In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wild-type p53 could be transiently phosphorylated at multiple sites

<p>Abstract</p> <p>Background</p> <p>Infected cells recognize viral replication as a DNA damage stress and elicit the host surveillance mechanism to anti-virus infection. Modulation of the activity of tumor suppressor p53 is a key event in the replication of many viruses. They could manipulate p53 function through phosphorylation modification for their own purpose. But there is rarely research about p53 phosphorylation status in the context of HPV-E6. Therefore, we investigated whether p53 could be phosphorylated by HPV-E6.</p> <p>Methods</p> <p>We used a mammalian green fluorescence protein (GFP) expression system to express HPV-18E6 with GFP fusion proteins (GFP-18E6) in wild-type (wt) p53 cell lines, such as 293T and MCF-7 cells to trace the traffic and subcellular location of E6 protein. By immunofluorescence technique and immunoblotting, we determined the positive phosphorylated sites of p53 and observed the distribution of phosphorylated p53 in the context of GFP-18E6.</p> <p>Results</p> <p>GFP-18E6 was predominantly located in nuclei of wt p53 cell lines, and it could induce transient phosphorylation of p53 at multiple sites, such as Ser¹⁵, Ser²⁰, and Ser³⁹². All the three sites of phosphorylated p53s were localized in nuclei together with GFP-18E6.</p> <p>Conclusion</p> <p>In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wt p53 could be transiently phosphorylated at multiple sites.</p

Arabidopsis blue light receptor phototropin 1 undergoes blue light-induced activation in membrane microdomains

Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and step-wise photobleaching analysis we demonstrated that in the dark phot1-GFP proteins remain in an inactive state and mostly present as a monomer. The phot1-GFP diffusion rate and its dimerization increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive phot1 -GFP, whereas it did enhance its dimerization, suggesting that phot1 dimerization is independent of its phosphorylation. Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) analysis revealed that the interaction between phot1-GFP and AtRem1.3-mCherry was enhanced along with increased time of BL treatment. However, the BL-dependent interaction was not obvious in plants co-expressing phot1 -GFP and AtRem1.3-mCherry, implicating that BL facilitated the translocation of functional phot1-GFP into AtRem1.3-labeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated phot1-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane microdomains act as an organizing platform essential for proper function of activated phot1 at the PM

Multi-platform Approach for Microbial Biomarker Identification Using Borrelia burgdorferi as a Model

The identification of microbial biomarkers is critical for the diagnosis of a disease early during infection. However, the identification of reliable biomarkers is often hampered by a low concentration of microbes or biomarkers within host fluids or tissues. We have outlined a multi-platform strategy to assess microbial biomarkers that can be consistently detected in host samples, using Borrelia burgdorferi, the causative agent of Lyme disease, as an example. Key aspects of the strategy include the selection of a macaque model of human disease, in vivo Microbial Antigen Discovery (InMAD), and proteomic methods that include microbial biomarker enrichment within samples to identify secreted proteins circulating during infection. Using the described strategy, we have identified 6 biomarkers from multiple samples. In addition, the temporal antibody response to select bacterial antigens was mapped. By integrating biomarkers identified from early infection with temporal patterns of expression, the described platform allows for the data driven selection of diagnostic targets

THE NUMERICAL SIMULATIONSTUDY SPANNING THE CONTINUUM/ATOMS SCALEON THE TYPE Ⅰ EDGE CRACK PROPAGATION PROCESS IN SINGLE CRYSTAL SILVERBASED ON THEDISPLACEMENT CONNECTION

The study on deformation and failure mechanism of materials,and the progressof NEMS/MEMStechnology and biotechnologypromote the development of multi-scale analysis method. However,Multi-scale connectionisthe key of multi-scale model. The finite element method（ FEM） and molecular dynamics simulation（ MD） were combined to simulate the multi-scale models,and the stress intensity factors（ SIF） and displacement mechanics parameters were adopted to connect and convertthecontinuum region and discrete molecular dynamics region,then three multi-scale model of two-dimensional silver crack propagationwasestablished to obtain the crack tip trajectory and the micro-scale crackpropagationprocess. The result is in good agreement with theory and experiment,and proves the rationality of the method

EXPERIMENTAL STUDY ON THE PLANE STRAIN DYNAMIC FRACTURE TOUGHNESS OF 20CRMNTI BASED ON IMPROVED HPB PLATFORM

To obtain the dynamic fracture toughness of 20 Cr Mn Ti,an impact loading test was conducted on three-point bending specimens with the improved Hopkinson pressure bar.Dynamic stress intensity factor was determined by the spring mass model.The onset time of crack initiation is experimentally detected using the strain gauge method.Dynamic fracture toughness of20 Cr Mn Ti with type I crack was obtained through the above experimental procedure.The geometrical dispersion distortion caused by lateral inertia effect is effectively inhibited by the wave shaping technique,to ensure the consistency of stress wave signal in measuring point and loading point is guaranteed.At the same time,the high-speed camera system was used to collect the image and the change of the surface strain field during the loading process was analyzed with GOM Correlate.The results show that samples before crack initiation has reached the dynamic strain balance,ensure the validity of the experiment

