious attenuation of these morphological lesions.The protein expression of TGF-β1 and desmin in the AN group was markedly up-regulated in contrast to that in the control group (P＜0.01),whereas that in the eplerenone-treated group was much lower than in the AN group (P＜0.05).It was concluded that eplerenone may ameliorate the proteinuria and the development of pathological alteration in adriamycin-induced nephropathy presumably via the inhibition of cytokine release,and restore the morphology of podocytes independent of its blood pressure-lowing effects.

Triptolide-induced Apoptosis by Inactivating Nuclear Factor-kappa B Apoptotic Pathway in Multiple Myeloma in vitro / 华中科技大学学报（医学）（英德文版）

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro,as well as the roles of nuclear factor-kappa B (NF-κB) and IκBα was investigated.The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay.The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose- and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells.Besides,we observed the inhibition of NF-κB/p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.

Albumin Modulates the Production of Matrix Metalloproteinases-2 and-9 in Podocytes / 华中科技大学学报（医学）（英德文版）

To investigate the effects of albumin on the production of matrix metalloproteinases-2 (MMP-2) and matrix metalioproteinases-9 (MMP-9) in podocytes. Podocytes were treated with bovine serum albumin (BSA) at the concentration of 0.1,0.5,1,2 g/L,respectively. Conditioned media were harvested 12,24,48 and 72 h after the treatment. The expression of MMP-2 and MMP-9 was assayed by gelatin zymography,RT-PCR and Western blotting analysis. Our results showed that in comparison with the control group,BSA increased the expression of MMP-2 and MMP-9 mRNA and protein in a dose-and time-dependent manner (P＜0.05). Meanwhile,the enzymatic activities of MMP-2 and MMP-9 in the culture supernatants of podocytes were also increased (P＜0.05). It is concluded that albumin up-regulated the expression of MMP-2 and MMP-9 at gene and protein levels in a time-and dose-dependent manner.

Protective Effect of Sulodexide on Podocyte Injury in Adriamycin Nephropathy Rats / 华中科技大学学报（医学）（英德文版）

This study examined the effect of sulodexide on podocyte injury in rats with adriamycin nephropathy (AN). A total of 36 healthy male SD rats were randomly assigned to three groups:control group,AN group and sulodexide treatment group. Rat models of AN were established by a single tail intravenous injection of adriamycin (6.5 mg/kg) in both AN group and sulodexide treatment group. Sulodexide (10 mg/kg) was administered the rats in the treatment group once daily by garage from the fast day of model establishment until the 14th day or the 28th day. Samples of 24-h urine and renal cortex tissues were harvested at day 14,28 after the model establishment. Excretion of 24-h urinary protein was measured by Coomassie brilliant blue method. The pathological changes in renal tissues were observed by light microscopy and electron microscopy respectively. Heparanase mRNA was detected by RT-PCR. Expressions of desmin,CD2AP and heparanase were determined by immunohistological staining. The results showed that the expressions of heparanase mRNA and proteinwere increased in the glomeruli of AN rats at day 14 and 28 after the model establishment,which was accompanied by the increased expression of desmin and CD2AP. The mRNA and protein expression of heparanase was decreased in the sulodexide-treated rats as compared with AN rats at day 14 and 28. And,the protein expression of desmin and CD2AP was reduced as with heparanase in the sulodexide-treated rats. Proteinuria and podocyte foot process effacement were alleviated in the AN rats after sulodexide treatment. There was a positive correlation between the expression of heparanase and the expression of desmin and CD2AP (as well as 24-h urinary protein excretion). It was concluded that increased heparanase is involved in podocyte injury. Sulodexide can maintain and restore podocyte morphology by inhibiting the expression of heparanase in AN.

A Rho-kinase Inhibitor, Fasudil, Attenuates Progressive Glomerulosclerosis Induced by Daunorubicin in Rats / 华中科技大学学报（医学）（英德文版）

Accumulating evidence suggests that the small G protein Rho and its downstream effector Rho kinase may play important roles in kidney biology. The present study examined the effects of a Rho-kinase inhibitor,fasudil,on daunorubicin-induced progressive glomerulosclerosis and explored the underlying mechanism by which fasudil ameliorates glomerulosclerosis. Thirty-six male SD rats were randomly allocated into sham-operation group (sham group,n=12),unilateral ncphrectomy (UNX)+daunorubicin (DRB) group (model group,n=12),UNX+DRB+Fasudil group (treatment group,n=12). Two to four weeks after the establishment of the animal model,6 rats in each group were taken randomly for the detection of 24-h urine protein excretion. Kidney sections were examined by HE and PAS staining,immunohistochemistry and transmission electric microscopy (TEM).The expression of Rho-kinase mRNA and P27 mRNA in kidney were detected by RT-PCR. It was found that the 24-h urine protein excretion in model group was increased significantly as compared with sham group (P＜0.01). But this increase was significantly suppressed by fasudil (P＜0.05). At 4 week,the foot process effacement in podocytes,mesangial proliferation and ECM accumulation were observed in model group,presenting as focal segmental glomerulosclerosis. But in the treatment group, the fasudil alleviated glomerular injury, with proliferating cell nuclear antigen (PCNA)-positive cell infiltration ameliorated and the expression of P27 increased. The expression of Rho-kinase mRNA was significantly enhanced in model group and was suppressed in treatment group. Moreover,fasudil up-regulated the mRNA expression of P27. Our study demonstrated that the glomerulosclerosis was substantially ameliorated by inhibiting the expression of Rho-kinase. It is suggested that Rho-kinase pathway is involved in the renal injury and the inhibition of Rho-kinase may constitute a therapeutic strategy for the treatment of renal injury.

Effect of Down-regulation of TRPC6 on the Puromycin Aminonucleoside-induced Apoptosis of Mouse Podocytes / 华中科技大学学报（医学）（英德文版）

poptosis induced by PAN.

The polymorphisms of bovine cocaine- and amphetamine-regulated transcripts and their associations with cattle (Bos taurus) growth traits.

We investigated the polymorphisms of bovine cocaine- and amphetamine-regulated transcripts (CART ). The coding and regulating regions of CART were screened in 7 cattle breeds by the single-stranded conformation polymorphism (SSCP) technique. The four loci (C1,C2, C3 and C4) studied were all polymorphic. Polymerase chain reaction (PCR) products representing different SSCP variants were sequenced and a total of 9 single-nucleotide polymorphisms (SNPs) were found. The associations between polymorphic loci and the growth traits of Nanyang cattle were analysed. The results indicated that genotype A1A1 of the C1 locus was associated with a higher body weight (P less than 0.05) than heterozygous A1B1. Genotype A2A2 of the C2 locus was associated with lower body weight and average daily weight gain (P < or = 0.001) than heterozygous A2B2. C3 and C4 loci had no significant effect on Nanyang cattle growth traits (P > 0.05).