A Chromosome-level Genome Assembly Provides Insights into the Local Adaptation of Tamarix austromongolica in the Yellow River Basin, China.

Tamarix austromongolica is endemic to the Yellow River Basin and has adapted to diverse ecological settings in the region, including the arid areas of northwestern China and the saline soil regions of the Yellow River Delta. However, the genetic basis of its local adaptation remains unclear. We report a chromosome-level assembly of the T. austromongolica genome based on PacBio high-fidelity sequencing and Hi-C technology. The 12 pseudochromosomes cover 98.44% of the 1.32 Gb assembly, with a contig N50 of 52.57 Mb and a BUSCO score of 98.2%. The genome comprises 913.6 Mb (68.83%) of repetitive sequences and 22,374 protein-coding genes. Genome evolution analyses suggest that genes under positive selection and significantly expanded gene families have facilitated T. austromongolica's adaptability to diverse environmental factors and high resistance to diseases. Using genotyping-by-sequencing, we conducted population structure and selection analyses of 114 samples from 15 sites. Two genetic groups were identified, and 114 and 289 candidate genes were assigned to the populations of the northwestern and eastern parts of the Yellow River, respectively. Furthermore, we discovered numerous candidate genes associated with high-altitude adaptability and salt tolerance. This research provides valuable genomic resources for the evolutionary study and genetic breeding of tamarisk.

In-situ evaluation the fluctuation of hypochlorous acid in acute liver injury mice models with a mitochondria-targeted NIR ratiometric fluorescent probe.

Acute liver injury (ALI) is a frequent and devastating liver disease that has been made more prevalent by the excessive use of chemicals, drugs, and alcohol in modern life. Hypochlorous acid (HClO), an important biomarker of oxidative stress originating mainly from the mitochondria, has been shown to be intimately connected to the development and course of ALI. Herein, a novel BODIPY-based NIR ratiometric fluorescent probe Mito-BS was constructed for the specific recognition of mitochondrial HClO. The probe Mito-BS can rapidly respond to HClO within 20 s with a ratiometric fluorescence response (from 680 nm to 645 nm), 24-fold fluorescence intensity ratio enhancement (I645/I680), a wide pH adaptation range (5-9) and the low detection limit (31 nM). The probe Mito-BS has been effectively applied to visualize endogenous and exogenous HClO fluctuations in living zebrafish and cells based on its low cytotoxicity and prominent mitochondria-targeting ability. Furthermore, the fluorescent probe Mito-BS makes it possible to achieve the non-invasive in-situ diagnosis of ALI through in mice, and provides a feasible strategy for early diagnosis and drug therapy of ALI and its complications.

A ratiometric fluorescent probe with a large Stokes shift for rapid and sensitive detection of Hg2+ in environmental water samples and its applications in living cells and zebrafish.

Detection of highly toxic mercury ions (Hg2+) in actual environmental and biological samples is of significant importance for protecting environment and human health. In this paper, a new ratiometric fluorescent probe BTIA was designed and synthesized from 3-pinone based on Internal Charge Transfer (ICT) mechanism. BTIA could selectively recognize Hg2+ over other competitive analytes with short reaction time (5 s), distinct ratiometric response, strong anti-interference ability, large Stokes shift (200 nm), and low detection limit (2.36 × 10-7 M). Furthermore, BTIA was applicable for detecting Hg2+ in actual water samples and it also performed an excellent imaging capability in living RAW264.7 cells, zebrafish and onion tissue.

Seasonal variations of organophosphate esters (OPEs) in atmospheric deposition, and their contribution to soil loading.