Distinguish Effect of Cu, Zn and Cd on Wheat’s Growth Using Nondestructive and Rapid Minolta SPAD-502

International audienceThis paper focused on the SPAD values response to different treatment levels of heavy metal Cu, Zn and Cd stress on growth of wheat. Random blocks design experiment has been carried out to simulate five concentration levels’ heavy metal Cu, Zn and Cd respectively. The in-situ SPAD values were measured for each treatment on December 8th 2014 (seedling stage), December 13th 2014 (tillering stage), January 8th 2015 (tillering stage), April 18th 2015 (elongation stage), April 30th 2015 (booting stage), May 12th 2015 (heading stage) and June 3rd 2015 (filling stage) respectively using Minolta SPAD-502. The standard deviation of SPAD in different between check plot (CK) and level i, Normalized SPAD values and their variation coefficients among five heavy metal treatments for Cd, Zn and Cu have been analyzed at different measurement time. SPAD values of CK treatment decreased slightly from seedling stage to tillering stage, followed by rapid increase to elongation stage, keeping a shoulder to heading stage, then rapid decrease to filling stage. With the increase of heavy metal treatment levels, standard deviations of SPAD values between CK and level i increased gradually, and the minimum of standard deviation appeared at booting and heading stage. The Normalized SPAD had been calculated by the SPAD ratio of heavy level i to CK. The higher Normalized SPAD, the lower Normalized SPAD value with the minimum at booting and heading stage. Variance coefficients of Normalized SPAD of Cd treatments at seedling stage and tillering stages lower than Zn and Cu treatments. Normalized SPAD among Cd, Cu and Zn treatments had lowest and similar variance at booting stage and heading stages

Dynamic analysis of Arabidopsis AP2 σ subunit reveals a key role in clathrin-mediated endocytosis and plant development

Clathrin-mediated endocytosis, which depends on the AP2 complex, plays an essential role in many cellular and developmental processes in mammalian cells. However, the function of the AP2 complex in plants remains largely unexplored. Here, we show in Arabidopsis that the AP2 sigma subunit mutant (ap2 sigma) displays various developmental defects that are similar to those of mutants defective in auxin transport and/or signaling, including single, trumpet-shaped and triple cotyledons, impaired vascular pattern, reduced vegetative growth, defective silique development and drastically reduced fertility. We demonstrate that AP2 sigma is closely associated and physically interacts with the clathrin light chain (CLC) in vivo using fluorescence cross-correlation spectroscopy (FCCS), protein proximity analyses and co-immunoprecipitation assays. Using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), we show that AP2 sigma-mCherry spots colocalize with CLC-EGFP at the plasma membrane, and that AP2 s-mCherry fluorescence appears and disappears before CLC-EGFP fluorescence. The density and turnover rate of the CLC-EGFP spots are significantly reduced in the ap2 sigma mutant. The internalization and recycling of the endocytic tracer FM4-64 and the auxin efflux carrier protein PIN1 are also significantly reduced in the ap2 sigma mutant. Further, the polar localization of PIN1-GFP is significantly disrupted during embryogenesis in the ap2 sigma mutant. Taken together, our results support an essential role of AP2 s in the assembly of a functional AP2 complex in plants, which is required for clathrin-mediated endocytosis, polar auxin transport and plant growth regulation

Tandem Histone-Binding Domains Enhance the Activity of a Synthetic Chromatin Effector

Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than DNA sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. We have previously reported the development and validation of the “polycomb-based transcription factor” (PcTF), a fusion protein that recognizes histone modifications through a protein–protein interaction between its polycomb chromodomain (PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3) at genomic sites. We demonstrated that PcTF activates genes at methyl-histone-enriched loci in cancer-derived cell lines. However, PcTF induces modest activation of a methyl-histone associated reporter compared to a DNA-binding activator. Therefore, we modified PcTF to enhance its binding avidity. Here, we demonstrate the activity of a modified regulator called Pc₂TF, which has two tandem copies of the H3K27me3-binding PCD at the N-terminus. Pc₂TF has a smaller apparent dissociation constant value <i>in vitro</i> and shows enhanced gene activation in HEK293 cells compared to PcTF. These results provide compelling evidence that the intrinsic histone-binding activity of the PCD motif can be used to tune the activity of synthetic histone-binding transcriptional regulators