SpyCatcher-NTEV: A Circularly Permuted, Disordered SpyCatcher Variant for Less Trace Ligation.

The SpyTag/SpyCatcher reaction has emerged as a powerful way for bioconjugation, but it leaves a folded complex in the product after the formation of the isopeptide bond. To vary the location of the reactive residue and reduce the size of the complex and its potential immunogenicity, we engineer two circularly permuted SpyCatcher variants, SpyCatcher-N and SpyCatcher-NTEV, the latter of which possesses a TEV-recognition site for removal of the fragment containing the catalytic site. Surprisingly, both variants are found to be disordered in solution, yet still retain the ability to form an ordered complex upon reaction with SpyTag with second-order rate constants of ∼10 M-1 s-1. Cellular expression of a telechelic protein bearing SpyCatcher-NTEV at the N-terminus and SpyTag at the C-terminus gives both cyclized and chain-extended products. Notably, the monomers exist almost exclusively in the cyclic form owing to its high reactivity in vivo. The fragment containing the catalytic site of SpyCatcher-NTEV can then be removed by TEV digestion, giving a circular protein with minimal trace from the ligation reaction. The plasticity of SpyTag/SpyCatcher reactive pair has promised an ever-expanding toolbox of genetically encoded peptide-protein reaction with versatile features.

A Versatile and Robust Approach to Stimuli-Responsive Protein Multilayers with Biologically Enabled Unique Functions.

Protein-based materials call for innovative processing techniques to integrate their unique biologically enabled functions with other materials of complementary features. Herein, we report the covalent protein layer-by-layer assembly via orthogonal "Tag-Catcher" reactions as a facile and robust approach to make entirely protein-based multilayers on a variety of substrates. Programmed assembly of native telechelic proteins not only endows the materials valuable stimuli-sensitive behaviors, but also unique properties unparalleled by any synthetic counterparts. As proof of concept, super uranyl-binding protein (SUP) is immobilized on silica gel by this method with tunable capacity and enhanced capability for uranyl sequestration. Not only is the capturing performance enhanced in the multilayer setup, it also confers resilience to recycling, allowing efficient harvest of uranyl with an average of ∼90% and ∼60% recovery rate in over 10 cycles from water and synthetic seawater, respectively. The approach is the first entirely protein-based multilayers covalently assembled by the layer-by-layer method. It provides a platform for immobilizing proteins with synergistic enhancement of function and resilience and expands the scope and capability of genetically encoded protein-based materials.

Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction.

Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody, and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to biosignals, such as Ca2+, trifluoperazine, and nicotinamide adenine dinucleotide phosphate (NADPH), and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multilayer setup exhibits a synergistic enhancement of DHFR activity with increasing numbers of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.

[Study on crystal growth and vibrational spectra of Yb(x) : KY(1-x) (WO4)2].

Yb(x) : KY(1-x)W (x = 0.05)and KYbW crystals were grown by TSSG method. Both of the structure and spectral properties were compared. The condition for the crystal growth is: the rotation rate 10-15 r x min(-1), the pulling speed 1-2 d(-1), the growing period 10-15 d, cooling growing speed 0.05-0.1 degrees C x h(-1), and the cooling speed 20 degrees C x h(-1). X-ray powder diffraction analysis was performed for the crystal powder. They belong to beta-KYW structure with low thermal phase. The cell parameters of the two crystals were calculated, and they are respectively a1 = 1.063 nm, b1 = 1.034 nm, c1 = 0.755 nm, beta1 = 130.75 degrees, Z1 = 4 and a2 = 1.061 nm, b2 = 1.029 nm, c2 = 0.749 nm, beta2 = 130.65 degrees and Z2 = 4. The infrared spectrum and Raman spectrum of crystal were measured. The sample of Yb(x) : KY(1-x) W (x = 0.05) had stronger infrared absorption peaks at 925, 891, 840, 777 and 749 cm(-1), which were caused by stretching vibration. The sample of KYW had stronger infrared absorption peaks at 484 and 437 cm(-1) caused by bending vibration. The vibration modes were analysed and vibrational frequencies of vibratory activity was assigned. The two crystals had strong Raman activity. The vibration of WOOW and WOW exists from 200 to 1000 cm(-1).

