Tanshinone IIA modulates cancer cell morphology and movement via Rho GTPases-mediated actin cytoskeleton remodeling.

Actin filaments form unique structures with robust actin bundles and cytoskeletal networks affixed to the extracellular matrix and interact with neighboring cells, which are crucial structures for cancer cells to acquire a motile phenotype. This study aims to investigate a novel antitumor mechanism by which Tanshinone IIA (Tan IIA) modulates the morphology and migration of liver cancer cells via actin cytoskeleton regulation. 97H and Huh7 exhibited numerous tentacle-like protrusions that interacted with neighboring cells. Following treatment with Tan IIA, 97H and Huh7 showed a complete absence of cytoplasmic protrusion and adherens junctions, thereby effectively impeding their migration capability. The fluorescence staining of F-actin and microtubules indicated that these tentacle-like protrusions and cell-cell networks were actin-based structures that led to morphological changes after Tan IIA treatment by retracting and reorganizing beneath the membrane. Tan IIA can reverse the actin depolymerization and cell morphology alterations induced by latrunculin A. Tan IIA down-regulated actin and Rho GTPases expression significantly, as opposed to inducing Rho signaling activation. Preventing the activity of proteasomes and lysosomes had no discernible impact on the modifications in cellular structure and protein expression induced by Tan IIA. However, as demonstrated by the puromycin labeling technique, the newly synthesized proteins were significantly inhibited by Tan IIA. In conclusion, Tan IIA can induce dramatic actin cytoskeleton remodeling by inhibiting the protein synthesis of actin and Rho GTPases, resulting in the suppression of tumor growth and migration. Targeting the actin cytoskeleton of Tan IIA is a promising strategy for HCC treatment.

[Association Between Dietary Knowledge and Food Preferences in Residents Aged 18 and Over in China Based on Latent Class Analysis].

Objective To investigate the food preferences and explore the potential association between dietary knowledge and food preferences in residents aged 18 and over in China,so as to provide a basis for promoting healthy diets.Methods The latent class analysis was carried out with the 2015 cross-sectional data of China health and nutrition survey to categorize the food preferences among 8 783 residents aged 18 and over.Multinomial Logistic regression was adopted to assess the association between and dietary knowledge and food preferences.Results The food preferences of the residents aged 18 and over in China were classified into preference for less vegetable(3.28%),lack of preference(11.20%),diverse preferences(4.19%),and preference for healthy diets(81.33%).The proportion of the adults with dietary knowledge was 36.87%(3 238/8 783).The dietary knowledge varied in the adults with different food preferences(all P<0.001).After adjusting for gender,age,urban and rural distribution,education background,and annual household income,for each point increase in the dietary knowledge score,there was an estimated reduction of 22% in the probability of preferring less vegetables(OR=0.78,95%CI=0.76-0.80, P<0.001),13% in the probability of lacking preference(OR=0.87,95%CI=0.86-0.89, P<0.001),and 3% in the probability of having diverse preferences(OR=0.97,95%CI=0.94-1.00, P=0.030).Compared with those lacking dietary knowledge,the individuals with dietary knowledge had a 77% less probability of preferring less vegetables(OR=0.23,95%CI=0.16-0.32, P<0.001),a 55% less probability of lacking preference(OR=0.45,95%CI=0.39-0.53, P<0.001),and a 23% less probability of having diverse preferences(OR=0.77,95%CI=0.61-0.96, P=0.023).Conclusions The residents aged 18 and over in China presented four food preferences,including preference for less vegetables,lack of preference,diverse preferences,and preference for healthy diets,the last of which had the highest proportion.The individuals with lower levels of dietary knowledge have higher probability of preferring unhealthy food.

[Comparison on Performance of Quantitative Ultrasound and Dual-Energy X-ray Absorptiometry in Evaluating Bone Health of Adults Aged 18-40 Years].

