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The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.
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Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.
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Breast cancer is one of the most common cancers in China. With increasing of the incidence and mortality, the harm to women is becoming much serious. Estrogen positive patients need to take 5-10 years′ endocrine drugs to prolong survival and prevent recurrence. Endocrine therapy has become one of the most common and effective methods in the treatment of breast cancer. In order to achieve the desired effect of cancer treatment, reduce cancer metastasis and recurrence rate, good compliance is essential. In conclusion, this review summarizes the evaluation of compliance, various evaluation tools and their use.
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This study was aimed to investigate the effect of Compound Danshen injection in treatment of dysphagia by electromyography (EMG) guided on new Renying point caused by bulbar paralysis after stroke.A total of 60 patients with dydphagia caused by cerebral palsy after stroke were randomly divided into the experimental group and the control group (30 patients in each group).Patients in the control group were conducted with routine treatment,local low frequency electrical stimulation and rehabilitation training.Patients in the experimental group were treated with acupoint injection therapy guided by EMG on the basis of treatment of the control group.Four weeks were one treatment course.Observations were made on changes of indexes,such as water swallow test,swallowing ability evaluation,and etc.between two groups.The results showed that the cure rate of the experimental group was 76.67％,which was higher than that of the control group (50.00％).The treatment efficiency of the experimental group (93.33％) was higher than that of the control group (80.00％),with statistical significance (P ＜ 0.05).It was concluded that clinical effects of EMG guided injection on new Renying point in the treatment of dysphagia caused by bulbar paralysis in the experimental group was better than that of the control group.It is worthy widely applied and distributed in clinical practices.
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Objective To investigate the differentially expressed genes of urine samples from renal fibrosis model and carry out bioinformatics analysis.Methods Rat renal fibrosis model was constructed with the method of unilateral ureteric obstruction.Urine were collected from the rats with unilateral obstructive nephropathy and sham group, respectively.Total RNA was extracted and sequencing library was established.Differential expression mRNA were analyzed by GO and KEGG pathway.Known pre-miRNA were detected and novel lncRNA family were classified.Results Compared with the sham group urine, 813 up-regulated mRNA/lncRNA and 213 down-regulated mRNA/lncRNA were collected from the urine of renal fibrosis rats.Conclusions There are significant differential expression profile in urine samples between renal fibrosis rat group and the shame group.With high-throughput transcriptome sequencing and bioinformatics analysis, the exciting possibility was raised for better understanding renal pathologies and development of new diagnostic biomarkers.
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Objective:To establish a headspace capillary gas chromatography method for the determination of residual solvents in erlotinib hydrochloride .Methods:A DB-624 capillary column (30 m ×0.53 mm, 3.0 μm) was used and the carrier gas was nitro-gen.The flow rate was 2.0 ml· min-1 and the inlet temperature was 190℃.The FID detector temperature was 230 ℃.The column temperature program was with the initial temperature of 35℃( maitaining 8 min) , risen to 170℃with the rate of 28℃· min-1 ( main-taining 8 min) , and then risen to 200℃with the rate of 32℃· min-1 ( maintaining 7 min) .The headspace vial temperature was 100℃and the time was 30 min.Results:Ethanol, isopropanol, methylene chloride and n-butanol had a good linear relationship within the range of 0.68-409.14 μg· ml-1 (r=0.999 8),0.67-404.88 μg· ml-1 (r=0.999 8),1.71-51.31μg· ml-1 (r=0.999 7) and 0.72-431.12 μg· ml-1(r=0.999 8), respectively.The average recovery was 99.0% (RSD=0.41%, n=9), 100.2%(RSD=0.52%, n=9),97.1%(RSD=1.75%, n =9) and 102.5% (RSD=1.08%, n=9), respectively.Conclusion: The method is simple and accurate , which can be used for the determination of four residual organic solvents in erlotinib hydrochloride .
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BACKGROUND:Mesenchymal stem cel s-released factors can form a microenvironment inhibiting malignant tumor cel proliferation, and moreover, salinomycin also inhibits the growth of tumor cel s. OBJECTIVE:To explore the effect of salinomycin with umbilical cord blood mesenchymal stem cel s in breast cancer MCF-7 cel s and its action mechanism, in order to seek a new target and therapeutic strategy to treat breast cancer. METHODS:Logarithmically growing MCF-7 cells were randomly divided into control, umbilical cord blood mesenchymal stem cell group and combination group. Cells in the control group were given no treatment, while those in the other two groups were given umbilical cord blood mesenchymal stem cell suspension or umbilical cord blood mesenchymal stem cell suspension combined with salinomycin, respectively. RESULTS AND CONCLUSION:Forty-eight hours after the intervention, the proliferation and invasion of MCF-7 cel s and expression of POSTN protein in cel s were significantly reduced in the combination group compared with the other two groups. These findings indicate that the combination of umbilical cord blood mesenchymal stem cel suspension and salinomycin effectively reduces the proliferation and migration of MCF-7 cel s, and this combined use provides a new insight into the treatment of breast cancer.
