Synthesis and Structure Elucidation of Two Essential Metal Complexes: In-Vitro Studies of Their BSA/HSA-Binding Properties, Docking Simulations, and Anticancer Activities.

Imidazole and tetrazole derivatives are widely used as clinical drugs since they possess a variety of pharmaceutical function. Zinc and iron are essential trace elements of the human body, with less toxicity and good biocompatibility. In this paper, two new essential metal mononuclear complexes [M(H2tmidc)2(H2O)2]·2H2O (M = Zn (1), Fe (2)) were synthesized through the reaction of 2-((1H-tetrazol-1-yl)methylene)-1H-imidazole-4,5-dicarboxylic acid (H3tmidc) and ZnSO4·7H2O or FeSO4·7H2O. The crystal structures were determined by means of the X-ray single crystal diffraction technique. Results from fluorescence investigations show that both complexes could interact with BSA as well as HSA through the static quenching mechanism. van der Waals forces and hydrogen bonds play important roles in the interaction of complexes and BSA/HSA since both ΔH and ΔS values are negative. The results of molecular docking are consistent with those in experimental studies. Furthermore, the anticancer activity of H3tmidc and both complexes against Eca-109 were preliminarily evaluated and the results show that both complexes have better anticancer activity than the corresponding ligand H3tmidc.

Analysis of lumbosacral sagittal balance parameter variation in minimally invasive transforaminal lumbar interbody fusion with real-time 3D navigation techniques / 中国骨伤

OBJECTIVE@#To explore the dynamic changes of lumbosacral sagittal parameters after real-time three-dimensional navigation assisted minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) and traditional open TLIF for treatment of lumbar degenerative disease.@*METHODS@#The clinical data of 61 patients with lumbar degenerative disease underwent single-segment surgery from September 2017 to September 2019 were retrospectively analyzed. Among them, 31 cases underwent MIS-TLIF with 3D navigation techniques (MIS-TLIF group) and another 30 cases underwent conventional open TLIF (traditional open TLIF group). The basic information, operative time and intraoperative blood loss were collected. The sagittal radiologic parameters were measured before surgery and 3 months after surgery, including lumbar lordosis (LL), segmental lordosis (SL), pelvic incidence (PI), pelvic tilt (PT), sacral slope (SS), anterior disc height (ADH), posterior disc height(PDH).And the average disc height(DH) and pelvic incidence to lumbar lordosis mismatch (PI-LL) were calculated.@*RESULTS@#Operative time and intraoperative blood loss in MIS-TLIF group were significantly less than in traditional open TLIF group(@*CONCLUSION@#Real-time navigation-assisted MIS-TLIF and traditional open TLIF can recover DH in a short term for lumbar degenerative diseases, improve LL and PI-LL, and make the arrangement of the sagittal plane of the lumbosacral region more coordinated after surgery. But only the navigation assisted MIS -TLIF can significantly improve SL. Compared with traditional open TLIF, real-time navigation assisted MIS-TLIF in the treatment of degenerative lumbar diseases has the advantages of short operation time and less intraoperative bleeding.

[Consumption Capacity of N2O in Paddy Soil and the Response Mechanism of nosZ-I-containing Communities].