Objective To compare the consistency of quantitative ultrasound(QUS)and dual-energy X-ray absorptiometry(DXA)in measuring bone mineral density(BMD)of adults aged 18-40 years in Guangzhou and evaluate the diagnostic value of QUS for identifying low bone mass.Methods DXA was employed to measure the BMD and QUS to measure the speed of sound(SOS)in 731 participants.The Bland-Altman analysis was performed to evaluate the consistency of Z scores between SOS and BMD.With the BMD Z ≤-2.00 as the diagnostic criterion for low bone mass,the receiver operating characteristics curve of QUS was established,and the area under the curve(AUC)and the sensitivity,specificity,and correct diagnostic index for the optimal cut-off of SOS Z score were calculated.Results The results of Bland-Altman analysis showed that the mean differences in the Z scores of SOS and BMD in males and females were 1.27(-0.94 to 3.47)and 0.93(-1.33 to 3.18),respectively.The AUC of SOS Z score in the diagnosis of low bone mass in males and females was 0.734(95%CI=0.380-0.788)and 0.679(95%CI=0.625-0.732),respectively.In males,the optimal cut-off of SOS Z score for low bone mass was -0.35,with the sensitivity,specificity,and correct diagnostic index of 64.1%,68.6%,and 0.327,respectively.In females,the optimal cut-off value of SOS Z scores for low bone mass was -1.14,with the sensitivity,specificity,and correct index of 73.9%,54.8%,and 0.285,respectively.Conclusion QUS and DXA show poor consistency in the diagnosis of BMD in the adults aged 18-40 years in Guangzhou,while QUS demonstrates an acceptable value in identifying low bone mass.

Comprehensive mapping of antigenic linear B-cell epitopes on K205R protein of African swine fever virus with monoclonal antibodies.

African swine fever virus causes an acute, highly contagious swine disease with high mortality, leading to enormous losses in the pig industry. The K205R, a nonstructural protein of African swine fever virus, is abundantly expressed in the cytoplasm of infected cells at the early stage of infection and induces a strong immune response. However, to date, the antigenic epitopes of this immunodeterminant have not been characterized. In the present study, the K205R protein was expressed in a mammalian cell line and purified using Ni-affinity chromatography. Furthermore, three monoclonal antibodies (mAbs; 5D6, 7A8, and 7H10) against K205R were generated. Indirect immunofluorescence assay and western blot results showed that all three mAbs recognized native and denatured K205R in African swine fever virus (ASFV)-infected cells. To identify the epitopes of the mAbs, a series of overlapping short peptides were designed and expressed as fusion proteins with maltose-binding protein. Subsequently, the peptide fusion proteins were probed with monoclonal antibodies using western blot and enzyme-linked immunosorbent assay. The three target epitopes were fine-mapped; the core sequences of recognized by the mAbs 5D6, 7A8, and 7H10 were identified as 157FLTPEIQAILDE168, 154REKFLTP160, and 136PTNAMFFTRSEWA148, respectively. Probing with sera from ASFV-infected pigs in a dot blot assay demonstrated that epitope 7H10 was the immunodominant epitope of K205R. Sequence alignment showed that all epitopes were conserved across ASFV strains and genotypes. To our knowledge, this is the first study to characterize the epitopes of the antigenic K205R protein of ASFV. These findings may serve as a basis for the development of serological diagnostic methods and subunit vaccines.

A modified LC-MS/MS method for the detection of whole blood tacrolimus and its clinical value in Chinese kidney transplant patients.