Organophosphate esters (OPEs) are ubiquitous in surface soil, and atmospheric deposition is considered to be the major pollution source. However, the research on the environmental transport behaviors of OPEs between atmospheric deposition and soil is very limited. In this study, we investigated the contamination levels and seasonal variations of OPEs in atmospheric deposition samples (n = 33) collected from an area of South China every month between February 2021 and January 2022, and evaluated the contribution of OPEs in atmospheric deposition to soil. The concentrations of ∑21target-OPEs ranged from 3670 to 18,600 ng/g dry weight (dw), with a mean of 8200 ng/g dw (median: 7600 ng/g dw). ∑21target-OPEs concentrations in all atmospheric deposition samples exhibited significant seasonal differences (p < 0.05) with higher concentrations observed in winter and lower concentrations in summer. Tris(2,4-di-tert-butylphenyl) phosphate (TDTBPP) was the most dominant target OPE in atmospheric deposition (4870 ng/g dw), and its seasonal variation trend was consistent with ∑21OPEs (p < 0.05). Simultaneously, in order to further explore the effect of atmospheric deposition on the levels of OPEs in soil of the study region, input fluxes and accumulation increments were estimated. Ten OPEs (including seven target OPEs and three suspect OPEs) exhibited high input flux means and accumulation increments, indicating that these compounds are prone to accumulate in soil via atmospheric deposition. It is noteworthy that the non-target phosphonate analyte bis(2,4-di-tert-butylphenyl) dibutyl ethane-1,2-diylbis(phosphonate) (BDTBPDEDBP) was detected at highest median concentration (8960 ng/g dw) in atmospheric deposition. Correspondingly, the average input flux and accumulation increment of BDTBPDEDBP were higher than those of all target and suspect OPEs. Collectively, this study quantifies the environmental transport behavior of OPEs between atmospheric deposition and soil, and provides new evidences for the fact that atmospheric deposition is the important pollution source of OPEs in soil.

Postovulatory Aging of Mouse Oocytes Impairs Offspring Behavior by Causing Oxidative Stress and Damaging Mitochondria.

Information on long-term effects of postovulatory oocyte aging (POA) on offspring is limited. Whether POA affects offspring by causing oxidative stress (OS) and mitochondrial damage is unknown. Here, in vivo-aged (IVA) mouse oocytes were collected 9 h after ovulation, while in vitro-aged (ITA) oocytes were obtained by culturing freshly ovulated oocytes for 9 h in media with low, moderate, or high antioxidant potential. Oocytes were fertilized in vitro and blastocysts transferred to produce F1 offspring. F1 mice were mated with naturally bred mice to generate F2 offspring. Both IVA and the ITA groups in low antioxidant medium showed significantly increased anxiety-like behavior and impaired spatial and fear learning/memory and hippocampal expression of anxiolytic and learning/memory-beneficial genes in both male and female F1 offspring. Furthermore, the aging in both groups increased OS and impaired mitochondrial function in oocytes, blastocysts, and hippocampus of F1 offspring; however, it did not affect the behavior of F2 offspring. It is concluded that POA caused OS and damaged mitochondria in aged oocytes, leading to defects in anxiety-like behavior and learning/memory of F1 offspring. Thus, POA is a crucial factor that causes psychological problems in offspring, and antioxidant measures may be taken to ameliorate the detrimental effects of POA on offspring.

Roles of Wettability and Wickability on Enhanced Hydrogen Evolution Reactions.

Bubble dynamics significantly impact mass transfer and energy conversion in electrochemical gas evolution reactions. Micro-/nanostructured surfaces with extreme wettability have been employed as gas-evolving electrodes to promote bubble departure and decrease the bubble-induced overpotential. However, effects of the electrodes' wickability on the electrochemical reaction performances remain elusive. In this work, hydrogen evolution reaction (HER) performances are experimentally investigated using micropillar array electrodes with varying interpillar spacings, and effects of the electrodes' wettability, wickability as well as bubble adhesion are discussed. A deep learning-based object detection model was used to obtain bubble counts and bubble departure size distributions. We show that microstructures on the electrode have little effect on the total bubble counts and bubble size distribution characteristics at low current densities. At high current densities, however, micropillar array electrodes have much higher total bubble counts and smaller bubble departure sizes compared with the flat electrode. We also demonstrate that surface wettability is a critical factor influencing HER performances under low current densities, where bubbles exist in an isolated regime. Under high current densities, where bubbles are in an interacting regime, the wickability of the micropillar array electrodes emerges as a determining factor. This work elucidates the roles of surface wettability and wickability on enhancing electrochemical performances, providing guidelines for the optimal design of micro-/nanostructured electrodes in various gas evolution reactions.

Comprehensive investigations of four ratiometric fluorescent chemosensors based on 4-(1H-imidazol-2-yl)benzaldehyde skeleton for malononitrile detection.