The long-term flooding anaerobic environment in paddy soils is conducive to denitrification, which is one of the most important reasons for N2O emissions. N2O can be transformed to nitrogen gas (N2) by bacteria and archaea containing nitrous oxide reductase (N2OR) encoded by the nosZ gene, which is the only known biological pathway of N2O consumption in soil. nosZ-I is known to be typical in denitrifying bacteria, which is one of the clades of the nosZ gene and is mainly possessed a Tat signal peptide motif. Although many researchers have studied N2O emission characteristics of paddy soil, the capacity of N2O consumption and the response mechanism of related functional microorganisms in paddy fields is not yet clear. To verify the effect of exogenous N2O on N2O consumption and nosZ-I gene, a pot trial experiment was performed under anaerobic conditions. We collected intact soil cores from flooding paddy fields at a 0-5 cm depth, and exogenous N2O gas was input through the bottom of flooding paddy soil cores. Meanwhile, a control treatment (CK) with no additional N2O gas was also performed. The dynamic characteristics of the added exogenous N2O concentration through the intact soil cores, the content of inorganic nitrogen, and DOC were systematically monitored. In addition, the change in the nosZ-I population diversity and community composition were investigated by high-throughput sequencing approaches, with the purpose of revealing the N2O uptake ability of flooded paddy soil and the response mechanism of the nosZ-I population. The results showed that 97.39% of exogenous N2O diffused into the soil cores, and only 0.72%-7.75% of exogenous N2O escaped from the soil surface. The N2O released in the headspace of soil cores could continue being absorbed and consumed by the flooding soil column. In addition, 67.10% of the N2O escaped to the headspace was consumed in exogenous N2O treatment after 192 h of incubation, which was higher than that in CK treatment, and the N2O consumption rate increased by 144.2% than that in CK treatment. Meanwhile, the consumption of NH4+-N, NO3--N, and DOC consumed during exogenous N2O addition treatment was 19.65%, 16.29%, and 8.41% higher than that in CK treatment, respectively. However, the diversity of the nosZ-I gene community had no significant difference; the community composition of nosZ-I-containing bacteria changed significantly after 192 h when exogenous N2O was input. The abundances of OTU5004, OTU5065, OTU960, and OTU1282 (Proteobacteria) significantly increased, which were the dominant bacterial strain of nosZ-I gene on the OTU level. Compared with the initial sample and CK, the abundance of the OTU5004 strain increased by 7.3% and 4.63%, and the abundance of the OTU5265 strain (Azoarcus sp.) increased by 0.33% and 0.15%, respectively. The result indicated that the flooding paddy soil column at the soil layer of 0-5 cm has a strong N2O absorption and consumption ability. In summary, compared with CK, the addition of exogenous N2O significantly accelerated the N2O consumption rate, improved the consumption potential of flooding paddy soil column, promoted carbon and nitrogen conversion, and changed nosZ-I community composition. These results would provide a new reference for reducing atmospheric N2O emissions.

[Impact of Dicyandiamide (DCD) and 3,4-Dimethylpyrazole Phosphate (DMPP) on Ammonia-oxidizing Bacteria and Archaea in a Vegetable Planting Soil].

Nitrification inhibitors (NIs) dicyandiamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP) showed significant effects in the inhibition of nitrification and the improvement of the utilization efficiency of nitrogen fertilizer in agricultural soils. However, the effects of different NIs on ammonia-oxidizing bacteria (AOB) and archaea (AOA) is still unclear. To verify the inhibitory effect of DCD and DMPP on AOB and AOA, a pot experiment was performed, including Urea, Urea+DCD, and Urea+DMPP treatments. The dynamics of NH4+-N and NO3--N and nitrification potential among different treatments were measured. In addition, real-time PCR and high-throughput sequencing approaches were applied to investigate the changes in the AOB and AOA population abundance and composition. The results revealed that the concentrations of NH4+-N in Urea+DCD and Urea+DMPP treatments were 213% and 675% higher than that in the CK treatment, respectively. However, the concentrations of NO3--N and the nitrification potentials were 13.3% and 37.2%, and 20.4% and 82.4% lower than that in CK treatment, respectively; Furthermore, the copy numbers of the bacterial and archaeal amoA gene were 51.2% and 56.5%, and 6.0% and 27.0% lower than that in the CK treatment, respectively. However, the diversity indexes of AOB and AOA communities, including evenness and richness, exhibited no significant differences after addition of DCD and DMPP. The nork-environmental-samples, unclassified-Nitrosomonadaceae, unclassified-Bacteria, and Nitrosospira, were the predominant genera of the AOB community. The no rank-Crenarchaeota, no rank-environmental-samples and Nitrososphaera were the predominant groups in the AOA community. Summarily, application of DCD and DMPP significantly delayed the transformation of NH4+-N, decreased the formation of NO3--N, inhibited the abundance and changed the composition of AOB and AOA communities. DMPP had a stronger inhibitory effect on nitrification, and on AOB and AOA than DCD. Therefore, compared with DCD, DMPP had a better application prospect regarding the improvement of the nitrogen utilization efficiency in vegetable soil.

