1.Effect of Oral Sodium Butyrate on Skeletal Muscle Atrophy via The Gut-muscle Axis in Antibiotic-pretreated CT26 Tumor-bearing Mice and Its Mechanism
Shu-Ling ZHANG ; Jun-Wei WANG ; Shi-Liang HU ; Tu-Tu WANG ; Shun-Chang LI ; Jia FAN ; Jun-Zhi SUN
Progress in Biochemistry and Biophysics 2026;53(3):724-739
ObjectiveTo explore the effect of oral sodium butyrate on skeletal muscle atrophy in CT26 tumor mice through the gut microbiota-skeletal muscle axis and its potential mechanism. MethodsSixty SPF BALB/c male mice aged 8 weeks were randomly divided into a normal control group (NC, n=18) and a ABX-depleted group (ABX, n=42). The ABX mice were pretreated with a quadruple antibiotic cocktail via oral gavage (0.2 ml per administration, once daily, 6 d per week, for 2 weeks), whereas NC received an equal volume of sterile water. The quadruple antibiotic cocktail consisted of metronidazole (1 g/L), vancomycin (0.5 g/L), ampicillin (1 g/L), and gentamicin (1 g/L). Following successful pretreatment, six mice from each group were randomly selected for gut microbiota sequencing analysis and designated as the Abx group and the NC0 group, respectively. Theremaining mice in ABX were subcutaneously inoculated in the dorsum with 0.2 ml of CT26 cell suspension (at a cell density of 1×107/ml). Then these mice were randomly allocated into three subgroups: a control tumor bearing model group (0_NaB, n=12), a tumor-bearing model group receiving low-dose oral sodium butyrate (L_NaB, n=12), a tumor-bearing model group receiving high-dose oral sodium butyrate (H_NaB, n=12). And mice in NC were inoculated at the same site with 0.2 ml of normal saline. The administration dose for L_NaB was 0.3 g/(kg·d), that for H_NaB was 0.5 g/(kg·d), while NC and 0_NaB were given the same volume of normal saline (0.2ml per time, once daily, 6 d per week, for 4 weeks). The general condition of mice was monitored, and forelimb grip strength gastrocnemius muscle mass and its muscle fiber cross-sectional area were measured for each group. The structural changes in gut microbiota were assessed by 16S rRNA sequencing of cecal contents. Pathological alterations in the intestinal wall were examined via HE staining. Serum and gastrocnemius muscle levels of TNF‑α, IL-6, IL-1β, and LPS were quantified using ELISA. The protein expression of ZO-1 and occludin in the small intestine, as well as proteins associated with the TLR4/MyD88/NF-κB signaling pathway in the gastrocnemius muscle, were detected by Western blot analysis. Results(1) The alpha-diversity in Abx was significantly lower than that in NC0 (P<0.01), a significant decrease of the mass and muscle fiber cross-sectional area of the gastrocnemius (P<0.01), with the majority of gut microbiota being effectively depleted. (2) Compared with NC, the subcutaneous tumors of mice in 0_NaB were prominent, a significant increase of the mass and muscle fiber cross-sectional area of the gastrocnemius, accompanied by a significant decrease in body weight at the end of the 3th and 4th week (P<0.05), and a significant weakening of the forelimb grasping strength at the 5th and 6th week (P<0.01). Compared with 0_NaB, the tumor mass of mice in L_NaB and H_NaB showed a significant decreasing trend, and the grip strength of the forelimbs significantly increased at the 5th and 6th week (P<0.05, P<0.01). (3) Compared with 0_NaB, the Shannon and Observed species indices in α diversity of L_NaB and H_NaB were significantly increased (P<0.05). At the genus level, compared with 0_NaB, L_NaB exhibited a significant decrease in the relative abundance of Parasutterella (P< 0.01), while H_NaB showed significant reductions in the relative abundances of both Escherichia-Shigella and Parasutterella (P < 0.01). (4) Compared with 0_NaB, the small intestinal tissue structure in L_NaB and H_NaB was more intact, the infiltration of inflammatory cells was significantly reduced, and the capillaries were slightly dilated. The expression levels of ZO-1 and occludin proteins in L_NaB were significantly increased (P<0.01). (5) The LPS concentration in the gastrocnemius muscle and the protein expression levels of TLR4, MyD88, p-IκBα, and p-NF‑κB p65 in L_NaB and H_NaB were significantly lower than those in 0_NaB (P<0.05). The serum TNF‑α concentration in H_NaB and TNF-α concentration in the gastrocnemius muscle of the L_NaB and H_NaB were significantly lower than those in 0_NaB (P<0.05, P<0.01, P<0.01). ConclusionOral administration of NaB can improve gut microbiota α diversity, adjusting its composition, improving intestinal mucosal barrier function, reducing the LPS-induced pro-inflammatory response, and delaying skeletal muscle atrophy. The underlying mechanism may involve down regulation of TLR4/MyD88/NF-κB signaling in skeletal muscle.
