Homo- or heterodimeric compounds that affect dimeric protein function through interaction between monomeric moieties and protein subunits can serve as valuable sources of potent and selective drug candidates. Here, we screened an in-house dimeric natural product collection, and panepocyclinol A (PecA) emerged as a selective and potent STAT3 inhibitor with profound anti-tumor efficacy. Through cross-linking C712/C718 residues in separate STAT3 monomers with two distinct Michael receptors, PecA inhibits STAT3 DNA binding affinity and transcription activity. Molecular dynamics simulation reveals the key conformation changes of STAT3 dimers upon the di-covalent binding with PecA that abolishes its DNA interactions. Furthermore, PecA exhibits high efficacy against anaplastic large T cell lymphoma in vitro and in vivo, especially those with constitutively activated STAT3 or STAT3^Y640F. In summary, our study describes a distinct and effective di-covalent modification for the dimeric compound PecA to disrupt STAT3 function.

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant metabolites renowned for their pharmacological properties. However, sustainable sources for these compounds remain limited. Consequently, researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches. CYP80B, a family of cytochrome P450 (CYP450) enzymes, plays a crucial role in BIA biosynthesis. Previously reported CYP80Bs are known to catalyze the 3'-hydroxylation of (S)-N-methylcoclaurine, with the N-methyl group essential for catalytic activity. In this study, we successfully cloned a full-length CYP80B gene (StCYP80B) from Stephania tetrandra (S. tetrandra) and identified its function using a yeast heterologous expression system. Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that StCYP80B could catalyze N-methylcoclaurine and coclaurine into their respective 3'-hydroxylated products. Notably, StCYP80B exhibited an expanded substrate selectivity compared to previously reported wild-type CYP80Bs, as it did not require an N-methyl group for hydroxylase activity. Furthermore, StCYP80B displayed a clear preference for the (S)-configuration. Co-expression of StCYP80B with the CYP450 reductases (CPRs, StCPR1, and StCPR2), also cloned from S. tetrandra, significantly enhanced the catalytic activity towards (S)-coclaurine. Site-directed mutagenesis of StCYP80B revealed that the residue H205 is crucial for coclaurine catalysis. Additionally, StCYP80B exhibited tissue-specific expression in plants. This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
Cytochrome P-450 Enzyme System/chemistry* ; Benzylisoquinolines/chemistry* ; Hydroxylation ; Plant Proteins/chemistry* ; Alkaloids/metabolism* ; Stephania tetrandra/genetics*

Objective:To investigate the prognostic value of myocardial flow reserve (MFR) measured by SPECT myocardial blood flow (MBF) quantitative technique in patients with intermediate stenoses of coronary arteries.Methods:From September 2019 to May 2021, patients with intermediate stenoses (50% to 80%) identified by invasive coronary angiography in Fuwai Hospital, Chinese Academy of Medical Sciences, Fuwai Center China Cardiovascular Hospital, and TEDA International Cardiovascular Hospital were prospectively included. All patients underwent a one-day rest/stress SPECT myocardial perfusion imaging (MPI) and SPECT MBF quantification. The radioactivity distribution of each segment of the MPI bullseye polar maps were obtained according to the standard 5-point method to obtain the summed stress score (SSS) and the summed difference score (SDS) to determine the existence of abnormality. ROC curve analysis was used to obtain the optimal prognostic cut-off value for MFR. The primary endpoint was defined as cardiovascular endpoint events. Survival and prognostic analyses were conducted by Kaplan-Meier method and Cox proportional hazard models. The difference of AUCs was analyzed by Delong test.Results:A total of 314 patients (194 males, 120 females; age (59.4±8.6) years) were enrolled. Over a median follow-up duration of 754 (range: 628-914) d, 54 patients had endpoint events. ROC curve showed that the prediction ability of MFR was significantly better than that of conventional MPI (AUCs: 0.713 and 0.512; z=3.76, P<0.001). The optimal prognostic cut-off value for MFR to predict endpoint events in patients with intermediate stenoses was 2.04. Cox multivariate analysis showed that MFR (hazard ratio ( HR)=0.434, 95% CI: 0.282-0.669, P<0.001) was an independent predictor of endpoint events in patients with intermediate stenoses. Kaplan-Meier survival analysis showed that the prevalence of endpoint events in patients with MFR≤2.04 was significantly higher than that in patients with MFR>2.04 (25.4%(43/169) vs 7.6%(11/145); χ2=21.27, P<0.001). Conclusion:The MFR measured by SPECT MBF quantitative technique has an independent predictive value for cardiovascular endpoint events in patients with intermediate stenoses.