Effects of microRNA-145 on epithelial-mesenchymal transition of TGF-β1-induced human renal proximal tubular epithelial cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effects of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) of TGF-β1-induced human renal proximal tubular epithelial (HK-2) cells.</p><p><b>METHODS</b>The gene sequence of miR-145 was synthesized and cloned into pCMV-myc to construct recombinant plasmid pCMV-miR-145. HK-2 cells were divided into four groups: control (untreated), TGF-β1 (treated with TGF-β1), blank+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with blank plasmid) and miR-145+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with pCMV-miR-145 recombinant plasmid). Expression of miR-145 was detected by real-time PCR (RT-PCR). TGF-β1, Smad3, Smad2/3, p-Smad2/3, α-SMA, FN and type I collagen (Col I) protein levels were detected by Western blot. Concentrations of fibronectin (FN) and Col I in cell culture supernatants were measured using ELISA.</p><p><b>RESULTS</b>pCMV-miR-145 recombinant plasmid was successfully transfected into HK-2 cells. Compared with the control group, the miR-145+TGF-β1 group showed a significant up-regulation in the expression level of miR-145 (P<0.01). However, the TGF-β1 and blank+TGF-β1 groups showed a significant down-regulation in the expression level of miR-145 compared with that in the control and miR-145+TGF-β1 groups (P<0.01). Compared with the TGF-β1 and blank+TGF-β1 groups, the miR-145+TGF-β1 group showed significantly reduced levels of the signal proteins TGF-β1, Smad3, Smad2/3 and p-Smad2/3 (P<0.05), as well as significantly reduced levels of the biomarkers α-SMA, FN and Col I (P<0.05). Meanwhile, concentrations of FN and Col I in cell culture supernatants also decreased (P<0.05).</p><p><b>CONCLUSIONS</b>miR-145 modulates the EMT of HK-2 cells treated with TGF-β1, possibly by inhibition of the activation of TGF-β-dependent Smad signaling pathway.</p>

Mammalian Metallothionein-2A and Oxidative Stress.

Mammalian metallothionein-2A (MT2A) has received considerable attention in recent years due to its crucial pathophysiological role in anti-oxidant, anti-apoptosis, detoxification and anti-inflammation. For many years, most studies evaluating the effects of MT2A have focused on reactive oxygen species (ROS), as second messengers that lead to oxidative stress injury of cells and tissues. Recent studies have highlighted that oxidative stress could activate mitogen-activated protein kinases (MAPKs), and MT2A, as a mediator of MAPKs, to regulate the pathogenesis of various diseases. However, the molecule mechanism of MT2A remains elusive. A deeper understanding of the functional, biochemical and molecular characteristics of MT2A would be identified, in order to bring new opportunities for oxidative stress therapy.

Replantation for complete amputated finger composite lateral tissues / 中国骨伤

<p><b>OBJECTIVE</b>To explore the clinical therapeutic effect and safety of application of Ilizarov technique combined with flap instant expansion technique in correcting tibia angular deformity combined with skin contracture by one stage.</p><p><b>METHODS</b>From January 2010 to January 2013, 30 cases of tibial deformity with skin contracture were corrected by Ilizarov technique combined with flap instant expansion technique at one stage, including 21 males and 9 females with an average age of(40.2±5.5) years ranging from 25 to 60 years. All patients underwent regular reexamination of X-ray. After removal of the Ilizarov external fixation, knee joint function were assessed by American Hospital for Special Surgery (HSS) scoring criteria, and the pain was evaluated by visual simulation score(VAS).</p><p><b>RESULTS</b>All patients were followed up for 6 to 35 months with an average of 22 months. Among them, the incision of 29 patients were primary healing, 1 patient had wound infection complicated by osteomyelitis, 2 patients complicated with fixed screw loosening, there were no expanded skin flap necrosis and neurovascular injury symptoms. The external fixators were removed at 4 to 7 months after operation with an average of(5.2±1.1) months. Correction angle was 10° to 35° degrees with an average of (25.5±3.5)°. HSS total score was 92.5±6.6 and the result was excellent in 25 cases, good in 4 cases, fair in 1 case; the VAS score was 1.2±1.5.</p><p><b>CONCLUSIONS</b>The application of Ilizarov technique combined with flap instant expansion technique is a good method for correction of tibial angular deformity with skin contracture by one stage, with a shorter time of external fixation frame, without skin necrosis and neurological symptoms, early load exercise and improve the limb function.</p>

