1.Effect and Mechanism of Icariin on Improving Spermatogenesis in Exercise-induced Fatigue Model Mice Through Regucalcin
Kunyang TANG ; Min XIAO ; Xiaocui JIANG ; Xiaoxue TAO ; Yue ZOU ; Chunchun ZHAO ; Zhipeng FANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(8):117-127
ObjectiveThis paper aims to investigate the effects of icariin on spermatogenesis in mice with exercise-induced fatigue and explore the underlying mechanisms. MethodsICR male mice were screened by swimming and randomly divided into normal group, model group, vitamin C group, icariin groups with low, medium, and high doses, and medium-dose icariin+N-nitro-L-arginine methyl ester (L-NAME) group, with 10 mice per group. Except for the normal group, all the other groups underwent weighted swimming training to establish an exercise-induced fatigue model. No gavage was administered during the first two weeks of the weighted training. From week three to four, the icariin groups with low, medium, and high doses received 0.03, 0.06, and 0.12 g·kg-1 icariin via gavage, respectively. The vitamin C group received 0.2 g·kg-1 vitamin C. The L-NAME group received 0.06 g·kg-1 icariin and 0.01 g·kg-1 L-NAME via intraperitoneal injection. The normal and model groups received equivalent physiological saline. After the experiment, body weight and the last exhaustive swimming time were recorded. Blood urea nitrogen (BUN), lactate (LA), lactate dehydrogenase (LDH), malondialdehyde (MDA), testicular testosterone (T), testicular Ca2+/Mg2+-adenosine triphosphatase (ATPase) (micro-assay), and the levels of testicular cyclic guanosine monophosphate (cGMP) were measured by using kits. Sperm CD46 levels were detected by flow cytometry. Testicular seminiferous tubules were observed via hematoxylin-eosin (HE) staining, and the testicular morphometric score (TMS) was used to evaluate the spermatogenic function. Protein expression of regucalcin (RGN, SMP30), cGMP-dependent protein kinase 1 (PKG), and cGMP-dependent protein kinase anchoring protein (GKAP1) was detected by Western blot. Testicular regucalcin expression was examined by immunofluorescence (IF). The epididymal sperm quality of mice was observed under a microscope. Fluorescence-stained sections of stimulated by retinoic acid gene 8 (STRA8), synaptonemal complex protein 3 (SCP3), and transition protein 1(TNP1) in testicular seminiferous tubules were assessed by immunohistochemistry (IHC). ResultsCompared with the normal group, the model group showed decreased body weight and exhaustive swimming time (P<0.01), significantly increased fatigue markers (LA, LDH, and BUN) and lipid peroxidation product MDA (P<0.01), reduced testicular RGN, PKG, GKAP1, testosterone, Ca2+/Mg2+-ATPase, and cGMP levels (P<0.01), decreased sperm motility, sperm count, and TMS scores, and downregulated the expression of STRA8, SCP3, and TNP1. Compared with the model group, the icariin group with high dose exhibited increased exhaustive swimming time (P<0.01), reduced LA, LDH, BUN, and MDA levels (P<0.01), elevated superoxide dismutase (SOD) (P<0.01), upregulated testicular RGN, PKG, GKAP1, testosterone, Ca2+/Mg2+-ATPase, and cGMP levels (P<0.01), improved sperm motility, sperm count, and TMS scores, and enhanced STRA8, SCP3, and TNP1 expression. Compared with the L-NAME group, the icariin group with medium dose showed increased expression of STRA8, SCP3, and TNP1 in the testicular tissue (P<0.01) and elevated cGMP and GKAP1 levels (P<0.01). ConclusionExercise-induced fatigue reduces the expression of RGN and cGMP/PKG/GKAP1 in mice, thereby causing abnormal spermatogenesis and impairing reproductive function in mice. Icariin ameliorates spermatogenic dysfunction in exercise-induced fatigue mice by promoting the expression of RGN and cGMP/PKG/GKAP1, thereby mitigating the damage of exercise-induced fatigue to the reproductive system.