Background: For patients who treated with tacrolimus after kidney transplant, therapeutic drug monitoring is essential to improve their prognosis. However, previous detection methods have limitations, such as the overestimation and unacceptable bias in the immunoassays. Precision medicine has been challenged. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is recognized as the gold standard due to its accuracy and specificity, but lack of throughput and complex process limits its clinical application. Therefore, an accurate, simple and high throughput method for tacrolimus monitoring is needed for clinical practice. Methods: A modified LC-MS/MS method was introduced and validated. Whole blood samples were prepared by a one-step protein precipitation method. Chromatographic separation was achieved using a Phenomenex Kinetex 2.6 µm XB-C18 2.1 × 50 mm column with a total run time of 3.5 min to avoid matrix effect. An electrospray ionization source (ESI) was used in positive ion multiple reaction monitoring (MRM) mode for mass spectrometric detection. In order to protect the mass spectrometer, only part of the sample after LC separation was allowed to enter the mass spectrum, through a two HPLC systems coupled one mass spectrometry design. In this way, the instrument throughput is also improved and realizing the detection of 2 samples within 3.5 min and carried out a shorter analyzing time for each sample of 1.75 min. Additionally, we calculated tacrolimus-intrapatient variant (Tac-IPV) based on this modified method and assessed the prognostic value of Tac-IPV in Chinese kidney transplant patients. Results: The LC-MS/MS was modified by streamlining the procedure and increasing the throughput. The method proved to be accurate and reproducible with all performance parameters suitably meeting the clinical requirements over a calibration ranged from 0.37 to 42.90 ng/mL. Parameters such as linearity, limit of quantification (LoQ) and dilution integrity were validated with a clinical reportable range from 0.37 to 343.20 ng/mL, which was particularly useful for high drug concentrations patients (rare but very serious). Both cross-contamination and matrix effects were negligible. Clinical data of 83 patients showed that Tac-IPV was associated with poor kidney transplant outcome in Chinese (Hazard Ratio (HR) = 3.96, 4.75; 95% Cl: 1.10-14.21, 1.23-18.36; P < 0.05). Conclusions: This modified LC-MS/MS method possessed high throughput and simple sample preparation, allowing it to meet daily clinical needs. At the same time, Tac-IPV based on this modified LC-MS/MS had excellent prognostic value in kidney transplantation. These advantages have great significance for the individualized treatment of Chinese kidney transplant patients and broad application of Tac-IPV.

Long Noncoding RNA RP11-732M18.3 Promotes Glioma Angiogenesis by Upregulating VEGFA.

Gliomas are the most aggressive and common type of malignant brain tumor, with limited treatment options and a dismal prognosis. Angiogenesis, a hallmarks of cancer, is one of two critical events in the progression of gliomas. Accumulating evidence has demonstrated that in glioma dysregulated molecules like long noncoding RNAs (lncRNAs), are closely linked to tumorigenesis and prognosis. However, the effects of and mechanisms of action of lncRNAs during tumor angiogenesis are poorly understood. The effect of lncRNA RP11-732M18.3 on angiogenesis was elucidated through an intracranial orthotopic glioma model, immunohistochemistry, and an in vitro angiogenesis assay. Co-culture experiments and cell migration assays were performed to investigate the function of lncRNA RP11-732M18.3 in vitro. lncRNA RP11-732M18.3 increased CD31+ microvessel density, and overexpression of lncRNA RP11-732M18.3 resulted in poor mouse survival. lncRNA RP11-732M18.3 promoted endothelial cell migration and tube formation. Nomogram and Kaplan-Meier survival analyses indicated that higher VEGFA is correlated with a poor prognosis. Mechanistically, lncRNA RP11-732M18.3 promotes angiogenesis by increasing the nuclear level of EP300 and facilitating the transcription and secretion of VEGFA. Our study contributes to the latest understanding of glioma angiogenesis and prognosis. lncRNA RP11-732M18.3 may be a potential treatment target in glioma.

Long non­coding RNA AL355711 promotes smooth muscle cell migration through the ABCG1/MMP3 pathway.