Effects of high glucose on expression of OPG and RANKL in rat aortic vascular smooth muscle cells.

OBJECTIVE: To explore effect of high glucose on expression of osteoprotegerin (OPG) and receptor activator of NF- κ B ligand (RANKE) in rat aortic vascular smooth muscle cells. METHODS: SD rats were intraperitoneally injected with streptozotocin, OPG and RANKL expression in rat thoracic aortas were detected by immunohistochemical staining. In cultured vascular smooth muscle cells (VSMCs) (A7r5), qRT-PCR and Western blot analysis were used to examine the mRNA and protein levels of OPG and RANKL. RESULTS: Our results demonstrated that OPG expression was increased in hyperglycemic rat aortic VSMCs, while RANKL expression was decreased. Besides, in vitro experiments high glucose induced OPG expression, but depressed RANKL expression by dose- and time-dependent manner in cultured A7r5. CONCLUSIONS: Our findings suggested that high glucose could promote the expression of OPG, and inhibit the expression of RANKL in VSMCs, which may be partly be the molecular mechanism of diabetic vascular calcification.

[The anti-apoptotic effect of cytoplasmic alpha-fetoprotein in hepatoma cells induced by all-trans retinoic acid involves activation of the PI3K/AKT signaling pathway].

OBJECTIVE: To explore the effect of alpha-fetoprotein (AFP) on transduction of the PI3K/ AKT signal in hepatocellular carcinoma cells and the role played by AFP in resistance to cytotoxicity of all-trans retinoic acid (ATRA). METHODS: The effects of ATRA of human liver cancer cells was assessed using the BEL-7402 cell line with the MTT assay (to evaluate proliferation), microscopy (to evaluate morphology), flow cytometry (to evaluate apoptosis), laser confocal microscopy and coimmunoprecipitation (co-IP; to evaluate co-localization and interaction of AFP with PTEN), Western blotting (to evaluate expression of phosphorylated-protein kinase B (pAKT) and Src, and RNA interference (RNAi)-mediated knockdown of AFP. Finally, application of the PI3K-specific inhibitor Ly294002 was used to monitor the influence of AFP in transduction of the PI3K signal pathway. RESULTS: The human hepatoma cell line BEL-7402 were resistant to ATRA cytotoxicity. PTEN and AFP co-localized in the cytoplasm, and co-IP indicated that AFP interacts with PTEN in BEL-7402 cells.RNAi knockdown of AFP expression led to reduced growth of BEL-7402 cells.BEL-7402 cells transfected with AFP-short interfering (si)RNA vectors showed enhanced sensitivity to ATRA and reduced expression of pAKT(Ser473) and Src; Ly294002 reduced the role of AFP in stimulating expression of pAKT(Ser473) and Src. CONCLUSION: AFP can activate transduction of the PI3K/AKT signal, and expression of AFP in hepatoma cells is a pivotal event for resisting ATRA-induced apoptosis.

Antigen 43/Fcε3 chimeric protein expressed by a novel bacterial surface expression system as an effective asthma vaccine.

