Synthesis and Properties of a Novel Reactive and Low-Migration-Resistant Antioxidant and Its Application in Rubber Composites.

The addition of antioxidants to rubber is one of the most economical and effective methods for delaying rubber aging. However, antioxidant migration can cause environmental pollution. To address this issue, a new reactive antioxidant was synthesized via the chemical bonding of glycidyl methacrylate (GMA) and p-aminodiphenylamine (PPDA). The product was characterized by Fourier-transform infrared spectroscopy, which confirmed the successful reaction between GMA and PPDA, resulting in a compound with the expected structure. The mixture was then combined with a composite of styrene-butadiene rubber and carbon black. The tensile strength, antioxidant properties, migration, and RPA of the resulting materials were tested and compared with those of the commercial antioxidants N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, N-isopropyl-N'-phenyl-1,4-phenylenediamine, and poly(1,2-dihydro-2,2,4-trimethylquinoline). After the glycidyl methacrylate antioxidant was grafted onto p-aminodiphenylamine, it became highly compatible with and dispersed in the rubber matrix. The antiaging and antimigration properties of the rubber antioxidants were enhanced without damaging the mechanical properties of the rubber matrix. The short-term and long-term antiaging and antimigration properties of this antioxidant are superior to those of commercially available antioxidants.

Mitochondrial Localization Signal of Porcine Circovirus Type 2 Capsid Protein Plays a Critical Role in Cap-Induced Apoptosis.

Porcine circovirus 2 (PCV2), considered one of the most globally important porcine pathogens, causes postweaning multisystemic wasting syndrome (PMWS). This virus is localized in the mitochondria in pigs with PMWS. Here, we identified, for the first time, a mitochondrial localization signal (MLS) in the PCV2 capsid protein (Cap) at the N-terminus. PK-15 cells showed colocalization of the MLS-EGFP fusion protein with mitochondria. Since the PCV2 Cap also contained a nuclear localization signal (NLS) that mediated entry into the nucleus, we inferred that the subcellular localization of the PCV2 Cap is inherently complex and dependent on the viral life cycle. Furthermore, we also determined that deletion of the MLS attenuated Cap-induced apoptosis. More importantly, the MLS was essential for PCV2 replication, as absence of the MLS resulted in failure of virus rescue from cells infected with infectious clone DNA. In conclusion, the MLS of the PCV2 Cap plays critical roles in Cap-induced apoptosis, and MLS deletion of Cap is lethal for virus rescue.

TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells.

Protein-protein interactions (PPIs) are critical for cellular activity regulation. Visualization of PPIs using bimolecular fluorescence complementation (BiFC) techniques helps to understand how PPIs implement their functions. However, current BiFC is based on fluorescent proteins and the brightness and photostability are suboptimal for single molecule tracking experiments, resulting in either low spatiotemporal resolution or incapability of tracking for extended time course. Here, we developed the TagBiFC technique based on split HaloTag, a self-labeling tag that could conjugate an organic dye molecule and thus offered better brightness and photostability than fluorescent proteins for PPI visualization inside living cells. Through screening and optimization, we demonstrated that the reconstituted HaloTag exhibited higher localization precision and longer tracking length than previous methods. Using TagBiFC, we reveal that the dynamic interactions of transcription factor dimers with chromatin DNA are distinct and closely related to their dimeric states, indicating a general regulatory mechanism for these kinds of transcription factors. In addition, we also demonstrated the advantageous applications of TagBiFC in single nucleosome imaging, light-burden imaging of single mRNA, low background imaging of cellular structures. We believe these superior properties of our TagBiFC system will have broad applications in the studies of single molecule imaging inside living cells.

PN-ImTLSM facilitates high-throughput low background single-molecule localization microscopy deep in the cell.

When imaging the nucleus structure of a cell, the out-of-focus fluorescence acts as background and hinders the detection of weak signals. Light-sheet fluorescence microscopy (LSFM) is a wide-field imaging approach which has the best of both background removal and imaging speed. However, the commonly adopted orthogonal excitation/detection scheme is hard to be applied to single-cell imaging due to steric hindrance. For LSFMs capable of high spatiotemporal single-cell imaging, the complex instrument design and operation largely limit their throughput of data collection. Here, we propose an approach for high-throughput background-free fluorescence imaging of single cells facilitated by the Immersion Tilted Light Sheet Microscopy (ImTLSM). ImTLSM is based on a light-sheet projected off the optical axis of a water immersion objective. With the illumination objective and the detection objective placed opposingly, ImTLSM can rapidly patrol and optically section multiple individual cells while maintaining single-molecule detection sensitivity and resolution. Further, the simplicity and robustness of ImTLSM in operation and maintenance enables high-throughput image collection to establish background removal datasets for deep learning. Using a deep learning model to train the mapping from epi-illumination images to ImTLSM illumination images, namely PN-ImTLSM, we demonstrated cross-modality fluorescence imaging, transforming the epi-illumination image to approach the background removal performance obtained with ImTLSM. We demonstrated that PN-ImTLSM can be generalized to large-field homogeneous illumination imaging, thereby further improving the imaging throughput. In addition, compared to commonly used background removal methods, PN-ImTLSM showed much better performance for areas where the background intensity changes sharply in space, facilitating high-density single-molecule localization microscopy. In summary, PN-ImTLSM paves the way for background-free fluorescence imaging on ordinary inverted microscopes.

Lighting Up Live Cells with Smart Genetically Encoded Fluorescence Probes from GMars Family.