Atherosclerosis and related cardiovascular diseases pose severe threats to human health worldwide. There is evidence to suggest that at least 50% of foam cells in atheromas are derived from vascular smooth muscle cells (VSMCs); the first step in this process involves migration to human atherosclerotic lesions. Long non­coding RNAs (lncRNAs) have been found to play significant roles in diverse biological processes. The present study aimed to investigate the role of lncRNAs in VSMCs. The expression of lncRNAs or mRNAs was detected using reverse transcription­quantitative polymerase chain reaction. The Gene Expression Omnibus datasets in the NCBI portal were searched using the key words 'Atherosclerosis AND tissue AND Homo sapiens' and the GSE12288 dataset. Gene expression in circulating leukocytes was measured to identify patients with coronary artery disease (CAD) or controls, and used to analyze the correlation coefficient and expression profiles. The protein level of ATP­binding cassette sub­family G member 1 (ABCG1) and matrix metalloproteinase (MMP)3 was determined using immunohistochemistry and western blot analysis. The analysis of mouse aortic roots was performed using Masson's and Oil Red O staining. The expression of lncRNA AL355711, ABCG1 and MMP3 was found to be higher in human atherosclerotic plaques or in patients with atherosclerotic CAD. The correlation analysis revealed that ABCG1 may be involved in the regulation between lncRNA AL355711 and MMP3 in atherosclerotic CAD. The knockdown of lncRNA AL355711 inhibited ABCG1 transcription and smooth muscle cell migration. In addition, lncRNA AL355711 was found to regulate MMP3 expression through the ABCG1 pathway. The expression of ABCG1 and MMP3 was found to be high in an animal model of atherosclerosis. The results indicated that lncRNA AL355711 promoted VSMC migration and atherosclerosis partly via the ABCG1/MMP3 pathway. On the whole, the present study demonstrates that the inhibition of lncRNA AL355711 may serve as a novel therapeutic target for atherosclerosis. lncRNA AL355711 in circulating leukocytes may be a novel biomarker for atherosclerotic CAD.

[Effect of transcutaneous electrical acupoint stimulation on epidural-related maternal fever in parturients undergoing epidural labor analgesia].

OBJECTIVE: To evaluate the therapeutic effect and safety of transcutaneous electrical acupoint stimulation (TEAS) on epidural-related maternal fever in parturients undergoing epidural labor analgesia. METHODS: A total of 198 primiparas with single birth, full-term pregnancy and head position were recruited and randomized into a TEAS group (98 cases) and a control group (100 cases). In the TEAS group, after epidural labor analgesia, TEAS was applied to bilateral Hegu (LI4) and Quchi (LI11), once an hour, for 30 min each time, till the end of childbirth. In the control group, after epidural labor analgesia, TEAS electrodes were attached to the same acupoints, but without electric stimulation. Maternal tympanic temperature and the score of Visual Analogue Score (VAS) were measured before analgesia, at 1, 2, 3, 4 and 5 h after analgesia and during labor respectively and maternal fever rate was evaluated in the parturients of two groups. Separately, before analgesia, 2 h after analgesia and during labor, the levels of serum interleukin (IL-6) and IL-1ß were determined in the parturients of two groups. The duration of labor, the mode of labor, oxytocin dosage, postpartum hemorrhage, neonatal Apgar scores, time of labor analgesia, labor analgesic consumption and adverse effects were recorded in the parturients of two groups. RESULTS: Maternal tympanic temperature increased progressively in two groups as analgesic time prolonged. Tympanic temperature at 3, 4 and 5 h after analgesia and du-ring labor, and maternal fever rate during labor in the TEAS group were all lower than those in the control group respectively (P<0.05). The levels of serum IL-6 and IL-1ß increased after analgesia in the parturients of two groups. The serum IL-6 level during labor and the level of IL-1ß at 2 h after analgesia and during labor in the parturients of the TEAS group were lower than those in the control group (P<0.05). The analgesic consumption in the TEAS group was less than that in the control group (P<0.05). The incidence of chills in the TEAS group was lower than that in the control group (P<0.05). The differences were not statistical in VAS score, duration of labor, mode of labor, oxytocin dosage, postpartum hemorrhage, time of labor analgesia and neonatal Apgar score, as well as the incidence of urine retention, nausea and vomiting and urinary retention between two groups (P>0.05). CONCLUSION: Transcutaneous electrical acupoint stimulation at LI11 and LI4 is conductive to relieving epidural-rela-ted maternal fever and reducing serum levels of IL-6 and IL-1ß in the parturients undergoing epidural labor analgesia. It is safe and effective in clinical application.

[Effects of Transcutaneous Electrical Acupoint Stimulation for Depression in Late Pregnancy and Impacts on Inflammatory Cytokines].

