Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.
Animals ; Mice ; Antibodies ; Nicotiana/genetics* ; Agrobacterium/genetics* ; Bioreactors ; Blotting, Western

Objective To investigate the clinical phenotype,sperm characteristics,genetic etiology and the outcomes of assisted repro-ductive technology of 2 minority patients(P1 Hui and P2 Uyghur)with globozoospermia.Methods The clinical data and various pa-rameters of semen examination in the 2 minority patients with globozoospermia were analyzed.The ultrastructure of their sperms was ob-served,and the genetic etiology of the patients was analyzed by whole exome sequencing(WES)and qPCR.Intracytoplasmic sperm in-jection(ICSI)combined with artificial oocyte activation(AOA)were applied for assisted reproduction thyrapy.The outcomes of assis-ted reproduction in the two couples were observed.Results Homozygous 109681 bp deletion in DPY19L2 gene was found in both the patients.The homozygous deletion of DPY19L2 gene in P2 patient came from his inbred parents.Low sperm motility and high sperm DNA fragmentation rate were observed in the P1 patient,the and the rate of round-headed sperm was 100％in morphology.The ab-sence of sperm acrosome and ultrastructural defects of plasma membrane,mitochondria and microtubules were found under the electron microscope.In the P2 patient,sperm motility and sperm DNA fragmentation rate patient were normal,and the sperm morphology was 100％round-headed sperm.Under the electron microscope,the main defects of sperm were small and round head with acrosomal ab-sence,structural damage,but the ultrastructural defects of organelles were rare,such as plasma membrane,mitochondria and microtu-bules.Both couples of the two patients received ICSI+AOA for assisted pregnancy,and the ICSI fertilization rate was 62.5％for P1 pa-tient and 75％for P2 patient.Both the patients successfully obtained clinical pregnancy.Conclusion The abnormal DPY19L2 gene is the main genetic cause in the globozoospermia patients with different ethnic backgrounds.Both structural damage and ultrastructural de-fects of organelles such as plasma membrane,mitochondrial and microtubule damage could be found in globozoospermia.ICSI combined with AOA should be an effective assisted reproductive thyrapy strategy for the patients with globozoospermia.

Background::T-cell-mediated immunity is crucial for the effective clearance of viral infection, but the T-cell-mediated immune responses that are induced by booster doses of inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in people living with human immunodeficiency virus (PLWH) remain unclear.Methods::Forty-five PLWH who had received antiretroviral therapy (ART) for more than two years and 29 healthy controls (HCs) at Beijing Youan Hospital were enrolled to assess the dynamic changes in T-cell responses between the day before the third vaccine dose (week 0) and 4 or 12 weeks (week 4 or week 12) after receiving the third dose of inactivated SARS-CoV-2 vaccine. Flow cytometry, enzyme-linked immunospot (ELISpot), and multiplex cytokines profiling were used to assess T-cell responses at the three timepoints in this study.Results::The results of the ELISpot and activation-induced marker (AIM) assays showed that SARS-CoV-2-specific T-cell responses were increased in both PLWH and HCs after the third dose of the inactivated SARS-CoV-2 vaccine, and a similar magnitude of immune response was induced against the Omicron (B.1.1.529) variant compared to the wild-type strain. In detail, spike-specific T-cell responses (measured by the ELISpot assay for interferon γ [IFN-γ] release) in both PLWH and HCs significantly increased in week 4, and the spike-specific T-cell responses in HCs were significantly stronger than those in PLWH 4 weeks after the third vaccination. In the AIM assay, spike-specific CD4 + T-cell responses peaked in both PLWH and HCs in week 12. Additionally, significantly higher spike-specific CD8 + T-cell responses were induced in PLWH than in HCs in week 12. In PLWH, the release of the cytokines interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-α), and IL-22 by peripheral blood mononuclear cells (PBMCs) that were stimulated with spike peptides increased in week 12. In addition, the levels of IL-4 and IL-5 were higher in PLWH than in HCs in week 12. Interestingly, the magnitude of SARS-CoV-2-specific T-cell responses in PLWH was negatively associated with the extent of CD8 + T-cell activation and exhaustion. In addition, positive correlations were observed between the magnitude of spike-specific T-cell responses (determined by measuring IFN-γ release by ELISpot) and the amounts of IL-4, IL-5, IL-2 and IL-17F. Conclusions::Our findings suggested that SARS-CoV-2-specific T-cell responses could be enhanced by the booster dose of inactivated COVID-19 vaccines and further illustrate the importance of additional vaccination for PLWH.

Ovary is a vital female gonadal organ.It not only provides gametes for reproduction,but also secretes hormones to support and maintain the function of multiple systems.The decline of ovarian function is characterized by low fertility,early menopause,etc.It may even lead to dysfunction of multiple organs,which seriously damage women's health.Trace elements(TEs)play an important role in maintaining the normal ovarian function.When the TEs are unbalanced and cannot keep the ho-meostasis of ovary,inducing hormone secretion disorder,ovulatory disorder,decreased enzyme activity and histological changes of ovary.These changes lead to diminished ovarian function.Therefore,in-depth study on the role and mechanism of TEs in the regulation of ovarian function is conducive to understand the physiological function and prevent ovarian dysfunction.In this re-view,in combination with the research on the application of TEs at home and abroad in recent years,the relationship between TEs(iron,zinc,copper,selenium,iodine,cobalt,chromium,molybdenum)and ovarian function,as well as the mechanism of TEs affecting ovarian function are summarized,which will provide some theoretical guidance for the study of TEs in ovarian function in the future.