As a special kind of delicate light-controllable genetically encoded optical device, reversibly photoswitchable fluorescent proteins (RSFPs) have been widely applied in many fields, especially various kinds of advanced nanoscopy approaches in recent years. However, there are still necessities for exploring novel RSFPs with specific biochemical or photophysical properties not only for bioimaging or biosensing applications but also for fluorescent protein (FP) mechanisms study and further knowledge-based molecular sensors or optical actuators' rational design and evolution. Besides previously reported GMars-Q and GMars-T variants, herein, we reported the development and applications of other RSFPs from GMars family, especially some featured RSFPs with desired optical properties. In the current work, in vitro FP purification, spectra measurements, and live-cell RESOLFT nanoscopy approaches were applied to characterize the basic properties and test the imaging performances of the selected RSFPs. As demonstrated, GMars variants such as GMars-A, GMars-G, or remarkable photofatigue-resistant GMars-L were found with beneficial properties to be capable of parallelized RESOLFT nanoscopy in living cells, while other featured GMars variants such as dark GMars-P may be a good candidate for further biosensor or actuator design and applications.

Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system.

Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.

[Experimental research on the prevention of rabbit postoperative abdominal cavity adhesion with PLGA membrane].

The aim of this paper is to explore the prevention of rabbit postoperative abdominal cavity adhesion with poly (lactic-co-glycotic acid) (PLGA) membrane and the mechanism of this prevention function. Sixty-six Japanese white rabbits were randomly divided into normal control group, model control group and PLGA membrane group. The rabbits were treated with multifactor methods to establish the postoperative abdominal cavity adhesion models except for those in the normal control group. PLGA membrane was used to cover the wounds of rabbits in the PLGA membrane group and nothing covered the wounds of rabbits in the model control group. The hematologic parameters, liver and kidney functions and fibrinogen contents were detected at different time. The rabbit were sacrificed 1, 2, 4, 6, 12 weeks after the operations, respectively. The adhesions were graded blindly, and Masson staining and immunohistochemistry methods were used to observe the proliferation of collagen fiber and the expression of transforming growth factor ß1 (TGF-ß1) on the cecal tissues, respectively. The grade of abdominal cavity adhesion showed that the PLGA membrane-treated group was significant lower than that in the model control group, and it has no influence on liver and kidney function and hematologic parameters. But the fibrinogen content and the number of white blood cell in the PLGA membrane group were significant lower than those of model control group 1 week and 2 weeks after operation, respectively. The density of collagen fiber and optical density of TGF-ß1 in the PLGA membrane group were significant lower than those of model control group. The results demonstrated that PLGA membrane could be effective in preventing the abdominal adhesions in rabbits, and it was mostly involved in the reducing of fibrinogen exudation, and inhibited the proliferation of collagen fiber and over-expression of TGF-ß1.

[Effects of PLGA absorbable membrane on preventing postoperative abdominal adhesion in rabbits].

OBJECTIVE: To investigate the effects of PLGA absorbable membrane in prevention of postoperative abdominal adhesion in rabbits. METHODS: 66 Japanese white rabbits were randomly divided into three groups: the normal control group n = 6, model control group n = 30 and PLGA group n = 30. Rabbits were received multifactor methods to establish postoperative abdominal adhesion models except for normal control group. The cecum wound was covered PLGA membrane in the PLGA group. At postoperative 1, 2, 4, 6 and 12 weeks, the abdominal cavities were reopened and the adhesive severity was graded blindly, and the hydroxyproline level in cecum tissue was measured and the cecum histopathology was observed. RESULTS: (1) the degree of adhesion and hydroxyproline level in model control group were significantly higher than those of normal control group (P < 0.05, P < 0.01), while the degree of adhesion and hydroxyproline level in PLGA group were significantly lower than those of model control group (P < 0.05). (2) HE staining showed that cecum serosa had obviously inflammatory cell infiltration and fibroblast proliferation, while PLGA could inhibit fibroblast proliferation and reduce the inflammatory cell infiltration and collagen. CONCLUSION: PLGA absorbable membrane can inhibit fibroblast proliferation and collagen to prevent the experimental postoperative peritoneal adhesions.

[Study on the differences on HA1 regions between epidemic strains and vaccine strains of influenza virus subtype A3 from 1988 to 2005].

OBJECTIVE: To study the differences between epidemic strains of influenza virus subtype A3 circulated in China and Occident in past 18 years, in genetic level, and vaccine strains recommended by WHO in corresponding time. METHODS: Amino acid sequences of HA1 regions of the epidemic strains, which circulated in China and Occident from 1988 to 2005, and the vaccine strains of influenza virus subtype A3 were compared by BioEdit and analyzed on the differences of HA1 and it's antigen determinants RESULTS: Differences between epidemic strains and vaccine strains recommended in corresponding year, both in HA1 and it' s antigen determinant regions, were obviously greater than that between epidemic strains and vaccine strains recommended in next round (P< 0.01). However, differences between epidemic strains in Occident and vaccine strains recommended in corresponding year were slightly greater than that between epidemic strains and vaccine strains recommended in next round and it was not marked (P >0.05). In addition, differences between epidemic strains and vaccine strains which being used for several years, whether in China or in Occident, constantly increased accompanying the used time prolonged. CONCLUSION: There was an obvious lag between vaccine strains recommended by WHO, analyzed in genetic level, and epidemic strains of influenza virus subtype A3 circulated in China.