Gene expression exhibits temporal and spatial patterns to response environmental changes and growth cycle. Gene expression is under strict control at different levels among which control at transcription level is the predominant mode, especially in prokaryotes. In this review, we summarized the new developments of methods used in transcriptional studies, including modifications and improvements of the classic methods, such as gel-shift assay, DNA foot printing, and in vivo reporter system. In addition, we introduced examples to apply new methods, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize protein-DNA, ligand-protein, and ligand-protein-DNA interactions. The collection of these methods and their application could guide and accelerate relevant studies.
Calorimetry ; DNA Footprinting ; Gene Expression ; Ligands ; Proteins ; Surface Plasmon Resonance ; Transcription, Genetic

Bacterial two-hybrid system is a newly developed method for studying protein-protein interactions. However, in our studies of the interaction of regulatory proteins in Streptomyces, it was found that the bacterial two-hybrid system is not sensitive enough by the blue-and-white selection on X-gal plate. To overcome this drawback, the reason of false positive clone was firstly determined, which was the disturbance of other direct or indirect regulation on lacZ promoter. Then the disturbance was diluted by introducing multicopy lacZ promoter, which drive another reporter gene gfp. By such design, the sensitivity of the modified bacterial two-hybrid system was significantly inproved and the two different reporters also help to decrease the rate of the false positive clones. Further the evaluation of the modifiedd bacterial two-hybrid system indicated that the sensitivity was significantly improved.
Bacteria ; Genes, Reporter ; Promoter Regions, Genetic ; Protein Interaction Mapping ; methods ; Two-Hybrid System Techniques

Objective To investigate the consistency of M RI detecting posterior ligamentous complex (PLC) injury associated with thoracolumbar factures.Methods MRI data of 170 cases of thoracolumbar fractures were reviewed retrospectively.Each case underwent MRI around one week postinjury.MRI data were analyzed and compared by three physicians respectively to discuss the consistency in MRI detection of PLC injury and the severity of PLC injury.Results Kappa coefficient was 0.846 between observer 1 and 2,0.768 between observer 1 and 3,and 0.793 between observer 2 and 3.Interobserver reliability was high and overall Kappa coefficient was 0.803.Severity of PLC injury was interrelated with spinal cord nerve injury (P ＜ 0.05).Conclusions Accurate detection of PLC injury in thoracolumbar fractures is beneficial to clear the mechanical stability of the spine.MRI detection of PLC injury is of high consistency and hence deserves wide use.

The transforming growth factor-β1 (TGF-β1)/Smad3 signal pathway is related to mutiple physiological and pathological generation mechanism of human being. Up to date, however, the spacial and time information on the phosphorylated Smad3 is still unclear. In this study, the process of Smad3 phosphorylation was observed under the physiological state in the living cells. Firstly, the ECFP-Smad3-Citrine (Smad3 biosensor) fusion protein expression vector was constructed and identified. Then the Smad3 biosensor was transfected into 293T cells. The transfection efficiency and the expressions of fusion proteins were observed in 24 hours. Thirdly, Smad3 biosensor flurorescence resonance energy transfer (FRET) was observed with the inversion fluorescence microscope and measured by the MetaFlour FRET 4. 6 software. Smad3 biosensor transfection efficiency was nearly 40% and the fusion protein was seen under the fluorescence microscope. The FRET ratio of Smad3 biosensor in living 293T cells was decreased after 10 minutes incubation with the ligand of TGF-β1. The period of decreasing CFP and enhancing Citrine signals was about 300 seconds. With the technology of FRET, the TGF-β1/Smad3 signal pathway could be real time monitored dynamically under the physiological condition in living cells.
Fluorescence Resonance Energy Transfer ; Genetic Vectors ; HEK293 Cells ; Humans ; Microscopy, Fluorescence ; Phosphorylation ; Signal Transduction ; Smad3 Protein ; metabolism ; Software ; Transfection ; Transforming Growth Factor beta1 ; metabolism

