Bi-phase CT radiomics nomogram for the preoperative prediction of pylorus lymph node metastasis in non-pyloric gastric cancer patients.

BACKGROUND: The expansion of function-preserving surgery became possible due to a more profound understanding of gastric cancer (GC), and T1N + or T2N + gastric cancer patients might be potential beneficiaries. However, ways to evaluate the possibility of function-preserving pylorus surgery are still unknown. METHODS: A total of 288 patients at Renji Hospital and 58 patients at Huadong Hospital, pathologically diagnosed with gastric cancer staging at T1 and T2 with tumors located in the upper two-thirds of the stomach, were retrospectively enrolled from March 2015 to October 2022. Tumor regions of interest (ROIs) were manually delineated on bi-phase CT images, and a nomogram was built and evaluated. RESULTS: The radiomic features distributed differently between positive and negative pLNm groups. Two radiomic signatures (RS1 and RS2) and one clinical signature were constructed. The radiomic signatures exhibited good performance for discriminating pLNm status in the test set. The three signatures were then combined into an integrated nomogram (IN). The IN showed good discrimination of pLNm in the Renji cohort (AUC 0.918) and the Huadong cohort (AUC 0.649). The verification models showed high values. CONCLUSION: For GC patients with T1 and T2 tumors located in the upper two-thirds of the stomach, a nomogram was successfully built for predicting pylorus lymph node metastasis, which would guide the surgical indication extension of conservative gastrectomies.

Surface Oxygen Vacancies and Corona Polarization of Bi4Ti3O12 Nanosheets for Synergistically Enhanced Sonopiezoelectric Therapy.

Sonopiezoelectric therapy, an ultrasound-activated piezoelectric nanomaterial for tumor treatment, has emerged as a novel alternative modality. However, the limited piezoelectric catalytic efficiency is a serious bottleneck for its practical application. Excellent piezoelectric catalysts with high piezoelectric coefficients, good deformability, large mechanical impact surface area, and abundant catalytically active sites still need to be developed urgently. In this study, the classical ferroelectric material, bismuth titanate (Bi4Ti3O12, BTO), is selected as a sonopiezoelectric sensitizer for tumor therapy. BTO generates electron-hole pairs under ultrasonic irradiation, which can react with the substrates in a sonocatalytic-driven redox reaction. Aiming to further improve the catalytic activity of BTO, modification of surface oxygen vacancies and treatment of corona polarization are envisioned in this study. Notably, modification of the surface oxygen vacancies reduces its bandgap and inhibits electron-hole recombination. Additionally, the corona polarization treatment immobilized the built-in electric field on BTO, further promoting the separation of electrons and holes. Consequently, these modifications greatly improve the sonocatalytic efficiency for in situ generation of cytotoxic ROS and CO, effectively eradicating the tumor.

Emerging connectivity of programmed cell death pathways and pulmonary vascular remodelling during pulmonary hypertension.

Pulmonary hypertension (PH) is a chronic progressive vascular disease characterized by abnormal pulmonary vascular resistance and pulmonary artery pressure. The major structural alteration during PH is pulmonary vascular remodelling, which is mainly caused by the imbalance between proliferation and apoptosis of pulmonary vascular cells. Previously, it was thought that apoptosis was the only type of programmed cell death (PCD). Soon afterward, other types of PCD have been identified, including autophagy, pyroptosis, ferroptosis and necroptosis. In this review, we summarize the role of the above five forms of PCD in mediating pulmonary vascular remodelling, and discuss their guiding significance for PH treatment. The current review could provide a better understanding of the correlation between PCD and pulmonary vascular remodelling, contributing to identify new PCD-associated drug targets for PH.

Interfacial Cooperative Assembly of Surfactants and Opposite Wettability Nanoparticles Stabilizes Water-in-Oil Emulsions at High Temperature.