COVID-19 is caused by coronavirus SARS-CoV-2. Current systemic vaccines generally provide limited protection against viral replication and shedding within the airway. Recombinant VSV (rVSV) is an effective vector which inducing potent and comprehensive immunities. Currently, there are two clinical trials investigating COVID-19 vaccines based on VSV vectors. These vaccines were developed with spike protein of WA1 which administrated intramuscularly. Although intranasal route is ideal for activating mucosal immunity with VSV vector, safety is of concern. Thus, a highly attenuated rVSV with three amino acids mutations in matrix protein (VSV_MT) was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern. It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently. VSV_MT indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein. With a single-dosed intranasal inoculation of rVSV_ΔGMT-S_Δ21, potent SARS-CoV-2 specific neutralization antibodies could be stimulated in animals, particularly in term of mucosal and cellular immunity. Strikingly, the chimeric VSV encoding S_Δ21 of Delta-variant can induce more potent immune responses compared with those encoding S_Δ21 of Omicron- or WA1-strain. VSV_MT is a promising platform to develop a mucosal vaccine for countering COVID-19.

Objective:To assess the clinical application of bedside X-ray photography assistor (Patent No. 202 023 219 898.1) in neonatal bedside photography.Methods:From April 2021 to February 2022, a total of 180 pediatric patients were selected who underwent bedside chest X-ray photography in neonatal intensive care unit (NICU) of Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology. These patients were divided into contrpol group, consisting of 48 males and 42 females aged at (3.3 ± 2.0) d (0-10 d), and experimental group, including 50 males and 40 females aged (3.1±2.2) d (0-12 d). For chest photography, routine workflow was followed in the control group while in experimental group bedside photography protection and body position fixing device was used. The examination time, reshoot rate and image quality were compared between the two groups.Results:The diagnostic physician score and patient comfort score in the experimental group were higher than those in the control group, with statistically significant differences ( t = 3.98, 3.82, P < 0.001). The success rate in the experimental group was higher than that in the control group, with statistically significant difference ( χ2= 7.84, P < 0.05). The average time of examination in the experimental group was not significantly different from in the control group ( P>0.05 ). Conclusions:The application of bedside X-ray photography assistor in neonatal bedside photography can significantly improve the success rate and image quality and reduce the radiation dose to pediatric patients without significantly increased examination time, which is worthy of clinical application and promotion.

Anaplastic lymphoma kinase (ALK) is the most common fusion gene involved in non-small cell lung cancer (NSCLC), and remarkable response has been achieved with the use of ALK tyrosine kinase inhibitors (ALK-TKIs). However, the clinical efficacy is highly variable. Pre-existing intratumoral heterogeneity (ITH) has been proven to contribute to the poor treatment response and the resistance to targeted therapies. In this work, we investigated whether the variant allele frequencies (VAFs) of ALK fusions can help assess ITH and predict targeted therapy efficacy. Through the application of next-generation sequencing (NGS), 7.2% (326/4548) of patients were detected to be ALK positive. On the basis of the adjusted VAF (adjVAF, VAF normalization for tumor purity) of four different threshold values (adjVAF < 50%, 40%, 30%, or 20%), the association of ALK subclonality with crizotinib efficacy was assessed. Nonetheless, no statistical association was observed between median progression-free survival (PFS) and ALK subclonality assessed by adjVAF, and a poor correlation of adjVAF with PFS was found among the 85 patients who received first-line crizotinib. Results suggest that the ALK VAF determined by hybrid capture-based NGS is probably unreliable for ITH assessment and targeted therapy efficacy prediction in NSCLC.
Humans ; Carcinoma, Non-Small-Cell Lung/pathology* ; Anaplastic Lymphoma Kinase/therapeutic use* ; Crizotinib/therapeutic use* ; Lung Neoplasms/pathology* ; Protein Kinase Inhibitors/pharmacology* ; Gene Frequency