Objective To investigate the damage sequence of posterior ligamentous complex (PLC) and its clinical significance in thoracolumbar fracture.Methods Data of 132 patients with spinal fracture evaluated with X-rays,CT and short-tau inversion-recovery (STIR) sequences in MRI were collected prospectively.Fracture morphology was classified using the AO classification.PLC components including interspinous ligament (ISL),supraspinous ligament (SSL),ligamentum flavum (LF) and facet capsules (FC) were assessed and classified as intact,edema,or tear.ISL edema was further subdivided depending on the extension (＞ 50％ or ≤50％).Correlation between MRI signal and AO progressive scale of morphological damage was analyzed.Results AO type A1/A2 fracture associated with only FC distraction.AO type A3 fracture showed additional ISL tear,usually less than 50％,with neither LF nor SSL tear.AO type B1 fracture showed FC distraction,ISL edema or disruption,and low rate of SSL/LF tear,but B2 fracture increased the rate of SSL/LF tear.AO type C fracture showed facet fracture or dislocation and ISL,SSL as well as LF tear.High correlation was found between AO progressive scale and MRI signal (P ＜ 0.01).Conclusions MRI study can well display the PLC damage and damage sequence.MRI correlates with AO progressive scale of morphological damage,which shows a progressive orderly rupture sequence among different PLC components as traumatic forces increase.

Objective To explore the effect of microRNA-613 (miR-613)/Wee1 axis on the radiosensitivity of colorectal cancer cells.Methods A total of 20 patients with radiosensitive colorectal cancer and 20 patients with radioresistance were selected from Yan'an Hospital Affiliated to Kunming Medical University from November 2016 to May 2017.Human colorectal cancer cell lines LoVo and HCT116 were selected and the radioresistant cell lines LoVo/R and HCT116/R were established for subsequent experiments.Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of miR-613 and Wee1 in colorectal cancer tissues and cell lines.The radioresistant cells were transfected by miR-613 mimic,and non-transfected cells were used as control group.The effects of miR-613 overexpression on the proliferation,invasion and cell cycle of radiation resistance of colorectal cancer cells at different radiation doses were evaluated by CCK-8 assay,Transwell assay and Western blotting,respectively.Furthermore,dual-luciferase reporter gene assay was used to verify whether Wee1 was a target gene of miR-613.si-Wee1 was transfected into radioresistant cells of colorectal cancer,or co-transfected with si-Wee1 and miR-613 inhibitor,and non-transfected cells were used as control group.The effects of miR-613/Wee1 axis on cell proliferation,invasion and cell cycle were detected by CCK-8,Transwell and Western blotting at different radiation doses.Results The expression of miR-613 was downregulated in the radiation resistance group of patients (1.54 ± 0.25 vs.2.64 ± 0.45;t =3.140,P =0.009) and radiation resistance cell lines (LoVo/R vs.LoVo:1.03 ± 0.12 vs.3.05 ± 0.15;t =8.944,P =0.006;HCT116/R vs.HCT116:1.01 ±0.11 vs.2.85 ±0.16;t =8.050,P =0.008).Overexpression of miR-613 was significantly inhibited the proliferation (LoVo/R:t6 Gy =6.018,P =0.013;HCT116/R:t6Gy =5.634,P =0.015) and invasion (LoVo/R:45.00 ± 8.95 vs.180.15 ± 6.95,t6 Gy =11.93,P =0.003;HCT116/R:49.97 ±6.21 vs.170.20 ±7.03,t6 Gy =12.82,P =0.006) of LoVo/R and HCT116/R cells and decreased the expression levels of G2-M phase cell cycle correlated proteins (CDK1 and cyclin B).Moreover,dual-luciferase reporter gene assay confirmed that Wee1 was a target of miR-613.Mechanistically,overexpression of miR-613 promoted the radiosensitivity of LoVo/R and HCT116/R cells through inhibiting cell proliferation (compared with si-Wee1 group,co-transfected with si-Wee1 and miR-613 inhibitor,and control group,LoVo/R:F8 Gy =40.742,P =0.007;HCT116/R:F8 Gy =28.958,P =0.011),invasion (LoVo/R:F8 Gy =55.413,P =0.004;HCT116/R:F8 Gy =65.634,P =0.003) and arresting cell at G2-M phase via downregulating Wee1.Conclusion miR-613/Wee1 axis plays a certain role in regulating the radiation resistance of colorectal cancer cells,overexpression of miR-613 may reverse the radiation resistance of colorectal cancer cells.

Neuroinflammation has been a key pathological process in many neurodegenerative disorders including Parkinson′s disease (PD), while the microglial activation is not only involved in but also regarded as a hallmark of the neuroinflammation. With PET, it has become possible to image in vivo the changes of microglia cells under different pathophysiological conditions. In this review, the basic theory of translocator protein (TSPO)-targeting PET (TSPO-PET) imaging is well described and its intriguing applications in PD are also elaborated.