[Long-term effect of intruded upper molars and dental implant restoration in 24 patients].

PURPOSE: To evaluate the long-term effectiveness of intruded upper molar with orthodontic mini-screw and the long-term stability of the lower molar restored with dental implant. METHODS: Twenty-four patients with excessive growing upper molar were chosen. The upper molars were intruded by orthodontic mini-screw to increase the bite distance, and then restored with dental implant. The height of molar and the height of bone around the implant were measured on the lateral cephalometric films and panoramic films to analyze the long-term effect of molar intrusion and implantation. The data were analyzed with SPSS18.0 software package. RESULTS: There was significant difference between the length of upper molar before and after intrusion, the upper molars were intruded (2.7±0.3)mm 3.5 months after orthodontic mini-screw; there was no significant difference after 1 year with retainer and implant restoration. The height of bone around the implant decreased 1.2 mm but without significant difference. CONCLUSIONS: With the upper molar intruded by orthodontic mini-screw , more bite space can be obtained to restore lower molar with dental implant, and the long-term effect of upper molar intrusion and lower molar implant are stable.

[Experimental study on osteogenesis of acellular porcine pericardium in guided bone regeneration].

PURPOSE: To observe the retention time and effect of acellular porcine pericardium (APP) in bone regeneration for rabbit femoral bone defects. METHODS: APP was prepared as follows: fresh porcine pericardium was chosen, high and low osmotic NaCl solution were used to soak the specimen alternately, trypsin+EDTA were used for digest, and then immersed with TritonX-100, cross-linked by using glutaraldehyde. 24 New Zealand rabbits were selected, and the area of bone defect was created 8 mm×8 mm×5 mm in size on bilateral distal femur. Stochastic method was used for grouping. One side of bone defect was covered with APP (experimental group); and the other side was covered without APP (control group). General observation, X-ray examination, histological examination, and bone mineral density (BMD) were performed 4 weeks, 8 weeks and 12 weeks after operation. SPSS18.0 software package was used for statistical analysis. RESULTS: After surgery, all wounds healed without complications. Under general observation, there were some fibrous cysts covered on the surface of APP. BMD (P<0.05) in the experimental group was significantly greater than that in the control group. On histological examination, the rate of osteogenesis in the experimental group was faster than that in the control group. CONCLUSIONS: Bone regeneration can be achieved using APP in the repair of rabbit bone defects. The retention time of APP can meet the needs of osteogenesis.

[Clinical study of transcrestal maxillary sinus floor elevation with the disk-up sinus reamer].

OBJECTIVE: To evaluate the clinical effect of the disk-up sinus reamer (DSR) applied to transcrestal maxillary sinus floor elevation with simultaneous placement of implants. METHODS: Thirty-seven patients underwent transcrestal maxillary sinus floor elevation with fifty-one implants placed simultaneously using the DSR. The residual bone height (RBH) was 3 to 8 mm, (5.61 ± 1.61) mm on average. The safety of this technique and the pain index during the operation was evaluated. The final prostheses were restored in 3-6 months postoperatively. The follow-up period was 3 to 24 months. The stability and osseointegration of the implants were clinically evaluated, and the endo-sinus bone gain around the implants were measured. RESULTS: The elevation height ranged from 2 to 8 mm, with an average of (4.75 ± 1.55) mm. There was no detectable sinus membrane perforation, no serious suffering or uncomfortable subjective sensation in any patients during operation with a pain index of (2.22 ± 0.98). During the follow-up period, no sinus complication was observed. Favorable osseointegration was obtained. There were no implants or prostheses which were loose or lost. The survival rate was 100%. The radiographic results demonstrated that the endo-sinus bone gain tended to reach stabilization after 6 months and the marginal bone loss was (1.20 ± 0.72) mm after 12 months. CONCLUSIONS: Transcrestal maxillary sinus floor elevation with simultaneous implant placement by DSR is a safe, invasive and handy technique, with higher elevation height, fewer clinical complications and less pain. It shows satisfactory clinical results in short term and a long-term observation is still needed.