2.Artificial intelligence-based quality control of hand hygiene for hospital-acquired infection
Xuchen YANG ; Jingwen LI ; Wan ZHANG ; Shasha FENG ; Min ZENG ; Jianan SHI ; Youqiong CHEN ; Tao ZHENG ; Xun YAO
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2026;33(02):241-247
Objective To explore an artificial intelligence (AI)-based method for automated hand hygiene monitoring and to compare the effectiveness of three algorithms (UniFormerV2, TDN, C3D) in recognizing hand hygiene steps in surgical settings, thereby aiding hospital infection control. Methods From April to October 2024, we non-invasively collected 641 video recordings of healthcare staff performing hand hygiene at four-bay scrub sinks in two tertiary hospitals using overhead HD cameras. The dataset was annotated by five trained experts for model training and validation. Results Following training on 385 samples, internal validation (n=119) showed the C3D model achieved 81% accuracy, 87% recall, and an 83% F1-score. The TDN model achieved 93%, 91%, and 92% for the same metrics. The UniFormerV2 model outperformed both, with an accuracy, recall, and F1-score of 93%—an improvement of over 10 percentage points compared to traditional CNNs (TDN, C3D). It also achieved an 84% accuracy in external validation, demonstrating strong generalization. Conclusion The UniFormerV2 model is more accurate than CNN-based models for hand hygiene step recognition and shows robust performance in external validation. It presents a viable tool for healthcare facilities to enhance hand hygiene management, ultimately improving medical quality and patient safety.
3.Epidemiological investigation of a pertussis outbreak in a kindergarten in Guangzhou
WANG Min, WU Jueyu, ZHU Zhijie, CAI Wenfeng, HE Peng, XIAO Jiali
Chinese Journal of School Health 2026;47(2):283-286
Objective:
To understand the epidemiological characteristics of a pertussis outbreak in Guangzhou, so as to provide references for outbreak response and prevention strategies.
Methods:
From April 5 to June 9, 2024, case screening was conducted among 246 preschool children, 35 staff members, and one full time school nurse in a kindergarten in Guangzhou based on case definition. Field epidemiological investigation methods were employed to collect relevant information, and screening samples were collected from individuals involved in the outbreak. The clinical manifestations, epidemiological characteristics, and risk factors for transmission of the outbreak were analyzed, with rate comparisons performed using the χ 2 test.
Results:
There were a total of 15 confirmed cases of pertussis in the kindergarten. The main clinical manifestations included intermittent cough in 14 cases ( 93.33 %), sputum production in 5 cases (33.33%), fever in 2 cases (13.33%), paroxysmal spasmodic cough in 1 case (6.67%), and vomiting in 1 case (6.67%). There was no statistically significant difference in the reporting rates of interrupted cough symptoms between pertussis cases (93.33%) and non pertussis cases (92.86%)( χ 2=3.74, P >0.05). The cases were aged 4-5 years, including 5 males and 10 females. The interval between symptom onset and diagnosis ranged from 2 to 25 days, with a median of 10 days. The outbreak involved two classes, with attack rates of 48.28% and 3.45%, respectively. Laboratory testing confirmed 14 close contacts positive for Bordetella pertussisnucleic acid. Among close contacts, only one received prophylactic medication as required.
Conclusion
The outbreak is a pertussis outbreak in a kindergarten caused by Bordetella pertussis infection, demonstrating distinct temporal and spatial clustering characteristics.