Objective:To explore the locations of the lumbosacral nerve roots by use of the magnetic stimulation.Methods:Thirty healthy subjects were studied. The projections of the right L 2 to S 1 intervertebral foramina on their body surfaces were determined manually with ultrasound assistance. Magnetic stimulation was applied to different nerve root segments to induce compound muscle action potentials (CMAP) in the vastus medialis, tibialis anterior, and gastrocnemius muscles of the lower limbs. The changes in latency, amplitude, and motor threshold were observed. Results:Magnetic stimulation on the L 2-L 3 segment resulted in a significant direct excitation of the vastus medialis. That on the L 5-S 1 segment evoked a significant direct excitatory effect on the tibialis anterior and gastrocnemius, with a motor threshold below 40%, an amplitude exceeding 1mV, and many effective responses. However, during the magnetic stimulation on the L 4 segment, the amplitude of the vastus medialis was above 1mV, with no significant differences in the number of effective responses among the muscle groups. Moreover, there was a stepwise change in the latency of effective muscle responses to magnetic stimulation at different segments. The CMAP latencies of 12+ ms for the tibialis anterior and 13+ ms for the gastrocnemius indicated activation of the L 5 and L 4 nerve roots, respectively, while those of 6+ ms, 7+ ms, and 8+ ms for the vastus medialis suggested activation of the L 4, L 3, and L 2 nerve roots, respectively. Conclusions:Based on the responses (CMAP latency, amplitude and motor threshold) of the vastus medialis, tibialis anterior and gastrocnemius to magnetic stimulation at different L 2 to S 1 segments, the spinal nerve root segments responsible for innervation can be inferred.

The self-healing microcapsule material is one of the smart materials that can self-identify and self-heal microcracks. Adding microcapsules to dental resin composites can resist generation of microcracks and prolong service life of resin restorations. In this literature, the overview of self-healing microcapsule materials, the combined application of self-healing microcapsule materials and other modified materials, the silanization treatment of dental self-healing microcapsules, the dynamic mechanics, fatigue and aging studies on self-healing dental resins were comprehensively presented and reviewed. In addition, prospects of dental self-healing microcapsule materials were also discussed.

OBJECTIVES: The current study aimed to investigate the bonding properties of a novel low-shrinkage resin adhesive containing expanding monomer and epoxy resin monomer after thermal cycling aging treatment. METHODS: Expanding monomer of 3,9-diethyl-3,9-dimethylol-1,5,7,11-tetraoxaspiro-[5,5] undecane (DDTU) as an anti-shrinkage additive and unsaturated epoxy monomer of diallyl bisphenol A diglycidyl ether (DBDE) as a coupling agent were synthesized. A blend of DDTU and DBDE at a mass ratio of 1∶1, referred to as "UE", was added into the resin matrix at the mass fraction of 20% to prepare a novel low-shrinkage resin adhesive.Then, the methacrylate resin adhesive without UE was used as the blank control group, and a commercial resin adhesive system was selected as the commercial control group. Moreover, the resin-dentin bonding and micro-leakage testing specimens were prepared for the thermal cycling aging treatment. The bonding strength was tested, the fracture modes were calculated, the bonding fracture surface was observed by scanning electron microscope (SEM), and the dye penetration was used to evaluate the tooth-restoration marginal interface micro-leakage. All the data were analyzed statistically. RESULTS: After aging, the dentin bonding strength of the experimental group was (19.20±1.03) MPa without a significant decrease (P>0.05), that of the blank control group was (11.22±1.48) MPa with a significant decrease (P<0.05) and that of the commercial control group was (19.16±1.68) MPa without a significant decrease (P>0.05). The interface fracture was observed as the main fracture mode in each group after thermal cycling by SEM. The fractured bonding surfaces of the experimental group often occurred on the top of the hybrid layer, whereas those of the blank and commercial control groups mostly occurred on the bottom of the hybrid layer. Micro-leakage rating counts of specimens before and after thermal cycling were as follows: the experimental group was primarily 0 grade, thereby indicating that a relatively ideal marginal sealing effect could be achieved (P>0.05); meanwhile, the blank control group was primarily 1 grade, and the penetration depth of dye significantly increased after thermal cycling (P<0.05); the commercial control group was primarily 0 grade without statistical difference before and after thermal cycling (P>0.05), while a significant difference was observed between the commercial control group and experimental group after thermal cycling (P<0.05). CONCLUSIONS The novel low-shrinkage resin adhesive containing 20%UE exhibited excellent bonding properties even after thermal cycling aging treatment, thereby showing a promising prospect for dental application.
Composite Resins ; Dental Bonding ; Dental Cements ; Surface Properties ; Resin Cements ; Dentin-Bonding Agents ; Dentin ; Materials Testing ; Microscopy, Electron, Scanning