Malononitrile is a very important chemical material and has wide application fields in production of medicines, pesticides, and extraction of gold. However, its nonnegligible hypertoxicity inspired researchers to develop more efficient analysis techniques to sensitively and selectively detect malononitrile. Nopinone derivatives initiated by our research group have been developed as a class of organic fluorescent chemosensors for identifying multiple analytes in recent years. Different heterocyclic compounds based on nopinone were designed and synthesized to be applied in the fields of environmental analysis, food detection and bioimaging. Nevertheless, the comparison research on the optical properties of fluorescent compounds containing the nopinyl matrix with other structural analogs including alkyl, cyclohexyl and phenyl groups was deficient. Herein, four 4-(1H-imidazol-2-yl)benzaldehyde-based ratiometric fluorescent chemosensors based on o-dimethyl cyclohexyl, phenyl and nopinyl units for recognizing malononitrile were designed and developed, and their differences in the optical properties and detection performances were investigated by using spectral analysis combined with theoretical calculations. Moreover, the nopinone-based 4-(1H-imidazol-2-yl)benzaldehyde fluorescent chemosensor NMZQ was successfully applied in the dual channel fluorescence bioimaging of malononitrile in living HeLa cells and zebrafish, which attributed to its outstanding spectral property and detection performance.

hsa_circ_0007919 promotes pancreatic cancer metastasis by modulating Sp1-mediated THBS1 transcription.

CircRNAs are abnormally expressed in various cancers and play an important role in the occurrence and development of cancers. However, their biological functions and the underlying molecular mechanisms in pancreatic cancer (PC) metastasis are incompletely understood. Differentially expressed circRNAs were identified by second-generation transcriptome sequencing in three pairs of PC tissues and adjacent tissues. The expression and prognostic significance of hsa_circ_0007919 were evaluated by qRT-PCR and Kaplan-Meier survival curves. Gain- and loss-of-function assays were conducted to detect the role of hsa_circ_0007919 in PC metastasis in vitro. A lung metastasis model and IHC experiments were conducted to confirm the effects of hsa_circ_0007919 on tumor metastasis in vivo. Mechanistically, RNA immunoprecipitation and chromatin immunoprecipitation assays were conducted to explore the interplay among hsa_circ_0007919, Sp1, and the THBS1 promoter. hsa_circ_0007919 was significantly upregulated in PC tissues and cells and was correlated with lymph node metastasis, TNM stage, and poor prognosis. Knockdown of hsa_circ_0007919 significantly suppressed the migration and invasion of PC cells in vitro and inhibited tumor metastasis in vivo. However, overexpression of hsa_circ_0007919 exerted the opposite effects. Mechanistically, hsa_circ_0007919 could recruit the transcription factor Sp1 to inhibit THBS1 transcription, thereby facilitating PC metastasis. hsa_circ_0007919 can promote the metastasis of PC by inhibiting THBS1 expression. hsa_circ_0007919 may be a potential therapeutic target in PC.

Role and action mechanisms of miR-149 and miR-31 in regulating function of pig cumulus cells and oocytes.

Understanding the mechanisms for oocyte maturation and optimizing the protocols for in vitro maturation (IVM) are greatly important for improving developmental potential of IVM oocytes. The miRNAs expressed in cumulus cells (CCs) play important roles in oocyte maturation and may be used as markers for selection of competent oocytes/embryos. Although a recent study from our group identified several new CCs-expressed miRNAs that regulate cumulus expansion (CE) and CC apoptosis (CCA) in mouse oocytes, validation of these findings and further investigation of mechanisms of action in other model species was essential before wider applications. By using both in vitro and in vivo pig oocyte models with significant differences in CE, CCA and developmental potential, the present study validated that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes. We demonstrated that miR-149 and miR-31 targeted SMAD family member 6 (SMAD6) and transforming growth factor ß2 (TGFB2), respectively, in the transforming growth factor-ß (TGF-ß) signaling. Furthermore, both miR-149 and miR-31 increased CE and decreased CCA via activating SMAD family member 2 (SMAD2) and increasing the expression of SMAD2 and SMAD family member 4. In conclusion, the present results show that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes by activating the TGF-ß signaling, suggesting that they might be used as markers for pig oocyte quality.

A slow feature analysis approach for the optimization of collective variables.