Pickering emulsions have promising applications in the development of unconventional oil and gas resources. However, the high-temperature environment of the reservoir is not conducive to the stabilization of Pickering emulsions. In addition, the preparation of Pickering emulsions under low-energy emulsification and low-concentration emulsifier conditions is a difficult challenge. Here, we report a high-temperature resistant water-in-paraffin oil Pickering emulsion, which is synergistically stabilized by polyglycerol ester (PGE) and nanoparticles with opposite wettability (lipophilic silica and hydrophilic alumina). This emulsion can be prepared under mild stirring (500 rpm) conditions and can be stable at 140 °C for at least 30 days. The synergistic effects of surfactant, silicon nanoparticles (MSNPs) with different wettability, and alumina nanoparticles (AONPs) on the stability of both emulsions and water-oil interfacial membranes were investigated through bottle experiments, cryogenic scanning electron microscopy (cryo-SEM), optical microscopy, fluorescence microscopy, etc. The results showed that both hydrophobic MSNPs and hydrophilic AONPs are adsorbed together at the water-oil interface to stabilize the W/O emulsion, which can be prepared by 500 rpm stirring. The stability of emulsions strongly depends on the wettability of MSNPs, and the MSNP with moderate hydrophobicity (for example, aqueous phase contact angle of 136°) makes the emulsion exhibit the highest stability against aggregation and settling at elevated temperatures. The emulsion stabilization mechanism was revealed in terms of the adsorption capacity of the surfactant by MSNPs, the adsorption morphology and desorption energy of nanoparticles at the water-oil interface adsorption layer, and emulsion rheology. These findings demonstrate a novel and simple strategy to prepare Pickering W/O emulsions with high-temperature stability at low shear strength.

Germline-targeting HIV vaccination induces neutralizing antibodies to the CD4 binding site.

Eliciting potent and broadly neutralizing antibodies (bnAbs) is a major goal in HIV-1 vaccine development. Here, we describe how germline-targeting immunogen BG505 SOSIP germline trimer 1.1 (GT1.1), generated through structure-based design, engages a diverse range of VRC01-class bnAb precursors. A single immunization with GT1.1 expands CD4 binding site (CD4bs)-specific VRC01-class B cells in knock-in mice and drives VRC01-class maturation. In nonhuman primates (NHPs), GT1.1 primes CD4bs-specific neutralizing serum responses. Selected monoclonal antibodies (mAbs) isolated from GT1.1-immunized NHPs neutralize fully glycosylated BG505 virus. Two mAbs, 12C11 and 21N13, neutralize subsets of diverse heterologous neutralization-resistant viruses. High-resolution structures revealed that 21N13 targets the same conserved residues in the CD4bs as VRC01-class and CH235-class bnAbs despite its low sequence similarity (~40%), whereas mAb 12C11 binds predominantly through its heavy chain complementarity-determining region 3. These preclinical data underpin the ongoing evaluation of GT1.1 in a phase 1 clinical trial in healthy volunteers.

Single-cell RNA sequencing identifies inherent abnormalities of adipose-derived stem cells from nonlesional sites of patients with localized scleroderma.

BACKGROUND: Localized scleroderma (LoS) is an autoimmune disorder that primarily affects the skin, and is often treated with autologous fat grafting (AFG). Nevertheless, the retention rate of AFG in patients with LoS is typically low. We hypothesize that the low retention rate may be partially attributed to the inherent abnormalities of adipose-derived stem cells (ASCs) from nonlesional sites of patients with LoS. METHODS: We performed a comparative analysis of the single-cell transcriptome of the SVF from nonlesional sites of patients with LoS and healthy donors, including cellular compositional analysis, differential expression analysis, and high-dimensional weighted gene coexpression network analysis. Experimental validation with fluorescence-activated cell sorting and bleomycin-induced skin fibrosis mice models were conducted. RESULTS: We found a significant reduction in the relative proportion of CD55high interstitial progenitors in ASCs under the condition of LoS. Differential expression analysis revealed inherent abnormalities of ASCs from patients with LoS, including enhanced fibrogenesis, reduced anti-inflammatory properties, and increased oxidative stress. Compared with CD55low ASCs, CD55high ASCs expressed significantly higher levels of secreted protein genes that had functions related to anti-inflammation and tissue regeneration (such as CD55, MFAP5, and METRNL). Meanwhile, CD55high ASCs expressed significantly lower levels of secreted protein genes that promote inflammation, such as chemokine and complement protein genes. Furthermore, we provided in vivo experimental evidence that CD55high ASCs had superior treatment efficacy compared with CD55low ASCs in bleomycin-induced skin fibrosis mice models. CONCLUSIONS: We found that the low retention rate of AFG may be partially ascribed to the reduced pool of interstitial progenitor cells (CD55high) present within the ASC population in patients with LoS. We demonstrated the potential for improving the efficacy of AFG in the treatment of LoS by restoring the pool of interstitial progenitors within ASCs. Our study has significant implications for the field of translational regenerative medicine.

