Enhanced detection of ligand-PPARγ binding based on surface plasmon resonance through complexation with SRC1- or NCOR2-related polypeptide.

A previous study reported the use of a biosensing technique based on surface plasmon resonance (SPR) for the ligand binding detection of peroxisome proliferator activator receptor gamma (PPARγ). This detection was designed based on the structural properties of PPARγ. Because of cross-linked protein inactivation and the low molecular weight of conventional ligands, direct ligand binding detection based on SPR has low stability and repeatability. In this study, we report an indirect response methodology based on SPR technology in which anti-His CM5 chip binds fresh PPARγ every cycle, resulting in more stable detection. We developed a remarkable improvement in ligand-protein binding detectability in vitro by introducing two coregulator-related polypeptides into this system. In parallel, a systematic indirect response methodology can reflect the interaction relationship between ligands and proteins to some extent by detecting the changes in SA-SRC1 and GST-NCOR2 binding to PPARγ. Rosiglitazone, a PPARγ agonist with strong affinity, is a potent insulin-sensitizing agent. Some ligands may be competitively exerted at the same sites of PPARγ (binding rosiglitazone). We demonstrated using indirect response methodology that selective PPARγ modulator (SPPARM) candidates of PPARγ can be found by competing for the binding of the rosiglitazone site on PPARγ, although they may have no effect on polypeptides and PPARγ binding.

Optimized application of the secreted Nano-luciferase reporter system using an affinity purification strategy.

Secreted Nano-luciferase (secNluc) is a newly engineered secreted luciferase that possesses advantages of high structural stability, long half-life, and glow-type kinetics together with high light emission intensity, and thus would become one of the most valuable tools for bioluminescence assays. However, like other secreted luciferases, secNluc has to mix with the components in the conditioned medium surrounding test cells, or in the biological samples such as blood or urine after being secreted. These components may interfere with secNluc-catalyzed bioluminescence reactions and thus limit the application of the secNluc reporter system. In this study, we first examined the effects of three factors, pH, serum and residual reagents, on secNluc-catalyzed bioluminescence reactions, finding that these factors could interfere with bioluminescence reactions and result in background signal. To resolve these problems, we applied a simple affinity purification strategy in which secNluc was fused with a FLAG-tag, and anti-FLAG magnetic beads were used to catch and transfer the fusion protein to PBST, an optimal buffer for secNluc-catalyzed bioluminescence reactions that was identified in this study. The results indicated that this strategy could not only negate the interferences from serum or residual reagents and enhance the stability of light emission but also greatly increase signal intensity through enzyme enrichment. This strategy may contribute to biomedical studies that utilize secNluc and other secreted luciferases, especially those requiring superior sensitivity, low background noise and high reproducibility.

[Enhancing hRANKL production in Escherichia coli by optimizing culture conditions and lysing program].

RANKL/RANK/OPG axis is important in bone metabolism regulation, and becomes a popular research area in bone diseases. RANKL is a critical part of RANKL/RANK/OPG axis, and widely required in bone metabolism research. However, the yield of recombinant soluble human RANKL (hRANKL) in Escherichia coli is much lower than mouse RANKL (mRANKL). In this study, by adjusting and stabilizing the pH value of LB medium at 7.5, lowering the inducing temperature to 16 ℃ and optimizing the lysis program, the yield of soluble hRANKL increased by approximately 5 to 12-fold over the non-adjusted group. Our experiment effectively enhanced soluble hRANKL expression in E. coli and might constitute a meaningful attempt to obtain soluble expression of recombinant protein in E. coli.

Minimally invasive unilateral pedicle screws and a translaminar facet screw fixation and interbody fusion for treatment of single-segment lower lumbar vertebral disease: surgical technique and preliminary clinical results.

