SSINCC: Simple separation of interacting nucleoprotein complex components

Protein-DNA binding assays have been used in a va-riety of applications from fundamental studies re-garding the binding process itself to serve as probes for the detection, quantification and separation of target analytes. Here we describe a novel method of analyzing and identifying intermolecular DNA interactions that allows for the simple separation of interacting nucleoprotein complex components (SSINCC), focusing specifically on DNA-DNA interactions using P1 plasmid active partition system nucleoprotein complexes as a model to demonstrate DNA sequence specificity and tolerance of composite factor complexity. Traditional and recent assays of protein-DNA interaction are summarized and compared with SSINC. Although SSINC is examined here employing P1 partition nucleoprotein complex as an example of DNA-DNA intermolecular association, universal applications of this methodology to nucleo-protein complex studies can be envisioned.

CAPPh A Cytoskeleton-Based Localization Assay Reports Protein-Protein Interaction in Living Cells by Fluorescence Microscopy

Dear EditorProbing protein-protein interaction has become a routine practice in the post genomic era. Multiple in vitro or in vivo techniques have been developed to detect or report direct or indirect interactions of functionally related proteins （Lalonde et al., 2008）. These techniques sometimes are technically challenging, however, because the readout would demand sophisticated detectors and/or complicated calculations. Besides, a common drawback of many of these techniques is they can render inherent false positives to various degrees so that an interaction often cannot be judged unambiguously.

Split-HaloTag imaging assay for sophisticated microscopy of protein–protein interactions in planta

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells.Split-GFP-based protein–protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization.Because of their simple protocols,they have become some of the most frequently used methods.However,standard fluorescent proteins present several drawbacks for sophisticated microscopy.With the HaloTag system,these drawbacks can be overcome,as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands.Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods.Therefore,we have established the Split-HaloTag imaging assay in plants,which is based on the reconstitution of a functional HaloTag protein upon protein–protein interaction and the subsequent covalent binding of an added fluorescent ligand.Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein–protein interaction at cellular structures:the anchoring of the molybdenumcofactor biosynthesis complex to filamentous actin.In addition,a specific interactionwas visualized in a more distinctivemannerwith subdiffractional polarizationmicroscopy,Airyscan,and structured illumination microscopy to provide examples of sophisticated imaging.Split-GFPand Split-HaloTag can complement one another,as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods.Therefore,this promising new Split-HaloTag imaging assay provides a unique and sensitive approach formore detailed characterization of protein–protein interactions using specific microscopy techniques,such as 3D imaging,single-molecule tracking,and super-resolution microscopy.

Interactomic study on interaction between lipid droplets and mitochondria

An increasing body of evidence shows that the lipid droplet,a neutral lipid storage organelle,plays a role in lipid metabolism and energy homeostasis through its interaction with mitochondria.However,the cellular functions and molecular mechanisms of the interaction remain ambiguous.Here we present data from transmission electron microscopy,fluorescence imaging,and reconstitution assays,demonstrating that lipid droplets physically contact mitochondria in vivo and in vitro.Using a bimolecular fluorescence complementation assay in Saccharomyces cerevisiae,we generated an interactomic map of protein-protein contacts of lipid droplets with mitochondria and peroxisomes.The lipid droplet proteins Erg6 and Pet10 were found to be involved in 75%of the interactions detected.Interestingly,interactions between 3 pairs of lipid metabolic enzymes were detected.Collectively,these data demonstrate that lipid droplets make physical contacts with mitochondria and peroxisomes,and reveal specific molecular interactions that suggest active participation of lipid droplets in lipid metabolism in yeast.

Cross electro-nape-acupuncture ameliorates cerebral hemorrhageinduced brain damage by inhibiting necroptosis