Comparative Efficacy and Acceptability of Endoscopic Methods for Rectal Neuroendocrine Neoplasms with Low Malignant Potential: A Network Meta-analysis.

BACKGROUND/AIMS:  Although endoscopic resection is an effective treatment of rectal neuroendocrine neoplasms (R-NENs) with low malignant potential, there is no consensus on the most recommended endoscopic method. This study aimed to assess the efficacy and acceptability of different endoscopic treatments for R-NENs with low malignant potential. MATERIALS AND METHODS:  We searched databases for studies on treatments of R-NENs using endoscopic resection. These studies comprised techniques such as endoscopic mucosal resection (EMR), endoscopic submucosal dissection (ESD), modified endoscopic mucosal resection (EMRM), modified endoscopic submucosal dissection (ESDM), and transanal endoscopic microsurgery (TEM). The primary outcomes assessed were histological complete resection (HCR). RESULTS:  Overall, 38 retrospective studies (3040 R-NENs) were identified. Endoscopic mucosal resection with a cap (EMRC), endoscopic mucosal resection with ligation (EMRL), ESD, ESDM, and TEM demonstrated higher resectability than did EMR in achieving HCR. Endoscopic mucosal resection, EMRC, EMRL, EMRP, EMRD, and EMRU required shorter operation times than did ESD. Endoscopic mucosal resection, EMRC, ESDM, and TEM incurred lower risks than did ESD. CONCLUSION:  Regarding R-NENs <20 mm with low malignant potential, ESD could be used as the primary treatment. However, TEM may be more effective if supported by economic conditions and hospital facility. With respect to R-NENs <16 mm with low malignant potential, EMRL could be used as the primary treatment. In regard to R-NENs <10 mm with low malignant potential, EMRL, EMRC, and ESD could be used as the primary treatment. However, EMRL and EMRC might be better when operational difficulties and economic conditions were considered.

Pd(II)-Catalyzed C4-Selective Alkynylation of Indoles by a Transient Directing Group.

With alanine as a transient directing group, Pd-catalyzed regioselective alkynylation at the indole C4-position was successfully established in a good yield. The total synthesis of the PAF antagonist demonstrated the synthetic utility of this protocol. The regioselectivity was explicitly proven by the prepared C4-selective palladacycle intermediate in the catalytic process and the DFT calculation of the energy barriers of C4- and C2-site-selective C-H activation of indole.

High-level and -yield orotic acid production in Escherichia coli through systematic modular engineering and "Chaos to Order Cycles" fermentation.

Orotic acid is widely used in healthcare and cosmetic industries. However, orotic acid-producing microorganisms are auxotrophic, which results in inefficient microbial production. Herein, a plasmid-free, uninduced, non-auxotrophic orotic acid hyperproducer was constructed from Escherichia coli W3110. Initially, the orotic acid degradation pathway was blocked and the carbamoyl phosphate supply was enriched. Subsequently, pyr operon from Bacillus subtilis F126 was heterologously expressed and precursors' supply was optimized. Thereafter, pyrE was dynamically regulated to reconstruct the non-auxotrophic pathway. Employing fed-batch cultivation, orotic acid titer, yield, and productivity of strain Ora21 reached 182.5 g/L, 0.58 g/g, and 3.80 g/L/h, respectively, the highest levels reported so far. Finally, a novel "Chaos to Order Cycles (COC)" fermentation was developed, which effectively increased the yield to 0.63 g/g. This research is a remarkable achievement in orotic acid production by microbial fermentation and has vast potential for industrial applications.

Triple gene mutations boost amylose and resistant starch content in rice: insights from sbe2b/sbe1/OE-Wxa mutants.