OBJECTIVE: To assess the safety and efficacy of transcutaneous electrical acupoint stimulation (TEAS) for depression in late pregnancy and impacts on inflammatory cytokines. METHODS: A total of 150 pregnant women with depression in late pregnancy were randomized into a high intensity group (n＝52), a low intensity group (n＝49) and a control group (n＝49). TEAS was applied at bilateral Neiguan (PC 6) and Shenmen (HT 7) for 6 weeks. The intensities of TEAS in the high intensity group, the low intensity group and the control group were 10, 5 and 0 mA, respectively. During the process of TEAS, the blood pressure, pulse, uterine contraction and the fetal heart rate were recorded. Depression was evaluated by 24-item Hamilton depression scale (HAMD) before TEAS and after 2-week, 4-week, 6-week treatment. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1 ß (IL-1 ß) and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA) before and after 6-week treatment. Delivery outcomes were observed. The correlation was analyzed between HAMD difference value and the diffe-rence values of TNF-α， IL-1 ß and IL-6, respectively. RESULTS: The blood pressure, pulse and fetal heart rate had no statistical significance before and after treatment in the three groups (P>0.05). The HAMD scores at all the time points were lower than that before treatment in the high intensity group (P<0.05), which were lower compared with those in the low intensity group and the control group (P<0.05). The HAMD score in the low intensity group decreased after 6-week treatment compared with that before treatment and was lower than that in the control group (P<0.05). The serum levels of IL-1 ß and IL-6 in the high intensity group decreased compared with those before treatment and were lower compared with those in the low intensity group and the control group after 6-week treatment (P<0.05). There was no significant difference in the deliver outcomes among the three groups (P>0.05). The variation of HAMD score did not have significant correlation with those of TNF-α， IL-1 ß and IL-6 (P>0.05). CONCLUSION: TEAS is safe and effective for depression in late pregnancy and can regulate the serum levels of IL-1 ß and IL-6 without influencing on delivery outcome.

Clinical Correlates and Reference Intervals for Cystatin C in a Han Population from Southeast China.

BACKGROUND: Cystatin C (CysC) is an endogenous filtration marker for estimation of kidney function. This study aimed to define the reference interval (RI) for serum CysC in a southeast Chinese adult population and to explore the variables that affect serum CysC levels. METHODS: 532 reference individuals (259 male, 273 female, aged 18 - 79 years) were recruited from Guangzhou, China. Multiple regression analysis (MRA) was used to investigate the association between serum CysC levels and clinical factors including age, gender, body mass index, lifestyle, and biochemistry parameters. Reference values were defined using a parametric method according to Clinical and Laboratory Standards Institute guideline (C28A3). RESULTS: The mean serum CysC levels were significantly lower in females than in males (p < 0.001). Serum CysC levels increased with age (~0.047 mg/L increase per decade). MRA demonstrated that serum CysC levels correlated significantly with serum creatinine (Cr), high density lipoprotein (HDL-C), alkaline phosphatase (ALP), albumin (ALB), and uric acid (UA) concentrations, although their relationships were less prominent than those of gender or age. The RIs for serum CysC levels were calculated at 0.73 - 1.17 mg/L for subjects aged 18 - 49 years and at 0.73 - 1.49 mg/L for those aged 50 - 79 years. CONCLUSIONS: The RIs for serum CysC were established in a southeast Chinese population. In addition to gender and age, serum Cr, HDL-C, ALP, ALB, and UA also influenced serum CysC levels.

[Ultrasonic analysis on the effect of strengthening exercise with strong tendon on lumbosacral multifidus muscles].