Molecular dynamics simulations have become increasingly important in understanding the microscopic mechanisms of various molecular systems. However, the high energy barriers in complicated molecules often make it difficult to observe events of interest within a reasonable timescale. To address this issue, researchers have developed a variety of enhanced sampling methods to explore configuration space by adding bias potentials along the slowly changing collective variables (CVs). In this study, we have developed a new tool that combines slow feature analysis and biasing-enhanced sampling methods to identify effective CVs and enhance the sampling efficiency of configuration space. We have demonstrated the effectiveness of this tool through three general examples.

A ratiometric fluorescent probe with a large Stokes shift for the detection of Hg2+ and its applications in environmental sample and living system analysis.

Toxic mercury ions (Hg2+) can cause serious environmental pollution and accumulate in living organisms via the food chain. Therefore, monitoring Hg2+ is crucial in ensuring the safety of ecosystems and organisms. In this work, a novel ratiometric fluorescent probe CMT (5-(4-(diphenylamino)phenyl)-1-(7-hydroxy-coumarin-3-yl)-4-pentene-1,3-dione) based on coumarin was developed for detecting Hg2+, which displayed obvious fluorescence changes, a low detection limit (2.24 × 10-7 M), good selectivity, and a large Stokes shift (255 nm). The CMT probe could detect Hg2+ in real environmental soil and water samples. Furthermore, the CMT probe enabled the naked-eye detection of Hg2+ using test paper experiments. CMT was also applied for fluorescence imaging in living zebrafish and plants. This work provides a highly efficient tool for monitoring Hg2+ in environmental samples and biological systems.

CA19­9 is a significant prognostic factor in stage III gastric cancer patients undergoing radical gastrectomy.

BACKGROUND: Due to the great heterogeneity of gastric cancer (GC), the prognosis of patients within a stage is very different. Therefore, it is necessary to identify the high risk factors for postoperative recurrence and metastasis and take appropriate therapeutic strategies to improve the prognosis of patients. In this study, we aimed to explore the prognostic significance of preoperative and postoperative serum carcinoembryonic antigen (CEA), carbohydrate antigen 19 - 9 (CA19-9) and carbohydrate antigen 72 - 4 (CA72-4) in patients with stage I, II and III GC who underwent radical gastrectomy. METHODS: A total of 580 patients who underwent curative surgical resection and had not received neoadjuvant chemotherapy were included in this study. The relationship between clinicopathological features and recurrence was analysed. Survival analysis was performed by Kaplan-Meier curve. Univariate and multivariate Cox regression analyses were performed to determine prognostic factors in GC patients. RESULTS: Among patients with stage III GC, the recurrence free survival (RFS) and overall survival (OS) of patients with CA19-9>35 U/mL were significantly lower than those with CA19-9 ≤ 35 U/mL; CA19-9 was always a significant independent marker. CEA and CA72-4 were sometime useful to predict RFS or OS alternatively in the pre- or postoperative period. The only other independent significant factors for prognosis in our study were lymph node metastases for RFS and postoperative adjuvant chemotherapy for OS. CONCLUSION: Preoperative and postoperative CA19-9 values are independent risk factors for predicting prognosis in stage III GC after curative gastrectomy.

Encephalitis is an Important Phenotype of Myelin Oligodendrocyte Glycoprotein Antibody-Associated Diseases: A Single-Center Cohort Study.

BACKGROUND: Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is considered a demyelinating disease of the central nervous system, but an increasing number of encephalitis cases associated with MOG antibodies have been reported recently. METHODS: This was a single-center, retrospective study. All data for pediatric patients with MOGAD diagnosed at Beijing Children's Hospital from January 2017 to January 2022 were collected. Clinical characteristics and outcomes were analyzed, and treatment responses were compared between the rituximab (RTX) and mycophenolate mofetil (MMF) groups. RESULTS: A total of 190 patients (age range: 5 months to 16 years; median age: 7.2 years; females: 97) were included in this study. The phenotypes of the first attack included acquired demyelinating syndromes (105 [55%]), encephalitis other than acute disseminated encephalomyelitis (82 [43%]), and isolated meningitis (3 [2%]). After a median follow-up of 30.4 months (interquartile range: 14.8-43.7), 64 (34%) patients had relapses. Fifty-one of the 64 (80%) patients who had relapse received maintenance therapy, including MMF (41), RTX (11), maintenance intravenous immunoglobulin (two), and tocilizumab (two). The annualized relapse rates decreased significantly after treatment in both the RTX and MMF cohorts (P < 0.05); however, there were no significant differences between the two groups (P = 0.56). A total of 178 (94%) patients had complete (175 patients) or almost complete (three patients) recovery (modified Rankin scale [mRS] < 2), and 12 had moderate to severe deficits (mRS ≥ 2). CONCLUSIONS: The spectrum of pediatric MOGAD is broader than previously reported and includes demyelinating syndromes and encephalitis. Encephalitis is an important initial phenotype observed in pediatric patients with MOGAD.