Previous studies have modified rice's resistant starch (RS) content by mutating single and double genes. These mutations include knocking out or reducing the expression of sbe1 or sbe2b genes, as well as overexpressing Wxa . However, the impact of triple mutant sbe2b/sbe1/OE-Wxa on RS contents remained unknown. Here, we constructed a double mutant with sbe2b/RNAi-sbe1, based on IR36ae with sbe2b, and a triple mutant with sbe2b/RNAi-sbe1/OE-Wxa , based on the double mutant. The results showed that the amylose and RS contents gradually increased with an increase in the number of mutated genes. The triple mutant exhibited the highest amylose and RS contents, with 41.92% and 4.63%, respectively, which were 2- and 5-fold higher than those of the wild type, which had 22.19% and 0.86%, respectively. All three mutants altered chain length and starch composition compared to the wild type. However, there was minimal difference observed among the mutants. The Wxa gene contributed to the improvement of 1000-grain weight and seed-setting rate, in addition to the highest amylose and RS contents. Thus, our study offers valuable insight for breeding rice cultivars with a higher RS content and yields.

[Antibiotic Pollution and Its Effects on the Spatiotemporal Variation in Microbial Community Structure and Functional Genes in Sediment of Baiyangdian Lake].

Microbial communities play an important role in water quality regulation and biogeochemical cycles in lakes, and their community structure and function are affected by environmental factors. Therein, antibiotics affect the abundance, diversity, composition, and function of microbial communities. In this study, Baiyangdian Lake was selected as the study area. Sediment samples of 16 sites were collected in August 2018 and April 2019, respectively. Ultra-high performance liquid chromatography-mass spectrometry （HPLC-MS/MS） was used to determine the content of typical antibiotics-quinolones （QNs） in sediment. Through high-throughput sequencing technology, the structure and function of microbial communities was analyzed in the sediments to explore the spatiotemporal variation. Thereinto, redundancy analysis （RDA） was used to identify the key influencing factors of spatiotemporal variation of microbial communities. The results showed that： ① From August to April, the average ω（QNs） showed an increasing trend, and its mean value changed from 3.91 ng·g-1 to 6.34 ng·g-1, with significant seasonal differences in oxolinic acid （OXO） and total QNs content （P < 0.05）. ② In terms of temporal variation, the dominant bacteria were Proteobacteria and Chloroflexi. The relative abundance of Proteobacteria showed a decreasing trend, whereas Chloroflexi showed an increasing trend； at the genus level, the dominant bacteria genera in August were norank_ f__Anaerolineaceae and Thiobacilus, and the dominant bacteria genera in April were Acinetobacter and norank_ f_Anaerolineaceae, and the dominant bacteria genera had significant seasonal differences （P < 0.05）. ③ In terms of temporal variation, the index of Simpson, Chao, Ace, and OTU number all showed an increasing trend, and the seasonal differences were significant （P < 0.05）. ④ In terms of spatial variation, there were no significant spatial differences among functional genes of COG. In terms of temporal variation, there were significant seasonal differences in functional genes of energy production and conversion, carbohydrate transport and metabolism, transcription, cell wall/membrane/envelope biogenesis, and signal transduction mechanisms （P < 0.01 and P < 0.05）. ⑤ Microbial community structure and functional genes were significantly correlated with QNs （P < 0.01 and P < 0.05）, and QNs were the main influencing factors. Therefore, QNs were the main factor affecting the changes in microbial community structure and functional genes in sediments of Baiyangdian Lake. Thus, comprehensive control of antibiotic pollution in sediments should be further strengthened.

Rapid, sensitive, and visual detection of swine Japanese encephalitis virus with a one-pot RPA-CRISPR/EsCas13d-based dual readout portable platform.

Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1 h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100 % concordance with real-time PCR results, underscoring the method's simplicity, sensitivity, and specificity. This efficacy confirms the platform's suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings.

Latent class analysis of healthcare workers' knowledge, attitudes, and practice levels, and risk factors regarding associated with occupational exposure to antineoplastic drugs: A multicenter cross-sectional study.