4.Preventive and therapeutic effect of low-dose corticosteroids on early acute lung injury after thoracoscopic lobectomy
Liqiang XU ; Shaoqiu LI ; Qiang LIU ; Min ZENG ; Weimin LUO
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2026;33(03):390-396
Objective To investigate the efficacy of early, short-term, low-dose corticosteroid administration for the prevention and treatment of early acute lung injury (EALI) in patients undergoing thoracoscopic lobectomy. Methods A retrospective analysis was conducted on the clinical data of patients who underwent thoracoscopic lobectomy at the Department of Thoracic and Cardiovascular Surgery, Taihe Hospital, Hubei University of Medicine, from January 2019 to January 2022. Patients were divided into an early steroid therapy group and an observation group based on whether they received corticosteroids in the early postoperative period. In the early steroid therapy group, in addition to standard postoperative care, patients received a low-dose intravenous push of methylprednisolone (80-120 mg/d) for 3 consecutive days. In the observation group, patients received standard postoperative care without intravenous corticosteroids for the first 3 days. Chest plain CT scans were performed on postoperative day (POD) 1 and POD 3 or 4 to evaluate lung injury. CT scores and the incidence of postoperative EALI were recorded. Results A total of 521 patients were included (268 males, 253 females; age range: 11-80 years). There were 318 patients in the observation group and 203 in the early steroid therapy group. On POD 1, the incidence of EALI was 16.0% in the observation group and 13.8% in the early steroid therapy group, with no statistical difference (P=0.486). Correspondingly, there was no statistical difference in chest CT scores among EALI-positive patients between the two groups (P=0.927). On POD 3-4, the incidence of EALI was significantly lower in the early steroid therapy group (22.7%) compared to the observation group (33.6%) (P=0.007). Although chest CT scores among EALI-positive patients were lower in the early steroid therapy group, the difference was not statistically significant (P=0.377). The overall incidence of EALI within the first 4 postoperative days was significantly lower in the early steroid therapy group (26.1%) than in the observation group (37.4%) (P=0.007). Radiological progression (defined as new-onset EALI or progression of existing EALI) occurred in 14.8% of the early steroid therapy group, significantly lower than the 28.9% in the observation group (P<0.001). The early steroid therapy group had a shorter postoperative length of stay (P<0.001), while there was no statistical difference in the incidence of poor wound healing between the groups (P=0.762). Conclusion Early postoperative corticosteroid use effectively reduces the incidence of EALI on POD 3-4, lowers the risk of radiological progression, and decreases the overall incidence of postoperative EALI. This is achieved without prolonging the length of stay or increasing the risk of poor wound healing. Therefore, early administration of low-dose corticosteroids is beneficial in suppressing the occurrence and progression of EALI. Its early use is recommended for patients at high risk for postoperative EALI.
5.Risk prediction models for delirium after adult cardiac surgery: A systematic review and meta-analysis
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2026;33(03):444-453
Objective To systematically evaluate the risk prediction models for postoperative delirium in adults with cardiac surgery. Methods The SinoMed, CNKI, Wanfang, VIP, PubMed, EMbase, Web of Science, and Cochrane Library databases were searched to collect studies on risk prediction models for postoperative delirium in cardiac surgery published up to January 29, 2025. Two researchers screened the literature according to inclusion and exclusion criteria, used the PROBAST bias tool to assess the quality of the literature, and conducted a meta-analysis of common predictors in the model using Stata 17.0 software. Results A total of 21 articles were included, establishing 45 models with 28733 patients. Age, cardiopulmonary bypass time, history of diabetes, history of cerebrovascular disease, and gender were the top five common predictors. The area under the curve (AUC) of the 45 models ranged from 0.544 to 0.98. Fourteen out of the 21 studies had good applicability, while the applicability of the remaining seven was unclear; 20 studies had a high risk of bias. Meta-analysis showed that the incidence of postoperative delirium in adults with cardiac surgery was 18.6% [95%CI (15.7%, 21.6%)], and age [OR=1.045 (1.036, 1.054), P<0.001], history of cerebrovascular disease [OR=1.758 (1.459, 2.057), P<0.001], gender [OR=1.732 (1.430, 2.034), P<0.001], mini-mental state examination score [OR=3.930 (1.859, 8.309), P<0.001], and length of ICU stay [OR=5.586 (4.289, 6.883), P<0.001] were independent influencing factors for postoperative delirium after cardiac surgery. Conclusion The risk prediction models for postoperative delirium after cardiac surgery have good predictive performance, but there is a high overall risk of bias. In the future, large-sample, multicenter, high-quality prospective clinical studies should be conducted to construct the optimal risk prediction model for postoperative delirium in adults with cardiac surgery, aiming to identify and prevent the occurrence of postoperative delirium as early as possible.