Self-Assembly of Epitope-Tagged Proteins and Antibodies for Delivering Biologics to Antigen Presenting Cells.

Inspired by the immune system's own strategy for macrophage activation, we describe here a simple self-assembly strategy for generating artificial immune complexes. The built-in recognition domains in the antibody, viz. the Fab and Fc domains, are judiciously leveraged for cargo conjugation to generate the nanoassembly and macrophage targeting, respectively. A responsive linker is engineered into the nanoassembly for releasing the protein cargo inside the macrophages, while ensuring stability during delivery. The design principles are simple and versatile to be applicable to a range of biologics, from small protein toxins to large enzymes, with high loading capacity. This self-assembly platform has the potential for delivering biologics to immune cells with implications in immunotherapy.

hsa_circ_0007919 induces LIG1 transcription by binding to FOXA1/TET1 to enhance the DNA damage response and promote gemcitabine resistance in pancreatic ductal adenocarcinoma.

BACKGROUND: Circular RNAs (circRNAs) play important roles in the occurrence and development of cancer and chemoresistance. DNA damage repair contributes to the proliferation of cancer cells and resistance to chemotherapy-induced apoptosis. However, the role of circRNAs in the regulation of DNA damage repair needs clarification. METHODS: RNA sequencing analysis was applied to identify the differentially expressed circRNAs. qRT-PCR was conducted to confirm the expression of hsa_circ_0007919, and CCK-8, FCM, single-cell gel electrophoresis and IF assays were used to analyze the proliferation, apoptosis and gemcitabine (GEM) resistance of pancreatic ductal adenocarcinoma (PDAC) cells. Xenograft model and IHC experiments were conducted to confirm the effects of hsa_circ_0007919 on tumor growth and DNA damage in vivo. RNA sequencing and GSEA were applied to confirm the downstream genes and pathways of hsa_circ_0007919. FISH and nuclear-cytoplasmic RNA fractionation experiments were conducted to identify the cellular localization of hsa_circ_0007919. ChIRP, RIP, Co-IP, ChIP, MS-PCR and luciferase reporter assays were conducted to confirm the interaction among hsa_circ_0007919, FOXA1, TET1 and the LIG1 promoter. RESULTS: We identified a highly expressed circRNA, hsa_circ_0007919, in GEM-resistant PDAC tissues and cells. High expression of hsa_circ_0007919 correlates with poor overall survival (OS) and disease-free survival (DFS) of PDAC patients. Hsa_circ_0007919 inhibits the DNA damage, accumulation of DNA breaks and apoptosis induced by GEM in a LIG1-dependent manner to maintain cell survival. Mechanistically, hsa_circ_0007919 recruits FOXA1 and TET1 to decrease the methylation of the LIG1 promoter and increase its transcription, further promoting base excision repair, mismatch repair and nucleotide excision repair. At last, we found that GEM enhanced the binding of QKI to the introns of hsa_circ_0007919 pre-mRNA and the splicing and circularization of this pre-mRNA to generate hsa_circ_0007919. CONCLUSIONS: Hsa_circ_0007919 promotes GEM resistance by enhancing DNA damage repair in a LIG1-dependent manner to maintain cell survival. Targeting hsa_circ_0007919 and DNA damage repair pathways could be a therapeutic strategy for PDAC.

Size Ranges of Effective Nucleation Cavities on Gas-Evolving Surfaces.