The IgE Fcε3 domain is an active immunotherapeutic target for asthma and other allergic diseases. However, previous methods for preparing IgE fusion protein vaccines are complex. Antigen 43 (Ag43) is a surface protein found in Escherichia coli that contains α and ß subunits (the α subunit contains multiple T epitopes). Here we constructed a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against IgE. The Ag43 system was constructed from the E. coli strain Tan109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43) expressing a partial Ag43 gene was introduced. The Fcε3 domain of the IgE gene was then subcloned into plasmid pETAg43, resulting in a recombinant plasmid pETAg43/Fcε3, which was used to transform Tan109 for Ag43/Fcε3 surface expression. Thereafter, Ag43/Fcε3 was investigated as an asthma vaccine in a mouse model. Ag43/Fcε3 was expressed on and could be separated from the bacterial surface by heating to 60° while retaining activity. Ag43/Fcε3, as a protein vaccine, produced neutralizing autoantibodies to murine IgE, induced significant anti-asthma effects, and regulated IgE and T helper cytokines in a murine asthma model. Data show that Ag43/Fcε3 chimeric protein is a potential model vaccine for asthma treatment, and that the Ag43 system may be an effective tool for novel vaccine preparation to break immune tolerance to other self-molecules.

p42/p44 mitogen-activated protein kinases inhibit atrial natriuretic peptide mRNA transcription in gp130-mediated hypertrophic ventricular myocytes.

OBJECTIVE: To understand the role of ANP mRNA transcription regulation in gp130-mediated cardiomyocyte hypertrophy, and the involved mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK, also called p42/p44 MAPK) signaling pathway. METHODS: Isolated neonatal ventricular myocytes were treated with different concentrations of CT-1 (10(-9), 10(-8)and 10(-7)mol/L). MTT was used to analyze the viability and RT-PCR was used to detect ANP mRNA levels in cardiomyocyte. To inhibit p42/p44 MAPK activity in hypertrophic cardiomyocytes, the cells were pretreated with a specific MEK1 inhibitor. RESULTS: CT-1 significantly induced ANP mRNA expression and the viability of cardiomyocytes in a dose- and time-dependent manner. Furthermore, blocking p42/p44 MAPK activity by the special MEK1 inhibitor upregulated the ANP mRNA. CONCLUSIONS: p42/p44 MAPK have an important role in suppressing ANP mRNA transcription and cell activity in gp130-mediated hypertrophic ventricular myocytes.

Bacterial surface display of endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis.

Various angiogenesis-related self-molecules have been considered to be therapeutic targets. However, the direct use of self-molecules as vaccines is not recommended because of the inherent ability of the host to develop immune tolerance. Antigen 43 (Ag43) is a surface protein found in E. coli and contains an α and a ß subunits, which contains multiple T epitopes in α subunit. Here we construct a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against self-molecules. The Ag43 system was constructed from an Escherichia coli strain Tan109, derived from JM109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43') expressing a partial Ag43 gene was introduced. The extracellular domain of angiogenesis-related endoglin gene was then subcloned into plasmid pETAg43', resulting in a recombinant plasmid pETAg43'/END(e) which was then used to transform Tan109 for protein expression. We found that Ag43 and endoglin chimeric protein (Ag43'/END(e) ) was expressed on the bacterial surface. The chimeric protein could be separated from the bacterial surface by heating to 60°C and yet retain activity. We used Ag43'/END(e) as a protein vaccine and found that it could disrupt immune tolerance against endoglin by inducing significant antitumor activities and inhibit angiogenesis in several tumor models without significant side effects. These data suggest that Ag43'/END(e) chimeric protein is a potential model vaccine for active tumor immunotherapy, and that Ag43 system could be an effective tool for novel vaccine preparation to break immune tolerance to other angiogenesis-related self-molecules for cancer therapy.

Recombinant adeno-associated virus-mediated transfer of shRNA against Notch3 ameliorates hepatic fibrosis in rats.