OBJECTIVE: To study effects of strengthening exercise with strong tendon on the lumbosacral multifidus muscle. METHODS: Among 30 healthy volunteers, there were 21 males and 9 females, with an average age of(31.30±6.48) years old(ranged, 25 to 55 years old). The mean BMI was (21.70±1.95) kg/m²(ranged, 18.1 to 24.9 kg/m²). The ultrasonic image was used to analyze the thickness of lumbosacral multifidus(LM) under the follow conditions: supine position, and supine position with leg lifting 30°, 60°, 90°; seat and hip flexion, flexion to limit position, front to limit position with both hands climbing and strengthening the kidneys and the waist. RESULTS: The average lumbosacral multifidus thickness was (16.867±2.460) mm, (19.010±2.510) mm, (22.477±2.220) mm, and(27.593±2.370) mm respectively in supine position with leg lifting 0°, 30°, 60°, 90°. There were statistical differences(F=423.619, P<0.05). The average lumbosacral multifidus thickness was (25.810±2.440) mm, (15.677±2.130) mm, and (15.533±2.110) mm respectively in seat and hip flexion, flexion to limit, front to limit positions with both hands climbing and strengthening the kidneys and the waist. There were statistical differences(F=597.789, P<0.05). CONCLUSIONS: When healthy volunteers in Shi's Orthopedics strengthen muscle exercises training, multifidus thickness is increased with the increasing of leg degree, reduced with the increasing of the flexion degree. It can change the stretching state of multifidus muscle by a specific training, so as to achieve the purpose of training the multifidus muscle.

[Effect of acupuncture therapy on patients with low back pain: a Meta-analysis].

OBJECTIVE: To systematically review the clinical efficacy of acupuncture on the patients with low back pain (LBP). METHODS: Randomized controlled trials (RCTs) about pure acupuncture therapy versus other treatments in treating LBP were electronically searched in PubMed, CBM, EMbase, The Cochrane Library, CNKI, VIP and Wanfang Data from January 2004 to May 2014. The observed index on the results were the changed scores of VAS, ODI, JOA and RMDQ. Two reviewers independently screened the literatures according to the inclusion and exclusion criteria, as well as the extracted data, and assessed the methodological quality. The results of Meta-analysis was conducted by RevMan 5.2 software. RESULTS: Ten RCTs involved 751 patients were finally included. The results of Meta-analysis indicated that the role of pure acupuncture group in improving the VAS score was better than that of the control group, and the combined effect size was RR = -.32, 95% CI (-1.41, -1.22); Z=27.28, P<0.00001; the role of pure acupuncture group in improving the ODI score was better than that of the control group, and the combined effect size was RR = -5.07, 95% CI (-7.50, -2.65); Z=4.10, P<0.0001; the role of pure acupuncture group on improved JOA score was better than that of the control group and the combined effect size was RR=2.83, 95% CI (2.02, 3.63), Z=6.90, P<0.00001. The role of pure acupuncture group in improving the RMDQ score was better than that of the control group, and the combined effect size was RR = -2.80, 95% CI (-3.49, -2.11), Z=7.95, P<0.00001. CONCLUSION: The result of meta-analysis demonstrates that pure acupuncture may have a favorable effect on self-reported pain and functional limitations in LBP patients.

[Relationship between lumbosacral multifidus muscle and lumbar disc herniation].

As a common disease in clinical, the treatment of lumbar disc herniation (LDH) focused on local intervertebral disc, such as surgery and other interventional therapy treatment, but postoperative complications and recurrence rate has been a difficult problem in the field of profession. With the development of spine biomechanics and anatomy, researches on lumbar herniation also increased. Researchers discovered that the incidence and prognosis of LDH were inseparable with local muscle and soft tissue. As the deep paraspinal muscles, multifidus muscle plays an important role to make lumbar stability. Its abnormal function could reduce the stable of lumbar spine, and the chronic lumbar disease could also lead to multifidus muscle atrophy.

Performance evaluation of the Sysmex Cystatin C assay on the Roche Modular P Analyzer.

OBJECTIVES: Serum Cystatin C (CysC) is a promising endogenous marker for estimation of glomerular filtration rate. This study evaluated the analytical performance of the Sysmex CysC assay on the Roche Modular P Chemistry Analyzer (Modular P). DESIGN AND METHODS: The evaluation of imprecision, linearity, recovery, and interference were performed in accordance with the relevant Clinical and Laboratory Standards Institute guidelines protocols, using quality controls and patient samples. Comparison studies were conducted against the Siemens and Roche assay for CysC. RESULTS: At CysC concentrations of 0.45 and 1.80mg/L, the within-run coefficient of variations (CVs) were 2.81% and 2.04%, respectively, and the between-run CVs were 3.14% and 3.26%. The CysC assay was proven throughout the measuring range from 0.10 to 8.00mg/L. Good correlations were achieved among the 3 CysC assays. The Sysmex assay appeared to report higher results than the Siemens assay. No interference was detected from hemoglobin 5g/L, bilirubin (free or conjugated) 200mg/L, or chyle 1500 formazin turbidity units. CONCLUSIONS: The Sysmex CysC assay performed well with regard to basic performance, including precision, dilution linearity, and effects of interfering substances, and is an acceptable assay for the determination of CysC on the Modular P.