Objective:To investigate the clinical effects of anterior cage inserting for old thoracolumbar fractures with kyphosis through facet joint approach.Methods:A retrospective analysis was conducted on 32 patients with old thoracolumbar fractures complicated with kyphosis admitted from January 2018 to December 2019, including 14 males and 18 females. The average age was 47.3±13.1 years (range, 26-70 years). Thoracolumbar injury classification (TLICS) scores of patients with initial injury were 3-5 points, with an average of 4.0 points. After 6.3±2.9 months (range, 3-16 months) conservative treatment, intractable thorax and lumbar or back pain still existed. Anterior cage inserting via articular protrusion was performed in 15 cases and posterior screw placement and bone grafting fusion of injured vertebrae was performed in 17 cases. Preoperative sagittal Cobb angle was 27.0°±3.9° and 26.8°±4.6° in the anterior cage inserting group and fixation on fractured vertebrae group ( t=0.07, P=0.946), respectively. Sagittal vertical axis (SVA) was 4.2±1.8 cm and 4.1±2.1 cm ( t=0.14, P=0.887), respectively. The number of patients with ASIA impairment scale (AIS) of the anterior cage inserting group before surgery was 1 in grade C, 4 in grade D and 10 in grade E. However, the number of that in fixation on fractured vertebrae group was 2 in grade C, 2 in grade D and 13 in grade E. There was no significant difference between the two groups (χ 2=1.34, P=0.520). Results:All 32 patients were followed up for 12.2±3.1 months in the anterior cage inserting group and 12.0±3.3 months in fixation on fractured vertebrae group. The operative duration of the anterior cage inserting group and fixation on fractured vertebrae was 128±24.5 min and 123±40.6 min ( t=0.42, P=0.681). The intraoperative blood loss was 485±12.6 ml and 478±16.3 ml ( t=0.13, P=0.894), respectively. At the last follow-up, the improvement rate of VAS score of the anterior cage inserting group was higher than that of fixation on fractured vertebrae group (90%±10% vs. 75%±20%, t=3.17, P=0.004). The height of anterior margin of injured vertebra in the two groups was increased by 1.02±0.10 cm and 0.29±0.14 cm, the change rate of anterior cage inserting group was higher than that of fixation on fractured vertebrae group (67.1%±31.5% vs. 19.0%±14.9%, t=16.29, P<0.001). The sagittal Cobb angle of the anterior cage inserting group was significantly lower than that of fixation on fractured vertebrae group (7.4°±1.5° vs. 11.6°±2.5°, t=-5.85, P<0.001). The SVA of anterior cage inserting group was lower than that of fixation on fractured vertebrae group (1.1±0.6 cm vs. 1.6±0.6 cm, t=2.35, P=0.025). There were 15 patients in AIS grade E in the anterior cage inserting group, while 1 patient in grade D and 16 patients in grade E in fixation on fractured vertebrae group without significant difference between the two groups (χ 2=0.83, P=0.706). Conclusion:The treatment of old thoracolumbar fractures with kyphosis through facet joint approach and anterior fixation could achieve satisfied effects and could relieve pain symptoms of thoracolumbar and back, compared with posterior fusion for injured vertebra with nail and bone grafting.

OBJECTIVE: To analyze the clinical phenotype and genetic basis for a Chinese pedigree suspected for branchiootic syndrome (BOS). METHODS: The proband was subjected to target-capture high-throughput sequencing to detect potential variant of deafness-associated genes. Candidate variants were verified by Sanger sequencing of the family members. RESULTS: The proband was found to harbor a c.1627C>T (p.Gln543Ter) nonsense variant of the EYA1 gene. Sanger sequencing confirmed that all of the 4 patients with the BOS phenotype from the pedigree have harbored the same heterozygous variant. Based on the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic (PVS1+PS+PP3+PP4). CONCLUSION The c.1627C>T (p.Gln543Ter) variant of the EYA1 gene probably underlay the BOS phenotype in this pedigree. Above finding has provided a basis for its clinical diagnosis.
Branchio-Oto-Renal Syndrome ; China ; Humans ; Intracellular Signaling Peptides and Proteins/genetics* ; Mutation ; Nuclear Proteins/genetics* ; Pedigree ; Protein Tyrosine Phosphatases/genetics*

The development of artificial intelligence is becoming more and more mature, and has penetrated into every field of clinical trials. Artificial intelligence has brought new development opportunities for clinical trials. However, the application of artificial intelligence in clinical trials is still in the exploratory stage, facing many ethical issues, including trial risk caused by data quality, privacy protection caused by data regulation, and contradiction between data authorization and informed consent. We should precisely position the realizable application of artificial intelligence in clinical trials, understand its practical ethical issues, and formulate corresponding coping strategies to ensure the maximum improvement of the whole process performance of clinical trials, including strengthening data quality management and reducing clinical trial risks; optimizing data monitoring mechanisms to ensure data security and privacy; building a data authorization platform and improving judicial protection of informed consent, etc.