Liver fibrosis, a wound healing process following all kinds of liver injuries, is characterized by excessive deposition of extracellular matrix (ECM). Our previous study revealed that Notch3 might participate in liver fibrogenesis by regulating the activation of hepatic stellate cells (HSCs). The aim of this study was to assess the effects of Notch3 shRNA on hepatic fibrosis in a rat model induced by carbon tetrachloride (CCl4) and to clarify the mechanisms underlying those effects. Recombinant adeno-associated virus type 1 (rAAV1) vector carrying Notch3 shRNA (rAAV1-Notch3-shRNA) was generated and transferred to rat livers via the tail vein. The expression of Notch3, Jagged1, Hes1 and α-SMA were detected by real-time RT-PCR and immunofluorescence. The effects of rAAV1-Notch3-shRNA on fibrosis was investigated by pathological and immunohistochemical examination. Our findings showed that Notch3, Jagged1, Hes1 and α-SMA were downregulated. This downregulation was accompanied by improved hepatic fibrosis after the inhibition of Notch3 in vivo. rAAV1-Notch3-shRNA treatment reversed the epithelial-mesenchymal transition (EMT) in fibrotic livers by decreasing the expression of transforming growth factor ß1 (TGF-ß1) and vimentin in a line with the increased expression of E-cadherin. The inhibition of Notch3 was not found to play a role in hepatocyte proliferation. Rather, it inhibited hepatocyte apoptosis in vivo to some extent. The results of the present study suggest that the inhibition of Notch3 can protect hepatocytes from undergoing apoptosis and attenuate liver fibrogenesis. This may be a viable therapeutic option for hepatic fibrosis.

Cytochalasin D promotes pulmonary metastasis of B16 melanoma through expression of tissue factor.

Cytochalasin D (CytD) targets actin, a ubiquitous protein in eukaryotic cells. Previous studies have focused mainly on the antitumor effects of CytD. We previously found CytD to promote lung metastasis in B16 melanoma cells, which we had not anticipated, and, therefore, in the present study we investigated the possible underlying mechanisms. B16 melanoma cells were co-cultured with CytD and other agents and used to establish a lung metastatic model. In this B16 melanoma metastatic model, significantly increased lung metastasis and lung weight were found in CytD-treated mice, which was almost completely suppressed by tissue factor (TF) RNA interference expressed via lentivirus. The results of northern and western blot, and real-time RT-PCR analysis showed that the expression of TF was significantly upregulated in B16 cells treated with CytD but was significantly inhibited by TF RNA interference. In addition, upregulation and phosphorylation of mitogen-activated protein kinase p38 were also found in the metastatic lung tissues treated with CytD and in the B16 cells co-cultured with CytD and factor VIIa (FVIIa), but not in cells cultured with CytD, dimethyl sulfoxide or FVIIa alone. These results indicate that CytD stimulates the expression of TF in B16 melanoma cells, activating both coagulation-dependent and -independent pathways via binding to FVIIa, eventually promoting lung metastasis. TF interference is a potential approach to the prevention of B16 melanoma metastasis.

Evaluation on the immune response induced by DNA vaccine encoding MIC8 co-immunized with IL-12 genetic adjuvant against Toxoplasma gondii infection.