6.Efficacy and safety of single-, two-, and three-port video-assisted thoracoscopic surgery for spontaneous pneumothorax: A systematic review and meta-analysis
Weirun MIN ; Wei CAO ; Haochi LI ; Jinlong ZHANG ; Xinchun DONG ; Xindong LUO ; Shengliang HE ; Yunjiu GOU
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2026;33(03):461-467
Objective To systematically evaluate the efficacy and safety of video-assisted thoracoscopic surgery (VATS) with different numbers of ports in the treatment of spontaneous pneumothorax. Methods We conducted a comprehensive search of CNKI, PubMed, The Cochrane Library, Web of Science, EMbase, Wanfang Data, and the Chinese Medical Journal Full-text Database for clinical controlled trials on VATS with different port numbers for spontaneous pneumothorax, from their inception to March 2023. Two researchers independently screened the literature and assessed its quality.The Newcastle-Ottawa Scale (NOS) was used to assess the methodological quality of cohort and case-control studies, and the Cochrane risk-of-bias tool was used to evaluate randomized controlled trials (RCT). Meta-analysis was performed using RevMan 5.4.1 software. Results A total of 107 studies were included, comprising 35 RCT, 2 cohort studies, and 70 case-control studies. All cohort and case-control studies included in the analysis had NOS scores≥7. The meta-analysis revealed that compared to two-port VATS (2P-VATS) and three-port VATS (3P-VATS), single-port thoracoscopic surgery (SPTS) was associated with less intraoperative blood loss (SMD=–1.58, 95%CI: –1.93 to –1.22, P<0.001; and SMD=–1.59, 95%CI: –2.03 to –1.14, P<0.001, respectively), shorter postoperative hospital stay (SMD=–1.05, 95%CI: –1.29 to –0.82, P<0.001; and SMD=–1.08, 95%CI: –1.39 to –0.77, P<0.001), shorter duration of postoperative chest tube drainage (SMD=–0.75, 95%CI: –1.00 to –0.50, P<0.001; and SMD=–1.23, 95%CI: –1.72 to –0.75, P<0.001), fewer postoperative complications (OR=0.34, 95%CI: 0.26 to 0.45, P<0.001; and OR=0.47, 95%CI: 0.33 to 0.68, P<0.001), and lower pain scores at 24, 48, and 72 hours after surgery (P<0.05). The operative time for SPTS was shorter than that for 2P-VATS (SMD=–0.53, 95%CI: –0.90 to –0.16, P=0.005) but showed no significant difference compared to 3P-VATS (P=0.21). When comparing 2P-VATS with 3P-VATS, 2P-VATS demonstrated less intraoperative blood loss (SMD=–1.02, 95%CI: –1.81 to –0.22, P=0.01), shorter postoperative hospital stay (SMD=–0.59, 95%CI: –1.11 to –0.06, P=0.03), shorter duration of chest tube drainage (SMD=–0.46, 95%CI: –0.85 to –0.08, P=0.02), fewer postoperative complications (OR=0.36, 95%CI: 0.22 to 0.59, P<0.001), and lower pain scores at 24, 48, and 72 hours after surgery (P≤0.05). Conclusion Both SPTS and 2P-VATS are effective and safe surgical options for spontaneous pneumothorax, deserving further promotion and application in clinical practice. However, due to limitations in the quantity and quality of the included studies, more large-sample, high-quality research is needed to validate these findings.