Objective:To analyze the epidemiological characteristics of reinfection of 2019-nCoV and influencing factors, and provide evidence for effective prevention and control of COVID-19 epidemic.Methods:The incidence data of COVID-19 in Ningbo from January 1, 2020 to November 30, 2022 were collected from the infectious disease surveillance system of Chinese information system for disease control and prevention. The incidence of reinfection of 2019-nCoV was investigated by using questionnaire. logistic regression analysis was used to analyze the influences of gender, age, time interval from the first infection, history of underlying disease, 2019-nCoV vaccination dose and disease severity on the reinfection.Results:A total of 897 previous 2019-nCoV infection cases were investigated, of which 115 experienced the reinfection of 2019-nCoV, the reinfection rate was 12.82%. The interval between the two infections M( Q1, Q3) was 1 052 (504, 1 056) days. Univariate analysis showed that age, 2019-nCoV vaccination dose, history of underlying disease, type of 2019-nCoV variant causing the first infection, time interval from the first infection and severity of the first infection were associated with the reinfection rate (all P<0.05). Multivariate logistic regression analysis showed that the risk for reinfection in age group 30- years was higher than that in age group ≥60 years ( OR=2.10, 95% CI: 1.11-3.97). No reinfection occurred in those with time interval from the first infection of <6 months, and the risk for reinfection was higher in those with the time interval of ≥12 months than in those with the time interval of 6- months ( OR=6.68, 95% CI: 3.46-12.90). The risk for reinfection was higher in the common or mild cases than in the asymptomatic cases ( OR=2.64, 95% CI: 1.18-5.88; OR=2.79, 95% CI: 1.27-6.11). Conclusion:The time interval from the first infection was an important influencing factor for the reinfection of 2019-nCoV, and the probability of the reinfection within 6 months was low.

Objective:To analyze the clinical reliability of neonatal bedside photography protection and body position fixing device during neonatal bedside X-ray photography.Methods:A mobile X-ray diagnostic machine was used to project the phantom of children. The samples were divided into group A with conventional bedside photography mode, and group B using neonatal bedside X-ray photography protection device. X-ray diagnostic level dosimeters were placed at the projection and radiation sensitive sites, respectively. The three parts of the chest, pelvis and skull were used as the projection center, and the radiation dose to the projection site and the radiation sensitive site were collected and recorded, and the statistical analysis was carried out.Results:When the chest was taken as the center of the projection, the radiation doses to the lens of the eye, thyroid and gonad in the body model group B of children were 94.4%, 96.9% and 96.7% lower than those in the non-injected part of group A, respectively ( t=-152.55, -445.16, -129.07, P<0.05). When the pelvis was taken as the projection center, the radiation doses to the lens, thyroid and thymus in the body model group B were 85.5%, 87.1% and 94.9% lower than those in the non-projection part of group A, respectively ( t=-50.68, -194.18, -535.94, P<0.05). When the head was taken as the projection center, the radiation doses to thyroid, thymus and gonad in the body model group B were 99.3 %, 97.4 % and 94.3 % lower than those in the non-projection position of group A, respectively ( t=-1 859.97, -542.08, -66.26, P< 0.05). Conclusions:The use of neonatal bedside photography protection and position fixing device during neonatal bedside X-ray photography can significantly reduce the radiation dose to children in non-projected areas under the premise of ensuring image quality. At the same time, it can fix and protect the children, improve the success rate of examination, being worthy of clinical promotion.

Objective:To study the amplification / overexpression of human epidermal growth factor receptor 2 (HER2) in patients with gallbladder cancer, and to analyze the correlation between amplification/overexpression of HER2 with clinicopathological features and survival in patients after R 0 resection. Methods:There were 14 males and 26 females, aged (60.3±8.7) years old and treated at the Cancer Hospital of Chinese Academy of Medical Sciences from January 2011 to December 2016 who met the inclusion criteria of the study. Immunohistochemistry and fluorescence in situ hybridization were used to detect amplification / expression of HER2 in resected tumor tissues. Patients were divided into two groups according to the HER2 gene expression: the HER2-negative group ( n=40) and the HER2-positive group ( n=10). The Chi-square test was used to analyze the relationship between amplification/expression of HER2 and clinicopathological parameters. Patients were followed up by telephone for prognosis. The Kaplan-Meier method was used for survival analysis. The Cox proportional hazard model was used to explore factors affecting prognosis of gallbladder cancer patients. Results:HER2 amplification/overexpression was found in 10 patients with gallbladder cancer, with a positive rate of HER2 being 20.0% (10/50). There was a significant difference in the proportion of patients with vascular tumor thrombus between the HER2 negative group and the HER2 positive group [7.5%(3/40) vs. 30.0%(3/10), P<0.05]. On follow-up, data of 46 patients was available. There were 36 patients in the HER2-negative group and 10 patients in the HER2-positive group. Compared with the HER2-negative group, the median recurrence-free survival (10.10 vs. 75.07 months) and the median overall survival (16.77 vs. 83.07 months) of the HER2-positive group were both significantly lower (both P<0.05) than the HER2-negative group. Univariate analysis showed HER2 positivity to be a risk factor for recurrence-free and overall survival in patients with gallbladder cancer after radical resection. Cox multivariable analysis revealed that lymph node metastasis was an independent risk factor for both recurrence-free ( HR=4.31, 95% CI: 1.92-9.68, P<0.001) and overall survival ( HR=3.44, 95% CI: 1.08-11.00, P=0.037). Conclusion:Amplification / overexpression of HER2 was associated with venous invasion and worse prognosis in patients with gallbladder cancer.