BACKGROUND: Conventional open transforaminal lumbar interbody fusion (TLIF) using unilateral pedicle screws and a translaminar facet screw has been performed for many years with good results. The outcomes of minimally invasive TLIF (MIS TLIF) are similar to the good outcomes of open TLIF, with the additional benefits of reducing iatrogenic injury, shortening hospital stays, and reducing the recovery duration. Instead of using small cuts on both sides, we performed MIS TLIF through a single cut using unilateral pedicle screws and a translaminar facet screw. The operative feasibility, efficacy safety, and benefits of single-level MIS TLIF of such techniques require further clarification. METHODS: A total of 60 patients with various single-segment lower lumbar vertebral diseases were treated in our department from January 2010 to March 2013. All the patients were initially performed single-level MIS TLIF using a hybrid construction of unilateral pedicle screws and a translaminar facet screw. Patient demographics and operative data were collected. The clinical outcomes were assessed before surgery and 3, 6, 12, and 24 months after surgery using the visual analog scale (VAS) for back and leg pain and the Oswestry Disability Index (ODI). Radiologic assessment of the lumbar spine with static and dynamic plain radiographs was performed 3, 6, 12, and 24 months after surgery. The fusion rates were assessed by an independent radiologist 2 years after surgery according to the Bridwell interbody fusion grading system. RESULTS: No patients experienced significant postoperative complications. Excepting two cases, 58 cases were followed up for 24-38 months, averaged 29.9 ± 4.1 months. The patients' average age was 46.6 ± 11.5 years, operative time 109.7 ± 17.8 min, intraoperative blood loss 67.3 ± 29.7 ml, length of incision 29.0 ± 3.2 mm, fluoroscopy time 31.1 ± 7.2 s, time to ambulation 20.3 ± 7.0 h, length of hospital stay 5.1 ± 1.1 days, and length of the translaminar facet screw 51.7 ± 3.4 mm. Screw position results: type I, 54 cases with 54 segments; type II, four cases with four segments. There were two (3.4%) translaminar facet screw failures, which were intraoperatively converted to a bilateral pedicle screw fixation procedure and excluded from the research. The postoperative images showed good positioning of the hybrid internal fixation, and all of the translaminar facet screws penetrated the facet joint. Two (3.6%) translaminar facet screws penetrated the lateral lamina and two (3.6%) translaminar facet screws penetrated the medial lamina without any serious neural complications. During the follow-up, there was no screw loosening or pedicle fracture observed. The VAS and ODI scores were significantly improved compared with the preoperative scores (P < 0.05), and the symptoms disappeared gradually. Fifty-one patients (87.9%) achieved grade I fusion radiographically at the final follow-up. CONCLUSIONS: MIS TLIF using a hybrid construction of unilateral pedicle screws and a translaminar facet screw is safe and effective in the treatment of single-segment lower lumbar vertebral disease, and it can be used as an optimal choice for fixation and fusion of some single-segment lower lumbar vertebral diseases.

Sortase A-aided Escherichia coli expression system for functional osteoprotegerin cysteine-rich domain.

As a natural inhibitor of the receptor activator of nuclear factor-кB ligand (RANKL), osteprotegerin (OPG) is considered a promising treatment for metabolic bone diseases. Typical approaches for preparing recombinant OPG or its derivatives employ eukaryotic expression systems. Due to the advantages of a prokaryotic expression system, which include its convenience, low cost, and abundant production, in this study, we establish a strategy for preparing functional OPG using the Escherichia coli expression system. After initial failures in preparation of OPG and its truncation, OPG cysteine-rich domain (OPG-CRD/OPGT) by using pET and pGEX vectors, we constructed a sortase A (SrtA)-aided E. coli expression system, in which the expressed protein was a self-cleaving SrtA fusion protein. Using this system, we successfully prepared the recombinant OPGT protein. The BIAcore analyses indicated that the prepared OPGT had high affinities in binding with RANKL and TRAIL. Cell experiments confirmed the inhibitory effects of the prepared OPGT on RANKL-induced osteoclast differentiation and TRAIL-induced tumor cell apoptosis. The sortase A-aided E. coli expression system for OPGT established in this study may contribute to further studies and commercial applications of OPG.

[Construction and verification of NF-κB luciferase reporter gene system].

To quantify the transcriptional activity of NF-κB and to screen drugs related to the regulation of NF-κB activation, we constructed a recombinant plasmid through deleting the original CMV promoter of retrovirus vector pQCXIP and inserting the NF-κB enhancer and NanoLuc luciferase sequence into the vector. Then, using the recombinant plasmid we constructed a cell line in which the expression of NanoLuc luciferase (NLuc) was regulated by NF-κB. The inserted sequences were verified by restriction endonuclease digestion and sequencing. Tumor necrosis factor-α (TNF-α), an NF-κB activator, acted on the constructed NLuc cell line and leaded to the specific luciferase reaction. The luciferase reaction showed a fine time and dose dependence to the TNF-α stimulation, indicating the successful construction of the NF-κB regulated NLuc-expressing cell line. Besides, the NF-κB inhibitor, triptolide, reduced the expression of NLuc in a dose-dependent way. The constructed reporter system in this study could be applied in the quantification of the NF-κB transcriptional activity and in the NF-κB regulation-related drug screening.