OBJECTIVE: To examine the immunoprotection effect induced by MIC8 DNA vaccine co-immunized with a plasmid encoding murine IL-12 (pcIL-12) as an adjuvant in mice against the challenge of Toxoplasma gondii. METHODS: The gene sequence encoding MIC8 of T. gondii RH strain was inserted into eukaryotic expression vector pcDNA3.1 to construct the pcMIC8 expression plasmid. The recombinant plasmid was transfected into HeLa cells to test its expression and the recombinant protein was then characterized by Western blotting. Eighty Kunming mice were randomly divided into 5 groups (16 per group): 3 control groups (PBS, pcDNA3.1, and pcIL-12), pcMIC8 group, and pcMIC8 plus pcIL-12 group. Mice in the pcMIC8 plus pcIL-12 group were co-injected intramuscularly at a dosage of 100 microl each of pcMIC8 and pcIL-12 suspended in 100 microg sterile PBS. Mice in other groups were inoculated with PBS, pcDNA3.1, pcIL-12, and pcMIC8 respectively following the same protocol. All the mice received three immunizations at 2-week intervals. Serum samples were collected on day 0, 13, 27, 41, and 55 before each inoculation for determining antibody IgG, IgG subclass IgG2a. Four weeks after the final immunization, IFN-gamma and IL-4 levels in splenocytes cultures from immunized mice were detected by ELISA. The mice were challenged with 10(3) tachyzoites of the virulent T. gondii RH strain three weeks after the last immunization to observe the survival time. RESULTS: Western blotting showed that the protein extracts in HeLa cells upon transfection with pcMIC8 were effectively expressed in cells. The levels of IgG (0.51 +/- 0.028) and IgG2a (0.261 +/- 0.04)(on day 55) in mice immunized with pcMIC8 plus pcIL-12 were higher than pcMIC8 group (497.65 +/- 98.15) and control groups (PBS 47.18 +/- 2.73, pcDNA3.1 50.08 +/- 4.62, pcIL-12 118.15 +/- 12.73) (P < 0.05). There was no significant difference in the level of IgG 1 and IL-4 among the five groups (P > 0.05). After a lethal challenge of T. gondii RH strain, the survival time in mice immunized with pcMIC8 plus pcIL-12 (15d) was prolonged in comparison to that of pcMIC8 (10d) and control groups (PBS 5 d, pcDNA3.1 6d, pcIL-12 8 d) (P < 0.05). CONCLUSION: The immune responses induced by the combined use of the recombinant plasmid encoding MIC8 of T. gondii with murine IL-2 gene adjuvant can be enhanced.

Treatment of unstable middle 1/3 of clavicular fractures with composite external fixator in adult patients / 中国骨伤

<p><b>OBJECTIVE</b>To explore methods and clinical effects of composite external fixtor in treating adult patients with unstable middle 1/3 of clavicular fractures.</p><p><b>METHODS</b>From March 2008 to March 2011,36 patients with unstable middle 1/3 of clavicular fractures were treated with composite external fixtor. There were 24 males and 12 females, aged from 18 to 55 years old with an average of 43 years old. Twenty cases on the left side and 16 cases on the right side. Time from injury to operation was 2 to 6 days (averaged 3.5 days). According to Robinson classification, there were 7 cases with type 2A2, 18 cases with type 2B1, and 11 cases with type 2B2. No vessels and nerve damage occurred before opreation. The clinical effects were evaluated according to Neer scoring.</p><p><b>RESULTS</b>All cases were followed up from 6 to 12 months with an average of 8 months. The mean Neer score was 88.3 +/- 6.2, which included pain 31.6 +/- 3.2, functional score 25.7 +/- 2.2, range of motion score 21.1 +/- 1.7, and anatomy score 8.8 +/- 0.8. There were 22 cases in excellent, good in 13, fair in 1. Two cases occurred pin tract infection.</p><p><b>CONCLUSION</b>Composite external fixtor is an optional method in treating unstable middle 1/3 of clavicular fracture, and can obtain a good clinical effects.</p>

Toxicarioside A inhibits tumor growth and angiogenesis: involvement of TGF-ß/endoglin signaling.

Toxicarioside A is a cardenolide isolated mainly from plants and animals. Emerging evidence demonstrate that cardenolides not only have cardiac effects but also anticancer effects. In this study, we used in vivo models to investigate the antitumor activities of toxicarioside A and the potential mechanisms behind them. Murine colorectal carcinoma (CT26) and Lewis lung carcinoma (LL/2) models were established in syngeneic BALB/c and C57BL/6 mice, respectively. We found that the optimum effective dose of toxicarioside A treatment significantly suppressed tumor growth and angiogenesis in CT and LL/2 tumor models in vivo. Northern and Western blot analysis showed significant inhibition of endoglin expression in toxicarioside A-treated human umbilical vein endothelial cells (HUVECs) in vitro and tumor tissues in vivo. Toxicarioside A treatment significantly inhibited cell proliferation, migration and invasion, but did not cause significant cell apoptosis and affected other membrane protein (such as CD31 and MHC I) expression. In addition, TGF-ß expression was also significantly inhibited in CT26 and LL/2 tumor cells treated with toxicarioside A. Western blot analysis indicated that Smad1 and phosphorylated Smad1 but not Smad2/3 and phosphorylated Smad2/3 were attenuated in HUVECs treated with toxicarioside A. Smad1 and Smad2/3 signaling remained unchanged in CT26 and LL/2 tumor cells treated with toxicarioside A. Endoglin knockout by small interfering RNA against endoglin induced alternations in Smad1 and Smad2/3 signaling in HUVECs. Our results indicate that toxicarioside A suppresses tumor growth through inhibition of endoglin-related tumor angiogenesis, which involves in the endoglin/TGF-ß signal pathway.

