ObjectiveTo evaluate the clinical efficacy of Huayu Tongluo Formula （化瘀通络方， HTF） in patients with mid-stage diabetic kidney disease of blood stasis syndrome and explore its potential mechanisms. MethodsA multi-center， randomized， double-blind， placebo-controlled clinical trial was conducted. Ninety patients of mid-stage diabetic kidney disease of blood stasis syndrome were divided into a control group of 46 cases and a treatment group of 44 cases. Both groups received conventional western medicine treatment， the treatment group additionally taking HTF， while the control group taking a placebo of the formula. The treatment was administered once daily for 24 weeks. The primary outcomes included 24-hour urine total protein （24 h-UTP）， serum albumin （Alb）， glycated hemoglobin （HbA1c）， and serum creatinine （Scr）.The secondary outcomes included changes in levels of endothelin-1 （ET-1）， nitric oxide （NO）， vascular endothelial growth factor （VEGF）， and traditional Chinese medicine （TCM） syndrome scores before and after treatment. Clinical efficacy was evaluated based on TCM syndrome scores and overall disease outcomes. Adverse reactions and endpoint events were recorded. ResultsIn the treatment group after treatment， 24 h-UTP， ET-1， and VEGF levels significantly decreased （P＜0.05）， Alb and NO levels significantly increased （P＜0.05）； while the TCM syndrome scores for edema， lumbar pain， numbness of limbs， dark purple lips， dark purple tongue or purpura， and thin， rough pulse all significantly decreased （P＜0.05）. In the control group， no significant changes were observed in any of the indicators after treatment （P＞0.05）.Compared with the control group， the treatment group showed significant reductions in 24 h-UTP， ET-1， and VEGF levels， and increases in Alb and NO levels （P＜0.05）. The TCM syndrome scores for edema， lumbar pain， dark purple tongue or purpura， and thin， rough pulse were all lower in the treatment group than in the control group （P＜0.05）. The total effective rate of TCM syndrome in the treatment group was 59.09% （26/44）， and the overall clinical effective rate was 45.45% （20/44）. In the control group， these rates were 15.22% （7/46） and 8.7% （4/46）， respectively， with the treatment group showing significantly better outcomes （P＜0.05）. A total of 7 adverse events occurred across both groups， with no significant difference （P＞0.05）. No endpoint events occurred during the study. ConclusionOn the basis of conventional treatment of Western medicine， HTF can further reduce urinary protein levels and improve clinical symptoms in patients with mid-stage diabetic kidney disease of blood stasis syndrome. The mechanism may be related to its effects on endothelial function.

Gene therapy, harnessing the power of CRISPR-Cas9 and/or DNAzyme systems, stands as a pivotal approach in cancer therapy, enabling the meticulous manipulation of genes pivotal to tumorigenesis and immunity. However, the pursuit of precise gene therapy encounters formidable hurdles. Herein, a near-infrared upconversion theranostic nanomachine is devised and tailors for CRISPR-Cas9/DNAzyme systems mediate precise gene therapy. An ingenious logic DNAzyme system consists of Chain 1 (C1)/Chain 2 (C2) and endogenous lncRNA is designed. We employ manganese modified upconversion nanoparticles for carrying ultraviolet-responsive C1-PC linker-C2 (C₂P) chain and Cas9 ribonucleoprotein (RNP), with outermost coats with hyaluronic acid. Upon reaching tumor microenvironment (TME), the released Mn²⁺ ions orchestrate a trifecta: facilitating endosomal escape, activating cGAS-STING signaling, and enabling T1-magnetic resonance imaging. Under near-infrared irradiation, Cas9 RNP/C₂P complex dissociates, releasing Cas9 RNP into the nucleus to perform gene editing of Ptpn2, while C1/C2 chains self-assemble with endogenous lncRNA to form a functional DNAzyme system, targeting PD-L1 mRNA for gene silencing. This strategy remodels the TME by activating cGAS-STING signaling and dual immune checkpoints blockade, thus realizing tumor elimination. Our theranostic nanomachine armed with the CRISPR-Cas9/DNAzyme logic systems, represents a resourceful and promising strategy for advancing cancer systemic immunotherapy and precise gene therapy.