Objective:To investigate the role of integrin-linked kinase (ILK)-small interfering RNA (siRNA)-adeno-associated virus (AAV) in scar formation after glaucoma filtering surgery in rat eyes.Methods:Forty-eight Sprague Dawley rats of SPF grade, aged 8 to 9 weeks old, were selected and divided into blank control group, ILK-siRNA-AAV group, NC-siRNA-AAV group and mitomycin C (MMC) group by random number table method, with 12 rats in each group.Left eyes of the rats were taken as experimental eyes, and no intervention was administered to fellow eyes.The bulbar conjunctival filtering bleb after glaucoma filtration surgery in rats was established by anterior chamber drainage tube implantation.One day after operation, phosphate buffer solution, ILK-siRNA-AAV, and NC-siRNA-AAV were injected into the filtering bleb of blank control group, ILK-siRNA-AAV group and NC-siRNA-AAV group, 5 μl each group, respectively.Cotton tablets containing 0.4 mg/ml MMC were placed under conjunctival flap for 5 minutes during operation in MMC group.Intraocular pressure (IOP) was measured with a handheld tonometer before surgery and at 1, 2, 3, 7, 14, 21, 28 days after surgery.Formation of filtering blebs in rats was observed with a surgical microscope at 1, 2, 3, 7, 14, 21, 28 days after operation, and the bleb survival time was calculated by Kaplan-Meier survival analysis.The mRNA and protein expression levels of ILK in conjunctival and subconjunctival tissues at the surgical sites were detected by reverse transcription PCR and Western blot, respectively, on the 28th day after operation.Silencing of ILK gene was identified.Effect of ILK gene silencing on the morphology of drainage pathway was observed by hematoxylin-eosin staining.Effect of ILK gene silencing on collagen fiber deposition in the bulbar conjunctiva at filtration area was examined by Masson staining, and the percentage of positive area of collagen fiber staining in the total tissue visual field was calculated.The use and care of the animals complied with the ARVO Statement.This research protocol was approved by an Ethics Committee of Xi'an Jiaotong University (No.2013-772). Results:There were statistically significant differences in IOP at different time points between before and after surgery among four groups ( Fgroup=76.84, P<0.001; Ftime=114.49, P<0.001). The IOP of ILK-siRNA-AAV group on the 1st, 7th, 14th and 28th day after operation and the IOP of MMC group on the 2nd, 3rd, 7th, 14th and 28th day after operation were lower than those of blank control group, and the differences were statistically significant (all at P<0.05). The IOP of ILK-siRNA-AAV group was lower on 7th, 14th, 21st and 28th day after operation than those of NC-siRNA-AAV group, with statistically significant differences (all at P<0.05). The bleb survival time of blank control group, NC-siRNA-AAV group, ILK-siRNA-AAV group and MMC group was (3.50±1.51), (5.00±3.41), (31.50±3.15) and (31.33±2.46) days, respectively, with a significant difference among them ( F=395.83, P<0.05). The bleb survival time of ILK-siRNA-AAV group and MMC group was higher than that of blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of ILK mRNA and protein among four groups ( F=222.32, 752.69; both at P<0.05), and the relative expression levels of ILK mRNA and protein were significantly lower in ILK-siRNA-AAV group than blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Proliferative fibrous connective tissue and a large number of cells at surgical sites were found in blank control group and NC-siRNA-AAV group, and the fibroblasts were of a high density and grew in clumps.In ILK-siRNA-AAV group, the bulbar conjunctiva was thin, and the arrangement of fibrous connective tissue was loose, and a few proliferative fibroblasts were found.In MMC group, the conjunctival fibrous layer was loose and thin, forming cavities, and scarce cells were found.There was statistically significant difference in the percentage of collagen fiber positive staining area among four groups ( F=741.66, P<0.05). The positive staining percentage of ILK-siRNA-AAV group and MMC group was significantly lower than that of blank control group, among which there was lower positive staining percentage in ILK-siRNA-AAV group than NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Conclusions:Silencing of ILK can inhibit the scar formation after glaucoma filtering surgery and maintain low IOP in rats.