Construction and characterization of a full-lengh cDNA library from non-fresh Giardia lamblia.

OBJECTIVE: To construct rapidly a full-length cDNA library from nanogram amounts total RNA of Giardia lamblia (G. lamblia) trophozoites stocked in RNA stabilization reagent. METHODS: Total RNA of Giardia was extracted using Trizol reagent. A full-length cDNA library of G. lamblia trophozoites was constructed by a long-distance PCR (LD-PCR) method. The recombinant rate and the coverage rate of full-length clones of the library were evaluated. The inserted fragments were identified and sequenced by PCR amplification. RESULTS: The titer of cDNA library was 3.85 × 10(7) pfu/mL. The length of inserted fragments ranged from 0.4 to 2.5 kb, and the recombination efficiency accounted for 100% (20/20). The coverage rate of full-length clones is high (17/20). CONCLUSIONS: The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature. The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA.

Inhibition of Pim-1 attenuates the proliferation and migration in nasopharyngeal carcinoma cells.

OBJECTIVE: To explore the role of proto-oncogene Pim-1 in the proliferation and migration of nasopharyngeal carcinoma (NPC) cells. METHODS: Pim-1 expressions in NPC cell lines CNE1, CNE1-GL, CNE-2Z and C666-1 were examined by RT-PCR, western blotting and immunoflucesence, respectively. After CNE1, CNE1-GL and C666-1 cells were treated with different concentrations of Pim-1 special inhibitor, quercetagetin, the cell viability, colony formation rate and migration ability were analyzed. RESULTS: Pim-1 expression was negative in well-differentiated CNE1 cells, whereas expressed weakly positive in poor-differentiated CNE-2Z cells and strongly positive in undifferentiated C666-1 cells. Interestingly, CNE1-GL cells that derived from CNE1 transfected with an Epstein Barr virus latent membrane protein-1 over-expression plasmid displayed stronger expression of Pim-1. Treatment of CNE1-GL and C666-1 cells with quercetagetin significantly decreased the cell viability, colony formation rate and migration ability but not the CNE1 cells. CONCLUSIONS: These findings suggest that Pim-1 overexpression contributes to NPC proliferation and migration, and targeting Pim-1 may be a potential treatment for anti-Pim-1-expressed NPCs.

[Antitumor effect of adriamycin conjugated with downsized anti-endoglin monclonal antibody].

OBJECTIVE: To study the antitumor efficacy of an immunoconjugate composed of adriamycin (ADM) and downsized Fab fragment of mouse anti-Endoglin monoclonal antibody. METHODS: The Fab fragment of mouse anti-Endoglin monoclonal antibody was downsized and conjugated with ADM by m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). The antitumor effect of the conjugate was tested in mice bearing subcutaneous injection of H22 tumor in vivo. RESULTS: The molecular ratio of Fab:ADM in conjugate was approximately 1:2. The Fab-ADM conjugate inhibited the growth of H22 by 91.94% on day 14 after injection at the dose of 0.4 mg/ kg, much higher the inhibition rate of 25.00% by the equivalent dose of free ADM. The median survival time of the mice treated with the conjugate was longer than those treated with free ADM. The Fab-ADM conjugate was significantly more effective than free ADM in tumor suppression and life span prolongation. CONCLUSION: Fab -ADM displayed more significant antitumor efficacy than free ADM in vivo and might be a novel candidate for cancer treatment.