OBJECTIVES: To explore the expression profile of circular RNAs (circRNAs) and their potential roles in prognosis and progression of Wilms' tumor (WT). METHODS: Four pairs of WT and adjacent tissues were collected for high-throughput circRNA sequencing to identify the differentially expressed circular RNAs. RT-qPCR was used to verify the expression levels of the top 6 candidate circRNAs in the clinical samples. hsa_circ_0001900 was selected for analysis of its correlation with clinicopathological features and prognosis in 34 patients with WT. Sanger sequencing and RNase R digestion experiments were used to verify the cycling site and structural stability of hsa_circ_0001900 molecule. RESULTS: A total of 23 978 circular RNA molecules were identified in WT tissues by high-throughput circular RNA sequencing, and among them 614 were differentially expressed in WT. hsa_circ_0001900 showed the highest expression level among the differentially expressed circRNAs, which was consistent with the findings in clinical tumor samples and the sequencing results. Correlation analysis showed that hsa_circ_0001900 expression level was positively correlated with WT volume, and the children with high hsa_circ_0001900 expression had a lowered recurrence-free survival rate. The results of Sanger sequencing verified the circular splice site sequence of the molecule, and Rnase R digestion assay confirmed its stable covalent structure. CONCLUSIONS This study presents a comprehensive expression profile of circular RNAs in WT, and the expression level of hsa_circ_0001900 is related to the size of WT and the patients' prognosis, suggesting its possible role as a key driving gene in WT progression.
Humans ; RNA, Circular ; Wilms Tumor/pathology* ; Prognosis ; High-Throughput Nucleotide Sequencing ; Kidney Neoplasms/genetics* ; Sequence Analysis, RNA ; Male ; Female

O-acetyl-L-homoserine (OAH) is a promising platform compound for the production of L-methionine and other valuable compounds, while its low yield and low conversion rate limit the industrial application. To solve these problems, we constructed a strain for high OAH production with the previously constructed L-homoserine producer Escherichia coli HS33 as the chassis by systematic metabolic engineering. Firstly, PEP accumulation, pyruvate utilization, and OAH synthesis pathway (overexpressing aspB, aspA, and thrA^C1034T) were enhanced to obtain an initial strain accumulating 13.37 g/L OAH. Subsequently, the co-factor synthesis genes were integrated to supply reducing power and energy, which increased the yield to 15.79 g/L. The OAH yield of the engineered strain OAH28 was further increased to 17.49 g/L by strengthening the acetic acid reuse pathway, improving the supply of acetyl-CoA, and regulating the expression of MetX from different sources. Finally, in a 5 L fermenter, OAH28 achieved an OAH titer of 47.12 g/L, with a glucose conversion rate of 32% and productivity of 0.59 g/(L·h). The results lay a foundation for increasing the OAH production by metabolic engineering and give insights into the industrial production of OAH.
Metabolic Engineering/methods* ; Escherichia coli/genetics* ; Homoserine/biosynthesis* ; Fermentation

Objective:To investigate the effect and mechanism of isorhynchophylline (IRN) on angiotensin Ⅱ(Ang Ⅱ)-induced cardiac hypertrophy.Methods:H9c2 cells were co-cultured with Ang Ⅱ and different concentrations of IRN (0, 5, 10, 25, 50 μmol/L). The cell surface area and mRNA levels of cardiac hypertrophy markers atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) were detected to elucidate the effect of IRN on myocardial hypertrophy and the most effective concentration. H9c2 cells were co-cultured with Ang Ⅱ and IRN (25 μmol/L) at different times (0, 6, 12, 24 h) to elucidate the most effective time of inhibition. The phosphorylation levels of the signaling pathway were detected, and the effects of IRN and Akt inhibitor MK2206 on the phosphorylation levels of the signaling pathway were further explored to elucidate the underlying mechanisms.Results:Compared with the control group, the surface area of H9c2 cells, and the mRNA expression of myocardial hypertrophy markers ANP, BNP and β-MHC were significantly increased (all P<0.05). Pretreated with different concentrations of IRN (5, 10, 25, 50 μmol/L) could inhibit the increase in cell surface area induced by AngⅡ (all P<0.05), especially at the concentration of 25 μmol/ L ( P<0.01). IRN could time-dependently inhibit AngⅡ-induced activation of ANP, BNP, β-MHC mRNA (all P<0.05). AngⅡ caused increased phosphorylation levels of Akt, GSK3β, mTOR and FOXO3a. IRN could block AngⅡ-induced phosphorylation of the Akt signaling pathway. Conclusion:IRN attenuates AngⅡ-induced cardiomyocyte hypertrophy by inhibiting the Akt signaling pathway.

Objective To identify the key genes differentially expressed in Wilms tumor and analyze their potential impacts on prognosis and immune responses of the patients. Methods High-throughput RNA sequencing was used to identify the differentially expressed mRNAs in clinical samples of Wilms tumor and paired normal tissues, and their biological functions were analyzed using GO, KEGG and GSEA enrichment analyses. The hub genes were identified using STRING database, based on which a prognostic model was constructed using LASSO regression. The mutations of the key hub genes were analyzed and their impacts on immunotherapy efficacy was predicted using the cBioPortal platform. RT-qPCR was used to verify the differential expressions of the key hub genes in Wilms tumor. Results Of the 1612 differentially expressed genes identified in Wilms tumor, 1030 were up-regulated and 582 were down-regulated, involving mainly cell cycle processes and immune responses. Ten hub genes were identified, among which 4 genes (TP53, MED1, CCNB1 and EGF) were closely related to the survival of children with Wilms tumor. A 3-gene prognostic signature was constructed through LASSO regression analysis, and the patients stratified into with high- and low-risk groups based on this signature had significantly different survival outcomes (HR=1.814, log-rank P=0.002). The AUCs of the 3-, 5-and 7-year survival ROC curves of this model were all greater than 0.7. The overall mutations in the key hub genes or the individual mutations in TP53/CCNB1 were strongly correlated with a lower survival rates, and a high TP53 expression was correlated with a poor immunotherapy efficacy. RT-qPCR confirmed that the key hub genes had significant differential expressions in Wilms tumor tissues and cells. Conclusion TP53 gene plays an important role in the Wilms tumor and may potentially serve as a new immunotherapeutic biomarker as well as a therapeutic target.