Bubble nucleation has a significant influence on mass transfer and energy conversion in electrochemical gas-evolving reactions. In this work, we establish a theoretical model for bubble nucleation from gas cavities on gas-evolving surfaces. Based on analyses of transient gas diffusion within the concentration boundary layer and supersaturation equation for stable bubble nuclei, we determined the size ranges of effective nucleation cavities on gas-evolving surfaces under different levels of supersaturation conditions. In addition, a criterion for the incipience of bubble nucleation on gas-evolving surfaces is proposed. We investigate the effects of the contact angle, cone angle, concentration boundary layer thickness, ambient pressure, and temperature on the size ranges of effective nucleation cavities, respectively. We demonstrate that a larger contact angle or a smaller cone angle can broaden the size range of effective cavities, thereby promoting bubble nucleation from cavities. We also show that increasing the concentration boundary layer thickness causes larger cavities to become effective nucleation sites, which significantly expands the size range of effective cavities. In contrast, increasing the ambient pressure enables smaller cavities to become effective nucleation sites, resulting in an expansion in the size range of effective cavities. Results of this work will contribute to the manipulation of bubble nucleation densities and the optimal design of gas-evolving electrodes in various electrochemical gas-evolving reactions.

Aldolase A promotes cervical cancer cell radioresistance by regulating the glycolysis and DNA damage after irradiation.

Radioresistance is the major obstacle that affects the efficacy of radiotherapy which is an important treatment for cervical cancer. By analyzing the databases, we found that aldolase A (ALDOA), which is a key enzyme in metabolic reprogramming, has a higher expression in cervical cancer patients and is associated with poor prognosis. We detected the expression of ALDOA in the constructed cervical cancer radioresistance (RR) cells by repetitive irradiation and found that it was upregulated compared to the control cells. Functional assays were conducted and the results showed that the knockdown of ALDOA in cervical cancer RR cells inhibited the proliferation, migration, and clonogenic abilities by regulating the cell glycolysis. In addition, downregulation of ALDOA enhanced radiation-induced apoptosis and DNA damage by causing G2/M phase arrest and further promoted radiosensitivity of cervical cancer cells. The functions of ALDOA in regulating tumor radiosensitivity were also verified by the mouse tumor transplantation model in vivo. Therefore, our study provides new insights into the functions of ALDOA in regulating the efficacy of radiotherapy and indicates that ALDOA might be a promising target for enhancing radiosensitivity in treating cervical cancer patients.

Thalidomide for Recurrent Bleeding Due to Small-Intestinal Angiodysplasia.

BACKGROUND: Recurrent bleeding from the small intestine accounts for 5 to 10% of cases of gastrointestinal bleeding and remains a therapeutic challenge. Thalidomide has been evaluated for the treatment of recurrent bleeding due to small-intestinal angiodysplasia (SIA), but confirmatory trials are lacking. METHODS: We conducted a multicenter, double-blind, randomized, placebo-controlled trial to investigate the efficacy and safety of thalidomide for the treatment of recurrent bleeding due to SIA. Eligible patients with recurrent bleeding (at least four episodes of bleeding during the previous year) due to SIA were randomly assigned to receive thalidomide at an oral daily dose of 100 mg or 50 mg or placebo for 4 months. Patients were followed for at least 1 year after the end of the 4-month treatment period. The primary end point was effective response, which was defined as a reduction of at least 50% in the number of bleeding episodes that occurred during the year after the end of thalidomide treatment as compared with the number that occurred during the year before treatment. Key secondary end points were cessation of bleeding without rebleeding, blood transfusion, hospitalization because of bleeding, duration of bleeding, and hemoglobin levels. RESULTS: Overall, 150 patients underwent randomization: 51 to the 100-mg thalidomide group, 49 to the 50-mg thalidomide group, and 50 to the placebo group. The percentages of patients with an effective response in the 100-mg thalidomide group, 50-mg thalidomide group, and placebo group were 68.6%, 51.0%, and 16.0%, respectively (P<0.001 for simultaneous comparison across the three groups). The results of the analyses of the secondary end points supported those of the primary end point. Adverse events were more common in the thalidomide groups than in the placebo group overall; specific events included constipation, somnolence, limb numbness, peripheral edema, dizziness, and elevated liver-enzyme levels. CONCLUSIONS: In this placebo-controlled trial, treatment with thalidomide resulted in a reduction in bleeding in patients with recurrent bleeding due to SIA. (Funded by the National Natural Science Foundation of China and the Shanghai Municipal Education Commission, Gaofeng Clinical Medicine; ClinicalTrials.gov number, NCT02707484.).