[Higher procalcitonin level in diabetic nephropathy patients compared with healthy volunteers].

OBJECTIVE: To investigate the difference of procalcitonin (PCT) level between uninfected diabetic nephropathy patients and healthy volunteers. METHODS: This study enrolled 76 patients with diabetes only [DM group, 24 h urinary micro albumin (mALB) < 30 mg/24 h], 81 patients with early DN (EDN group, mALB 30-300 mg/24 h), 87 DN patients (DN group, mALB > or = 300 mg/24 h), and 82 age- and sex-matched healthy volunteers. All the patients were free of systemic infection. PCT levels and various laboratory parameters including metabolic and kidney functions as well as inflammatory element profiles were assessed. RESULTS: The PCT level of DN group was significantly higher than that of healthy control group, DM group and EDN group (P < 0.001 or P < 0.05). Spearman's test showed a significant positive correlation between PCT and serum lactate dehydrogenase (LDH, r = 0.541, P < 0.01), Urine acid (UA) (r = 0. 320, P < 0.01), Urea (r = 0.324, P < 0.01), creatinine (Cr) (r= 0.403, P < 0.01), alpha-hydroxybutyrate dehydrogenase (alpha-HBD) (r = 0.791, P < 0.01) and C-reactive protein (CRP) (r = 0.694, P < 0.001) in diabetic nephropathy patients respectively. CONCLUSION: Serum PCT level of patients with diabetic nephropathy is higher than that of healthy volunteers, which may be associated with minimal inflammation and kidney function damage.

[Bone marrow-derived dendritic cells rather than spleen-derived dendritic cells can generate regulatory T cells].

There are many evidences that dendritic cells (DC) can establish and maintain immunological tolerance through inducing the differentiation of regulatory T cells Treg. This study was purposed to explore the possibility to gene-rate Treg from bone marrow-derived DC (BM-DC) or spleen-derived DC (spDC) generated CD4(+) CD25(+) FOXP3(+) Treg by induction. Bone marrow immature DC (imDC) induced from bone marrow precursor cells of C57BL/6 mice by GM-CSF and IL-4; after culture for 6 day, imDC were stimulated by LPS for additional 16 hours and the mature DC (mDC) have been got; the spDC were collected from spleen of C57BL/6 mice by MACS. Co-culturing fresh BALB/c mouse CD4(+) T cells with these three sorts of DC above mentioned respectively was performed to generate CD4(+) CD25(+) FOXP3(+) Treg. The expression of FOXP3 in CD4(+) T cells was detected by flow cytometry, and the capacity of different DC generated CD4(+) CD25(+) FOXP3(+) Treg was evaluated. The results showed that stimulated by C57BL/6 immature or mature DC, the positive rate of FOXP3 in BALB/c CD4(+) T cells increased from (8.57 ± 1.14)% to (15.80 ± 1.35)%, (17.93 ± 1.45)% respectively (P < 0.01); while stimulated by spDC, the positive rate of FOXP3 in BALB/c CD4(+) T cells decreased from (8.57 ± 1.14)% to (3.95 ± 0.79)% (P < 0.05). It is concluded that the BM-DC but not spDC can generate Treg from CD4(+) T cells, BM-DC may mediate immune tolerance rather than the immune response.

Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats.