The study investigated the health care workers' knowledge, attitudes, and practice levels regarding occupational protection against antineoplastic drugs (ADs) via analysis of latent classes and their influencing factors. A convenience sampling method was used to select healthcare workers from 7 hospitals in southern China between April and August 2023. A questionnaire based on literature analysis, brainstorming, and Delphi method was used to investigate the knowledge, practice, and attitudes of healthcare workers exposed to ADs for appropriate occupational protection intervention, followed by latent class analysis. The factors influencing latent classes were identified via single-factor analysis and multiple logistic regression. A total of 322 healthcare workers from departments using ADs were surveyed. The knowledge score associated with occupational protection against ADs was 31.95 ±â€…7.38. The attitude score was 21.08 ±â€…2.729, while the practice score was 36.54 ±â€…9.485. The overall score was 89.57 ±â€…15.497. The healthcare workers were divided into 4 latent classes based on their knowledge, attitude, and practice associated with occupational protection measures against ADs. Healthcare workers in the 4 categories showed significance differences based on professional title, marital status, educational background, and frequency of exposure to ADs (P < .05). The knowledge, attitude, and practice levels of healthcare workers engaged in ADs at work can be divided into 4 latent classes. Despite their increased awareness of the hazards associated with ADs and their attitudes toward protection, the healthcare workers displayed poor knowledge and implementation of occupational protection measures.

Amino-Induced Modulation of Electronic State and Neighboring Site Distance through Second Shell Boosted Catecholase-Mimicking Activity of Electron-Rich Cu Center.

Boosting the biomimetic catalytic activity of nanozyme is important for its potential application. One common strategy to achieve this goal mainly focused on manipulating the electronic state of metal site through the first coordination shell to modulate the adsorption/desorption strength of related reactant, intermediate and/or product, but remained challenging. Taking Cu-based catecholase-mimicking nanozyme for example, this work herein reports a different strategy involving amino-induced modulation of electronic state through the second shell to raise the electron density of Cu site, which further triggers the repulsion effect between neighboring geminal Cu centers to increase the Cu─Cu distance. The resulting nanozyme with electron-rich Cu site (DT-Cu) presents a lower work function and an upshifted d-band center in comparison with its counterpart (i.e., relatively electron-deficient TA-Cu), which promotes the electron transfer and enhances the adsorption strengths of Cu site for O2, catechol and H2O2 intermediate. The longer Cu─Cu distance of DT-Cu accelerated the O─O bond dissociation of H2O2 intermediate. This expedites the oxygen reduction process during catecholase-like catalysis, which together with the enhanced O2/H2O2/catechol adsorption corporately boosts the catecholase-like activity of DT-Cu.

MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4.

Aging-related cardiac fibrosis represents the principal pathological progression in cardiovascular aging. The Muscleblind-like splicing regulator 2 (MBNL2) has been unequivocally established as being associated with cardiovascular diseases. Nevertheless, its role in aging-related cardiac fibrosis remains unexplored. This investigation revealed an elevation of MBNL2 levels in the aged heart and senescent cardiac fibroblasts. Notably, the inhibition of MBNL2 demonstrated a capacity to mitigate H2O2-induced myofibroblast transformation and aging-related cardiac fibrosis. Further mechanistic exploration unveiled that aging heightened the expression of SENP1 and impeded the SUMO1 binding with KLF4, and SUMOylation of KLF4 effectively increased by the inhibition of MBNL2. Additionally, the inhibition of TGF-ß1/SMAD3 signaling attenuated the impact of over-expression of MBNL2 in inducing senescence and cardiac fibrosis. MBNL2, by orchestrating SUMOylation of KLF4, upregulating the TGF-ß1/SMAD3 signaling pathway, emerges as a significant promoter of aging-related cardiac fibrosis. This discovery identifies a novel regulatory target for managing aging-related cardiac fibrosis.

Glycnsisitin A: A promising bicyclic peptide against heart failure that facilitates TFRC-mediated uptake of iron in cardiomyocytes.