7.Relationship between gender and efficacy of immune checkpoint inhibitors for esophageal cancer patients: A systematic review and meta-analysis
Jinlong ZHANG ; Haochi LI ; Zhaohao LIN ; Wei CAO ; Weirun MIN ; Xindong LUO ; Xinchun DONG ; Yunjiu GOU
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2026;33(04):639-645
Objective To systematically evaluate the efficacy of immune checkpoint inhibitors (ICIs) in treating esophageal cancer patients of different genders. Methods Computer searches were conducted on PubMed, Cochrane Library, and Embase databases to collect randomized controlled trial (RCT) on ICIs treatment for esophageal cancer patients from the establishment of the databases to January 25, 2024. Two researchers independently screened the literature and extracted data according to the inclusion and exclusion criteria. The outcome indicators were overall survival (OS) and progression-free survival (PFS), and RevMan 5.4 software was used for meta-analysis. The modified Jadad scoring scale was used to evaluate the quality of the included literature. Results A total of 10 RCT involving 5364 esophageal cancer patients were included in this study, with 2684 patients in the trial group and 2680 patients in the control group. The Jadad scores of the included literature were all ≥6 points, indicating high-quality RCT. Meta-analysis results showed that female esophageal cancer patients receiving ICIs treatment [HR=0.72, 95%CI (0.59, 0.87), P<0.001] had a more significant median OS prolongation than male patients [HR=0.73, 95%CI (0.68, 0.78), P<0.001]; while male patients [HR=0.57, 95%CI (0.52, 0.64), P<0.001] had a more significant PFS prolongation than female patients [HR=0.72, 95%CI (0.55, 0.94), P=0.01]. Female patients treated with ICIs alone [HR=0.66, 95%CI (0.50, 0.87), P=0.003] had a more significant median OS prolongation than male patients [HR=0.79, 95%CI (0.72, 0.87), P<0.001]; while male patients receiving ICIs combined with chemotherapy [HR=0.67, 95%CI (0.61, 0.74), P<0.001] had a more significant median OS prolongation than female patients [HR=0.77, 95%CI (0.59, 1.01), P=0.06]. Conclusion Female patients receiving ICIs have a slight advantage in OS compared to male patients, while male patients have an advantage in PFS. Male patients receiving ICIs combined with chemotherapy have better survival benefits than female patients, while female patients using ICIs monotherapy have better survival benefits than male patients.
8.Mechanical Loading Improves Qi-Blood Nourishment in "Sinew Wei (痿)"via Mitochondrial Regulation
Xili CHANG ; Sipeng HUANG ; Wuquan SUN ; Mengni SHI ; Chengheng YOU ; Min FANG ; Qingguang ZHU
Journal of Traditional Chinese Medicine 2026;67(7):725-729
This study focuses on the core pathology of sinew wei (痿), which is mainly characterized by the fai-lure of qi and blood to nourish the sinews. A mechanical-biological response framework is constructed with mitochondria as a key component, explaining the modern interpretation of the disease location of sinew transmitting to qi and blood pathology. Mechanical loading, as a physical stress stimulus applied to the body, manifests primarily as passive loading formed by external forces such as massage, and active loading resulting from voluntary muscle contractions, such as dao yin (导引). Mechanical loading can regulate mitochondrial function through two pathways, mechanical signal transduction and metabolic demand-driven regulation. Skeletal muscle mitochondrial dysfunction is regarded as the core microscopic basis of qi imbalance in sinew wei, highlighting the intrinsic connection between qi and mitochondrial energy metabolism, as well as between blood and microcirculatory efficiency. Accordingly, distinct regulatory patterns of mechanical loading are identified. Wei associated with qi stagnation may correspond to mitochondrial network fragmentation and can be treated by regulating qi through passive loading, such as tuina, to restore mitochondrial dynamics. In contrast, wei caused by qi deficiency is attributed to insufficient mitochondrial biogenesis and may be treated by tonifying qi through active loading, such as dao yin, to promote mitochondrial biogenesis. This framework reveals the biological differences in mitochondrial regulation induced by distinct mechanical loading modalities and provides a microscopic mechanism-based explanation for the principle of "treating the same disease with different methods" in sinew wei.