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Objective:To establish a method for the determination of diisopropyl sulfate in ezetimibe. Methods:Diisopropyl sul-fate was determined by GC with a DB-FFAP capillary column (30 m × 0. 32 mm, 0. 5 μm) and an FID. The carrier gas was nitrogen and the flow rate was 2. 0 ml·min-1 . The temperature program was as follows: the initial column temperature was 40 ℃, and then raised to 180 ℃ at a rate of 25℃·min-1 ,and maintained for 2 min. Results:Diisopropyl sulfate had a good linear relationship with-in the range of 4. 040-13. 466 μg·ml-1(r=0. 999 8). The average recovery was 97. 57%and RSD was 2. 37% (n=9). Conclu-sion:The method is specific and reproducible with high sensitivity, which can be used to determine the content of diisopropyl sulfate in ezetimibe.
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Objective To study the effect of psychological counseling for patients with thoracic surgery recovery period.Methods Ono hundred patients with lobectomy were selected,they were divided into experimental group and control group with 50 cases each according to the treatment method.Two groups of patients were in the ward completed indwelling catheter,inserted into the right double lumen endotracheal tube by high seniority anesthesiologists,postoperative given perfect analgesia.Two group patients were treated with routine operation before the visit,assess the condition and signed the anesthesia informed consent,the test group were given psychological support therapy,explain in detail postoperative recovery period may be uncomfortable,eliminate the anxiety of patients,establish the confidence to overcome the disease,Two groups of patients after surgery by the recovery room nurse to give psychological counseling,complete extubation and record the restlessness,24 h after the patient satisfaction questionnaire.Results In test group patients were able to follow instructions and coordinate nurse smooth tube removal,15 cases of patients need to brake,additional sedatives and dehyed extubation,the difference between the two groups has statistical significance (P ＜ 0.05).The satisfaction questionnaire in test group was significantly higher than that in control group:100％ (50/50) vs.82％ (41/50),there was statistical difference (P ＜ 0.05).Conclusion Psychological counseling can effectively avoid agitation in recovery period of patients with postoperative chest tube removal,smooth.
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Objective:To establish a method for the determination of trace nickel in agomelatine. Methods:An inductive coupled plasma atomic emission spectrometry method was applied in the determination at 221. 648 nm. The sample solution was prepared by the ignited residue of agomelatine. The content of nickel was determined using the standard curve. Results:The linear range was 0. 025-1. 000 μg·ml-1(r=0. 999 8). The RSD of precision was 0. 63%. The detection limit was 0. 000 8μg·ml-1. The quantitative limit was 0. 003μg·ml-1 . The average recovery was 99. 4% with RSD of 2. 20%. The RSD of repeatability was 1. 33%. Conclusion:The method is simple, sensitive and accurate in the determination of trace nickel in agomelatine.
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Group II chaperonins, which assemble as double-ring complexes, assist in the refolding of nascent peptides or denatured proteins in an ATP-dependent manner. The molecular mechanism of group II chaperonin assembly and thermal stability is yet to be elucidated. Here, we selected the group II chaperonins (cpn-α and cpn-β), also called thermosomes, from Acidianus tengchongensis and investigated their assembly and thermal stability. We found that the binding of ATP or its analogs contributed to the successful assembly of thermosomes and enhanced their thermal stabilities. Cpn-β is more thermally stable than cpn-α, while the thermal stability of the hetero thermosome cpn-αβ is intermediate. Cryo-electron microscopy reconstructions of cpn-α and cpn-β revealed the interwoven densities of their non-conserved flexible N/C-termini around the equatorial planes. The deletion or swapping of their termini and pH-dependent thermal stability assays revealed the key role of the termini electrostatic interactions in the assembly and thermal stability of the thermosomes.
Subject(s)
Acidianus , Metabolism , Adenosine Triphosphate , Metabolism , Amino Acid Sequence , Cryoelectron Microscopy , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutation , Nucleotides , Metabolism , Protein Binding , Protein Folding , Protein Stability , Protein Structure, Quaternary , Sequence Alignment , Static Electricity , Temperature , Thermosomes , Chemistry , Genetics , MetabolismABSTRACT
BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. We have showed previous administration of a recombinant adeno-associated virus (rAAV) vector expressing soluble TRAIL results in an efficient suppression of human tumor growth in nude mice. In the present study, we introduced Tet-On gene expression system into the rAAV vector to control the soluble TRAIL expression and evaluate the efficiency of the system in cancer gene therapy. METHODS: Controllability of the Tet-On system was determined by luciferase activity assay, and Western blotting and enzyme-linked immunoabsorbent assay. Cell viability was determined by MTT assay. The breast cancer xenograft animal model was established and recombinant virus was administrated through tail vein injection to evaluate the tumoricidal activity. RESULTS: The expression of soluble TRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of human cancer cell lines with rAAV-TRE-TRAIL&rAAV-Tet-On under the presence of inducer doxycycline resulted in a considerable cell death by apoptosis. Intravenous injection of the recombinant virus efficiently suppressed the growth of human breast carcinoma in nude mice when activated by doxycycline. CONCLUSION: These data suggest that rAAV-mediated soluble TRAIL expression under the control of the Tet-On system is a promising strategy for breast cancer therapy.