Objective:To understand the composition of respiratory virus spectrum in hospitalized children with acute respiratory infections in Guilin City from 2021 to 2022, and to explore the epidemic characteristics of different respiratory viruses.Methods:Information of a total of 638 hospitalized children with acute respiratory infections (ARI) syndrome under the age of 14 years in Guilin city, Guangxi from September 2021 to October 2022 was collected as research data. The collected pharyngeal swabs from pediatric patients were subjected to real-time fluorescence reverse transcription polymerase chain reaction for nucleic acid testing, screening for 15 common respiratory viruses, and analyzing the virus spectrum characteristics of hospitalized pediatric patients with acute respiratory infections.Results:Among the 638 specimens, 365 were tested positive for at least one virus, with a positive rate of 57.21% (365/638). The detection rate for two or more viruses was 12.85% (82/638). There were 12 types of viruses detected, and the difference in virus detection rates among different seasons was statistically significant (P<0.002). The virus positive detection rate of samples from different age groups was the highest in the 0-2-year old group (40.66%), followed by the 3-5-year old group (34.80%) and the 6-year and older group (24.54%).Conclusions:During the period from September 2021 to October 2022, all 12 respiratory viruses were prevalent in Guilin area, with obvious summer peak as characteristics. It is suggested that corresponding prevention and control measures should be focused on pathogens that may cause acute respiratory infections in children during the local summer to prevent and control the spread and prevalence of respiratory infections.

Objective:To understand the prevalence and characteristics of Rhinovirus (HRV) infection in influenza-like Illness (ILIs) patients in Mianyang, Sichuan province, China.Methods:Throat swabs were collected from patients of ILIs in sentinel hospitals in Mianyang during 2021—2022. Real-time fluorescence quantitative PCR was used to detect 16 common pathogens. The VP4/VP2 coding region genes of HRV positive samples were amplified by nest PCR. The phylogeny, consistency and amino acid variation of different serotypes were analyzed and compared with reference sequences from GenBank database.Results:A total of 332 ILIs′ samples were collected with a virus detection rate of 58.73% (195/332) in Mianyang. Among them, 23 samples (23/332) were HRV-positive, and 18 VP4/VP2 sequences of HRV strains were successfully amplified. It was found that 13 HRV serotypes were detected in ILIs samples in Mianyang, which belonged to three genotypes, namely HRV-A (12 strains), HRV-B (5 strains) and HRV-C (1 strain).Conclusions:HRV was one of the pathogens of ILIs cases in Mianyang during 2021—2022, with HRV-A types as the dominant strains.

Objective·To summarize the clinical features,imaging features,and diagnosis and treatment experience of primary mediastinal yolk sac tumor(YST).Methods·Data of 29 patients with primary mediastinal YST,who attended Shanghai Chest Hospital,Shanghai Jiao Tong University School of Medicine from September 2016 to May 2023,were collected and comprehensively analyzed,including imaging examination results,serum indicators,pathology reports and treatment methods.Results·There were 22 cases of pure YST and 7 cases of mixed YST comprising 28 males and 1 female.The mean age of onset was(24.5±5.9)years.The initial symptoms were chest tightness(34.5%),chest pain(27.8%),cough(34.5%),expectoration(34.5%)and no specific symptoms(24.1%).Chest computerized tomography(CT)enhancement showed that all the 29 lesions were located in the anterior mediastinum.The maximum diameter of the lesions ranged from 5.6 cm to 18.2 cm.The lesions were irregular in shape,uneven in density,partially cystic and solid in density.The enhancement scan showed the solid part was slightly and moderately enhanced,and the low-density area was not enhanced.Tumor boundary was not clear because tumors often compressed and invaded surrounding tissues.Among the 29 newly diagnosed patients,serum alpha-fetoprotein(AFP)was significantly increased in 28 cases(1 case was not tested).Patients received multidisciplinary comprehensive treatment,including chemotherapy(25/29),surgery(26/29),and radiotherapy(8/29).Seven patients directly received surgery after diagnosis.Nineteen patients received chemotherapy first and then surgery;16(84.2%)cases were evaluated as lesion shrinkage after chemotherapy.After surgery,73.1%(19/26)patients had a significant decrease in serum AFP.After chemotherapy,56.0%(14/25)patients had decreased serum AFP.Conclusion·Primary mediastinal YST usually occurs in middle-aged and young men,with certain clinical and radiographic features and elevated serum AFP,which requires multidisciplinary comprehensive treatment.