BACKGROUND Receptor interacting protein kinase 1(RIPK1)-mediated cell death,including apoptosis and necroptosis,belongs to programmed cell death.It has been reported that RIPK1-mediated necroptosis exists in lesions of cerebral hemorrhage(CH).Electroacupuncture,a treatment derived from traditional Chinese medicine,could improve neurological impairment in patients with brain injury.AIM To investigate the protective role of cross electro-nape acupuncture(CENA)in CH,and clarify the potential mechanism.METHODS CH rat models were established,and CENA was applied to the experimental rats.Neurological functions and encephaledema were then measured.Necrotic cells in the brain of rats with CH were evaluated by propidium iodide staining.Necroptosis was assessed by immunofluorescence.Activation of the necroptosisrelated pathway was detected by western blot.Extraction of brain tissue,cerebrospinal fluid and serum samples was conducted to measure the expression and secretion of inflammatory cytokines by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay.RESULTS The necroptotic marker p-MLKL was detectable in the brains of rats with CH.Next,we found that CENA could ameliorate neurological functions in rat models of CH.Moreover,the upregulation of RIPK1-mediated necroptosis-related molecules in the brains of rats with CH were inhibited by CENA.Further investigation revealed that CENA partially blocked the interaction between RIPK1 and RIPK3.Finally,in vivo assays showed that CENA decreased the expression of the inflammatory cytokines tumor necrosis factor-α,interleukin-6 and interleukin-8 in CH rat models.CONCLUSION These findings revealed that CENA exerts a protective role in CH models by inhibiting RIPK1-mediated necroptosis.

Study on Binding Affinity of a Glutathione <i>S</i>-Transferase (GST) Fusion Protein to DNA Probe

The aims of this work were to: i) purify GST-fusion protein from bacterial cell extracts of Escherichia coli;ii) quantify the protein by SDS PAGE and Bradford assay;iii) determine protein-DNA interaction of the purified protein by Electrophoretic Mobility Shift Assay. Bacterial culture prepared by inoculation of a single E. coli colony that had a GST fusion protein (gst: six-X10 hd) constructed by ligation of the six-7-hd (X10) sequence into the BamHI and EcoRI sites of the vector pGEX-2T, grown overnight, was sonicated using Cole-Palmer Ultrasonic Homogenizer. Fusion protein was eluted from the beads with Tris-glutathione buffer (50 mM Tris [pH 8.1], 20 mM glutathione), which contained reduced Glutathione. SDS-PAGE was used to calculate the extracted bound protein. Total protein quantification was then estimated by the Bradford assay. Bovine Serum Albumin (BSA) absorbance values were used to plot the standard curve used to calculate the concentrations of the sample proteins. Nylon membrane was used for the electrophoretic transfer;membrane was cross linked and detected by Pierce’s Chemiluminescent Nucleic Acid Detection module. Results showed that X10 gave a strong band shift observed in Lanes 6 and 7 for both 200 ng and 400 ng elute 1 samples;however, there was no shift in the bands for the wild-type, positive control. The concentration of the elute 1 was obtained by the Bradford assay as 242.52 ng/μl and that of elute 2 was 106.30 ng/μl. Similarly, the result obtained by gel analysis was 300 ng/μl (0.3 μg/μl) and 150 ng/μl (0.15 μg/μl) for elutes 1 and 2 respectively.

Synthesis of Novel Hydrophobic Media and Purification of Recombinant HBsAg

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Prey-mediated effects of mCry51Aa2-producing cotton on the predatory nontarget bug Orius majusculus(Reuter)

Genetically engineered(GE)cotton,MON 88702,is protected against cer-tain sucking pests,such as plant bugs and thrips,by producing mCry51Aa2,a mod-ified protein from Bacillus thuringiensis(Bt).Predatory pirate bugs(Orius spp.),natural enemies contributing to biological pest control,are also sensitive to the insecti-cidal protein when exposed continuously to high concentrations.We evaluated effects of MON 88702 on Orius majusculus when fed prey types with different mCry51Aa2 concen-trations.When neonates were provided exclusively Tetranychus urticae spider mites reared on MON 88702(high mCry51Aa2 content),adverse effects on predator survival and de-velopment were confirmed,compared with specimens fed prey from near-isogenic non-Bt cotton.When fed a mixture of T.urticae and Ephestia kuehniella eggs(mCry51Aa2-free),predator life table parameters were similar to the treatment where eggs were fed exclu-sively.When mCry51Aa2-containing spider mites were provided for a limited time at the beginning or the end of juvenile development,effects were less pronounced.While pirate bug nymphs showed similar consumption rates for prey from Bt and non-Bt cotton,choice experiments revealed a preference for E.kuehniella eggs over spider mites.Lepidopteran larvae(Spodoptera littoralis,high mCry51Aa2 content)or cotton aphids(Aphis gossypii,mCry51Aa2-free)reared on MON 88702 as alternative prey did not result in adverse ef-fects on O.majusculus.Our study suggests limited risk of mCry51Aa2-producing cotton for O.majusculus,because its sensitivity for the Bt protein is relatively low and its natural food consists of diverse prey species with varying concentrations of Bt protein.