Zhigancao decoction is a traditional prescription for treating irregular pulse and palpitations in China. As the monarch drug of Zhigancao decoction, the bioactive molecules of licorice against heart diseases remain elusive. We established the HRESIMS-guided method leading to the isolation of three novel bicyclic peptides, glycnsisitins A-C (1-3), with distinctive C-C and C-O-C side-chain-to-side-chain linkages from the roots of Glycyrrhiza uralensis (Licorice). Glycnsisitin A demonstrated stronger cardioprotective activity than glycnsisitins B and C in an in vitro model of doxorubicin (DOX)-induced cardiomyocyte injury. Glycnsisitin A treatment not only reduced the mortality of heart failure (HF) mice in a dose-dependent manner but also significantly attenuated DOX-induced cardiac dysfunction and myocardial fibrosis. Gene set enrichment analysis (GSEA) of the differentially expressed genes indicated that the cardioprotective effect of glycnsisitin A was mainly attributed to its ability to maintain iron homeostasis in the myocardium. Mechanistically, glycnsisitin A interacted with transferrin and facilitated its binding to the transferrin receptor (TFRC), which caused increased uptake of iron in cardiomyocytes. These findings highlight the key role of bicyclic peptides as bioactive molecules of Zhigancao decoction for the treatment of HF, and glycnsisitin A constitutes a promising therapeutic agent for the treatment of HF.

RAG1 and RAG2 non-core regions are implicated in leukemogenesis and off-target V(D)J recombination in BCR-ABL1-driven B-cell lineage lymphoblastic leukemia.

The evolutionary conservation of non-core RAG regions suggests significant roles that might involve quantitative or qualitative alterations in RAG activity. Off-target V(D)J recombination contributes to lymphomagenesis and is exacerbated by RAG2' C-terminus absence in Tp53-/- mice thymic lymphomas. However, the genomic stability effects of non-core regions from both Rag1c/c and Rag2c/c in BCR-ABL1+ B-lymphoblastic leukemia (BCR-ABL1+ B-ALL), the characteristics, and mechanisms of non-core regions in suppressing off-target V(D)J recombination remain unclear. Here, we established three mouse models of BCR-ABL1+ B-ALL in mice expressing full-length RAG (Ragf/f), core RAG1 (Rag1c/c), and core RAG2 (Rag2c/c). The Ragc/c (Rag1c/c and Rag2c/c) leukemia cells exhibited greater malignant tumor characteristics compared to Ragf/f cells. Additionally, Ragc/c cells showed higher frequency of off-target V(D)J recombination and oncogenic mutations than Ragf/f. We also revealed decreased RAG cleavage accuracy in Ragc/c cells and a smaller recombinant size in Rag1c/c cells, which could potentially exacerbate off-target V(D)J recombination in Ragc/c cells. In conclusion, these findings indicate that the non-core RAG regions, particularly the non-core region of RAG1, play a significant role in preserving V(D)J recombination precision and genomic stability in BCR-ABL1+ B-ALL.

Postoperative QRS duration to left ventricular end-diastolic diameter ratio as a predictor for the risk of postoperative atrial fibrillation in cardiac surgery: A single-center prospective study.

Background and aims: Postoperative atrial fibrillation (POAF) is a frequent complication following cardiac surgery and is associated with adverse clinical outcomes. Our study aimed at determining the clinical and echocardiographic predictors of POAF in patients with cardiac surgery and management of this group of patients may improve their outcome. Methods: We prospectively enrolled patients from the department of cardiovascular surgery in the Second Hospital of Tianjin Medical University from October 23, 2020 to October 30, 2022, without a history of atrial fibrillation. Cox regression was used to identify significant predictors of POAF. Results: A total of 217 patients (79 [36.41 %] were female, 63.96 ± 12.32 years) were included. 88 (40.55 %) patients met the criteria for POAF. Cox regression showed that preoperative left atrial diameter (LAD) (HR: 1.040, 95 % CI 1.008-1.073, p = 0.013) and postoperative QRS/LVEDD (HR: 0.398, 95 % CI 0.193-0.824, p = 0.013) and E/e' (HR: 1.029, 95 % CI 1.002-1.057，p = 0.033) were predictors of POAF. Conclusion: Preoperative LAD and postoperative QRS/LVEDD and E/e' were predictors of POAF in patients undergoing cardiac surgery. Trial registration site: http://www.chictr.org.cn. Registration number: ChiCTR2200063344.