Antibodies produced in animals vaccinated using live attenuated vaccines against Brucella spp. are indistinguishable using current conventional serological tests from those produced in infected animals. One potential approach is to develop marker vaccines in which specific genes have been deleted from parental vaccine strains that show good immunogenicity and vaccine efficacy. Corresponding methods of detection for antibodies raised by the marker vaccine should also be developed. A specific fragment of the bp26 gene of Brucella melitensis M5-90 was cloned into vector pQE32 to construct the recombinant plasmid (pQE32-rΔbp26). It was used to transform Escherichia coli M15 (pREP4) host cells, which expressed the rΔbp26 protein. Subsequently, the recombinant protein was purified by immobilized metal affinity chromatography and size-exclusion chromatography. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified rΔbp26 protein was represented by only one band, with a molecular weight of 14 kDa, and it showed good antigenic specificity on western blot and enzyme-linked immunosorbent assay (ELISA). The purified rΔbp26 protein was intended to be used as an antigen to develop a novel ELISA to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains.

Identification and characterization of a virus-specific continuous B-cell epitope on the PrM/M protein of Japanese Encephalitis Virus: potential application in the detection of antibodies to distinguish Japanese Encephalitis Virus infection from West Nile Virus and Dengue Virus infections.

BACKGROUND: Differential diagnose of Japanese encephalitis virus (JEV) infection from other flavivirus especially West Nile virus (WNV) and Dengue virus (DV) infection was greatly hindered for the serological cross-reactive. Virus specific epitopes could benefit for developing JEV specific antibodies detection methods. To identify the JEV specific epitopes, we fully mapped and characterized the continuous B-cell epitope of the PrM/M protein of JEV. RESULTS: To map the epitopes on the PrM/M protein, we designed a set of 20 partially overlapping fragments spanning the whole PrM, fused them with GST, and expressed them in an expression vector. Linear epitope M14 (105VNKKEAWLDSTKATRY120) was detected by enzyme-linked immunosorbent assay (ELISA). By removing amino acid residues individually from the carboxy and amino terminal of peptide M14, we confirmed that the minimal unit of the linear epitope of PrM/M was M14-13 (108KEAWLDSTKAT118). This epitope was highly conserved across different JEV strains. Moreover, this epitope did not cross-react with WNV-positive and DENV-positive sera. CONCLUSION: Epitope M14-13 was a JEV specific lineal B-cell epitpe. The results may provide a useful basis for the development of epitope-based virus specific diagnostic clinical techniques.

[Expression of recombinant extracellular region of human interleukin-1 receptor type I in Pichia pastoris].

OBJECTIVE: To construct pPICZalphaA-soluble interleukin-1 receptor type I (sIL-1RI) recombinant expression vector containing the gene fragment encoding the extracellular domain of sIL-1RI for its expression in Pichia pastoris. METHODS: sIL-1RI gene was amplified by RT-PCR and inserted into the yeast expression vector pPICZalphaA by digestion ligation. The recombinant plasmid pPICZalphaA-sIL1RI was transformed into E.coli Stb13, and the positive clones were analyzed by PCR and DNA sequencing. The pPICZalphaA-sIL1RI recombinant plasmid was electroporated into GS115 cells and the transformants were analyzed by PCR. After phenotype identification, the recombinant strains were induced by methanol to express the target protein, which was analyzed by Western blotting of the cell extract and supernatant. RESULTS: The recombinant plasmid pPICZalphaA-sIL-1RI was constructed successfully, and the results of Western blotting showed that yeast induced by methanol expressed a protein of about 39 kD. CONCLUSION: sIL-1RI protein has been successfully expressed in P.pastoris expression system, which provides the basis for further study of sIL-1RI.

[Expression of porcine gamma-interferon in recombinant baculovirus and determination of its antiviral activity].

The full-length porcine interferon gamma(PoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBac -PoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-gamma. Recombinant baculovirus, rBac-PoIFN-gamma, was generated for expressing PoIFN-gamma, by transfecting rBacmid-PoIFN-gamma with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-gamma in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-gamma expressed by the rBac-PoIFN-gamma can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-gamma can be specifically blocked by anti-PoIFN-gamma mouse serum. The antiviral titer of culture supernatant of insect cells infected by rBac-PoIFN-gamma is 2 x 10(4) IU/mL. The results demonstrat that the rBac-PoIFN-gamma can express rPoIFN-gamma efficiently and rPoIFN-gamma has high antiviral activity.