9.Comparative analyses of the detection performance of five multiplex polymerase chain reaction nucleic acid detection kits for respiratory pathogens
Fang YUAN ; Lei BI ; Jiajing LIU ; Huanru WANG ; Jun FENG ; Yuan ZHUANG ; Min CHEN ; Zheng TENG
Shanghai Journal of Preventive Medicine 2026;38(2):165-169
ObjectiveTo evaluate the detection specificity for clinical samples and the detection capability for standard substances of five commercially available multiplex polymerase chain reaction (PCR) nucleic acid detection kits (hereinafter referred to as the kits) for respiratory pathogens, and to provide a reference for selecting appropriate detection kits for multi-pathogen nucleic acid testing of respiratory infections. MethodsA total of 60 respiratory pathogen-positive clinical samples with known redults were selected and tested using the five kits (labeled as A, B, C, D, and E). The detection rates and Kappa coefficients were calculated to evaluate the consistency between the results from these kits and those from single-pathogen PCR kits. According to the limit of detection (LOD) provided by the kits, standard substances of respiratory pathogens (including 12 types such as influenza virus, Mycoplasma pneumoniae, and Bordetella pertussis) were diluted to four concentrations (250, 500, 1 000, and 2 000 copies·mL⁻¹). All five kits were used for detection to evaluate their respective detection capabilities. ResultsCompared with the results from single-pathogen PCR kits, the five tested kits demonstrated good consistency (all Kappa >0.80). Among them, Kit A had the highest detection rate (100.00%), followed by Kits C and E (98.33%), and then Kits B and D (95.00%). All five kits showed a relatively low false negative rate (FNR) for samples with a cycle threshold (Ct) value ≤35 (≤2.38%). However, for samples with Ct values>35, the FNR increased accordingly(average FNR=6.67%, P=0.029). Kit C exhibited the highest detection sensitivity for the tested standard substances (average LOD: 458.33 copies·mL⁻¹), followed by Kit D, then Kits A/E, and finally Kit B. ConclusionThe five multiplex PCR kits showed good consistency with single-pathogen detection results, but each had its own performance emphasis. Kit A, with the highest detection rate and high throughput, is suitable for targeted viral screening. Kit B, covering the broadest pathogen spectrum (including fungi/bacteria), is suitable for comprehensive respiratory pathogen screening. Kits C, D and E, are applicable for rapid detection. It is important to note that the detection efficacy of all kits decreases for low viral load samples with Ct values >35. In practical application, selection should be based on specific screening objectives, throughput requirements, and sample types.
10.Therapeutic Effect and Mechanism of Solanum nigrum on Hepatic Fibrosis Induced by Carbon Tetrachloride in Rats
Min WU ; Zhenxiang AN ; Yuanli HE ; Weinong WEN ; Qiang SU ; Song HE
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):117-125
ObjectiveTo investigate the therapeutic effect and mechanism of Solanum nigrum on hepatic fibrosis induced by carbon tetrachloride (CCl4) in rats. MethodsSixty SD rats were randomly allocated into blank, model, low-, medium-, and high-dose (0.9, 1.8, 3.6 g·kg-1, respectively) S. nigrum, and silibinin capsules (18.9 mg·kg-1) groups. Except the blank group, the other groups were subjected to intraperitoneal injection of 40% CCl4 solution for the modeling of hepatic fibrosis. After 4 weeks of gavage, blood was collected from the abdominal aorta following intraperitoneal anesthesia. The rats were sacrificed, and the liver was separated. The pathological changes were observed by hematoxylin-eosin staining and Masson staining. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and liver fibrosis indexes [type Ⅲ procollagen (PCⅢ), type Ⅳ collagen (Col Ⅳ), laminin (LN), and hyaluronic acid (HA)] in the rat serum were determined. The mRNA and protein levels of B cell lymphoma-2 (Bcl-2)/Bcl-2-associated X protein (Bax)/cysteinyl aspartate-specific proteinase-3 (Caspase-3) pathway-related factors were determined by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) and Western blot, respectively. ResultsCompared with the blank group, the model group exhibited significant hepatocyte edema, infiltration of inflammatory cells, connective tissue proliferation, and collagen fiber deposition in the liver tissue. Compared with the model group, low-, medium-, and high-dose S. nigrum and silymarin capsules significantly improved the structure of liver cells and alleviated the edema, inflammatory cell infiltration, connective tissue proliferation, and collagen fiber deposition. Compared with those in the blank group, the serum levels of ALT, AST, PCⅢ, Col Ⅳ, LN, and HA were elevated in the model group (P<0.01). Compared with the model group, the serum levels of ALT, AST, PCⅢ, Col Ⅳ, LN, and HA were reduced in all the treatment groups (P<0.05). Real-time PCR and Western blot results showed that compared with the blank group, the model group had up-regulated mRNA and protein levels of Bcl-2 and down-regulated mRNA and protein levels of Bax and Caspase-3 (P<0.01). Compared with the model group, all the treatment groups showed down-regulated mRNA and protein levels of Bcl-2 and up-regulated mRNA and protein levels of Bax and Caspase-3 (P<0.05), with the high-dose S. nigrum group showing the best therapeutic effect. ConclusionS. nigrum modulates the progression of hepatic fibrosis in rats by regulating apoptosis through the Bcl-2/Bax/caspase-3 pathway.


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