Design of a zinc finger protein binding a sequence upstream of the A20 gene

<p>Abstract</p> <p>Background</p> <p>Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys₂-His₂zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells.</p> <p>Results</p> <p>In order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity.</p> <p>Conclusion</p> <p>It is feasible to design specific zinc finger proteins by bioinformatics methods.</p

Configuration of the active region for the Ge-on-Si photodetector based on carrier mobility

The design of vertical and lateral PIN Ge-on-Si photodetectors was motivated by the disparity in electron and hole mobilities. In the case of vertical PIN junction detectors, configuring the slab region as n-type doping leads to a notable increase in the bandwidth of approximately 20 GHz compared to utilizing p-type doping for the slab. For lateral PIN junction detectors, we determined that setting the length of the n-type slab region to be 2.8 times that of the p-type slab region, based on the carrier saturation drift rate ratio, does not compromise the bandwidth. This configuration enhances the bandwidth while minimizing light absorption loss from the electrode. The proposed design in this study enhances the performance of Ge-on-Si photodetectors without adding complexity to the fabrication process. The principles applied in this study serve as instructive references for the conceptualization of other photonic or electronic devices, reinforcing the widespread applicability of these design strategies

Tyrosine phosphorylation of the N-Methyl-D-Aspartate receptor 2B subunit in spinal cord contributes to remifentanil-induced postoperative hyperalgesia: the preventive effect of ketamine

<p>Abstract</p> <p>Background</p> <p>Experimental and clinical studies showed that intraoperative infusionof remifentanil has been associated with postoperative hyperalgesia. Previous reports suggested that spinal N-methyl-D-aspartate (NMDA) receptors may contribute to the development and maintenance of opioid-induced hyperalgesia. In the present study, we used a rat model of postoperative pain to investigate the role of tyrosine phosphorylation of NMDA receptor 2B (NR2B) subunit in spinal cord in the postoperative hyperalgesia induced by remifentanil and the intervention of pretreatment with ketamine.</p> <p>Results</p> <p>Intraoperative infusion of remifentanil (0.04 mg/kg, subcutaneous) significantly enhanced mechanical allodynia and thermal hyperalgesia induced by the plantar incision during the postoperative period (each lasting between 2 h and 48 h), which was attenuated by pretreatment with ketamine (10 mg/kg, subcutaneous). Correlated with the pain behavior changes, immunocytochemical and western blotting experiments in our study revealed that there was a marked increase in NR2B phosphorylation at Tyr1472 in the superficial dorsal horn after intraoperative infusion of remifentanil, which was attenuated by pretreatment with ketamine.</p> <p>Conclusions</p> <p>This study provides direct evidence that tyrosine phosphorylation of the NR2B at Tyr1472 in spinal dosal horn contributes to postoperative hyperalgesia induced by remifentanil and supports the potential therapeutic value of ketamine for improving postoperative hyperalgesia induced by remifentanil.</p

Large animal models in the study of gynecological diseases

Gynecological diseases are a series of diseases caused by abnormalities in the female reproductive organs or breast, which endanger women’s fertility and even their lives. Therefore, it is important to investigate the mechanism of occurrence and treatment of gynecological diseases. Animal models are the main objects for people to study the development of diseases and explore treatment options. Large animals, compared to small rodents, have reproductive organs with structural and physiological characteristics closer to those of humans, and are also better suited for long-term serial examinations for gynecological disease studies. This review gives examples of large animal models in gynecological diseases and provides a reference for the selection of animal models for gynecological diseases

Acute effects of different load intensities and rest intervals on muscle strength endurance in male college athletes

The purpose of this study was to compare the effects of different load intensities with rest intervals between sets on heart rate, rating of perceived exertion (RPE), power output, and blood lactate concentration during a squat strength endurance training protocol. A total of 4 sets of strength endurance tests were performed on 15 national Level 2 or above athletes with different load intensities (30% of 1 repetition maximum (1RM), 50% 1RM) and different rest intervals between sets (1 min, 2 min), 20 reps per set. Mean power (P-mean), mean heart-rate (HRmean) and RPE variations during the exercise were collected by using a linear position transducer, heart rate monitor, and Borg 6–20 scale. Besides, finger blood was collected before and after the exercise, and analyzed by using a blood lactate analyzer. HRmean, P-mean and RPE values were significantly higher at 50% 1RM load intensity than at 30% 1RM (p < 0.01), HRmean was significantly higher at 1 min rest interval than at 2 min between sets, while P-mean was significantly higher at 2 min rest interval than at 1 min between sets (p < 0.05); at 30% 1RM loading intensity, blood lactate concentrations were significantly higher at the completion of exercise for the 1 min rest interval between sets than for the 2 min (p < 0.01). However, at 50% 1RM loading intensity, blood lactate increased similarly at the completion of training in multiple sets, independent of the rest interval between sets. From a practical point of view, the results suggest that a 1 min rest interval between sets may be sufficient in a strength endurance training protocol when the load intensity is 30% 1RM. However, when the load intensity is 50% 1RM, we suggest that a 2 min rest interval between sets is required for optimal recovery and maintenance of power output

Construction of an artificial recombinant bicistronic plasmid DNA vaccine against porcine rotavirus

The attenuated Salmonella typhimurium χ4550 strain was used to harbour a reconstructed bicistronic DNA vaccine against porcine rotavirus, which carried the rotavirus nonstructural protein 4 (NSP4) and VP7 genes simultaneously. Using a balanced lethal system, the kanamycin resistance gene of expressing eukaryotic plasmids pVAX1 and pVAXD were replaced by the aspartate β-semialdehyde dehydrogenase (asd) gene. The NSP4 cleavage product (259–525) of rotavirus OSU strain and VP7 full-length genes were amplified by reverse transcription polymerase chain reaction and then inserted into the eukaryotic single-expression plasmid, pVAX1-asd, and the eukaryotic dual-expression plasmid, pVAXD-asd, respectively. The recombinant plasmids pVAX1-asd-NSP4, pVAX1-asd-VP7 and pVAXD-asd-NSP4-VP7 were transformed into the attenuated S. typhimurium χ4550 strain by electrotransformation. An indirect immunofluorescence assay of the expressed COS-7 cell suggested that the recombinant S. typhimurium χ4550 strain was constructed successfully. The recombinant S. typhimurium χ4550 strain was orally administered to BALB/c mice. The group immunised with dual- expression plasmids produced a significantly higher level of serum Immunoglobulin G (IgG) and intestinal Immunoglobulin A (IgA) than the group immunised with single-expression plasmids. These results indicated that eukaryotic bicistronic plasmid DNA vaccines could be successfully constructed to enhance humoural, mucosal and cellular immune response against rotavirus infection