2.Study on Spatial Distribution Characteristics and Influencing Factors of the Potential Suitable Habitats for Cremastrae Pseudobulbus Pleiones Pseudobulbus Based on Maximum Entropy Model Combined with Geographical Detector
Shaoyang XI ; Gonghan TU ; Huaqian GONG ; Fei CHEN ; Xudong GUO ; Ling JIN
Chinese Journal of Information on Traditional Chinese Medicine 2025;32(2):7-13
Objective To analyze the spatial distribution patterns of the potential suitable habitats for Cremastrae Pseudobulbus Pleiones Pseudobulbus,including Cremastra appendiculata(D.Don)Makino,Pleione bulbocodioides(Franch.)Rolfe and Pleione yunnanensis Rolfe under current climatic conditions,and the factors influencing the spatial differentiation of their habitats.Methods Based on the maximum entropy(MaxEnt)model,a species distribution model was established using the screened species distribution data and environmental variable data.The factor detection and interaction detection of geographical detector were applied to quantify the factors influencing the spatial differentiation of the suitable areas.By overlaying the suitable areas with land cover types,the distribution characteristics of forest land and arable land in the potential suitable areas were quantified.Results Under the current climatic scenario,environmental variables such as solar radiation intensity,precipitation and temperature were closely related to the spatial distribution of Cremastrae Pseudobulbus Pleiones Pseudobulbus.The potential suitable areas for Cremastra appendiculata(D.Don)Makino,Pleione bulbocodioides(Franch.)Rolfe and Pleione yunnanensis Rolfe were 2.20×106 km2,2.75×106 km2 and 7.22×105 km2,respectively.Considering land cover types,the actual possible suitable areas for Cremastra appendiculata(D.Don)Makino,Pleione bulbocodioides(Franch.)Rolfe and Pleione yunnanensis Rolfe were 1.86×106 km2,2.20×106 km2 and 5.77×105 km2,respectively.Among these,the area of forest land was 1.17×106 km2,1.34×106 km2 and 3.67×105 km2,respectively,and the area of arable land was 6.95×105 km2,8.64×105 km2 and 2.10×105 km2,respectively.Conclusion This study can provide a basis for the protection and sustainable utilization of wild resources of the original plant materials of Cremastrae Pseudobulbus Pleiones Pseudobulbus.
3.Study on Tongue Manifestations of Patients with Different Syndromes in Non-Small Cell Lung Cancer and Their Correlation with Laboratory Indicators
Jiayi LIU ; Liping TU ; Yulin SHI ; Yu WANG ; Ling XU ; Yun YANG ; Wen JIAO ; Changle ZHOU ; Jiatuo XU
Chinese Journal of Information on Traditional Chinese Medicine 2025;32(3):136-143
Objective To study the tongue manifestation of patients with different syndromes in non-small cell lung cancer(NSCLC)and the correlation between tongue characteristics of different syndromes and tumor markers and coagulation indicators.Methods Totally 497 patients with NSCLC were grouped according to syndrome differentiation,and the differences in tongue characteristics of different syndromes were compared.Bivariate correlation analysis was used to study the correlation between tongue characteristics and serum tumor markers and coagulation indicators in patients with NSCLC of different syndromes.Results Compared with healthy people of different syndromes,in TB-a,yin deficiency and phlegm-heat syndrome>healthy group>qi-yin deficiency syndrome>spleen deficiency and phlegm-dampness syndrome>lung stagnation and phlegm-stasis syndrome(P<0.001).In TB-L,healthy group>spleen deficiency and phlegm-dampness syndrome>qi-yin deficiency syndrome>lung stagnation and phlegm-stasis syndrome>yin deficiency and phlegm-heat syndrome(P<0.001).In TB-b,yin deficiency and phlegm-heat syndrome>qi-yin deficiency syndrome>spleen deficiency and phlegm-dampness syndrome>healthy group>lung stagnation and phlegm-stasis syndrome(P<0.001).Yin deficiency and phlegm-heat syndrome had the highest TB-a and the lowest Per-all.Spleen deficiency and phlegm-dampness syndrome had the highest TB-L and Per-all.Lung stagnation and phlegm-stasis syndrome had lower TB-b and TC-b than other groups,lower TB-a than the healthy group,and a high Per-all index(P<0.05).In terms of tumor markers,Per-all in spleen deficiency and phlegm-dampness syndrome was positively correlated with Ca199,Ca50 and Ca242(P<0.05).In terms of coagulation indicators,the tongue texture index of lung stagnation and phlegm-stasis syndrome had a high correlation with the coagulation indicator Fg(P<0.01).Conclusion Different TCM syndromes of NSCLC have their own typical tongue characteristics.Tongue manifestations of different syndromes are correlated with tumor markers and coagulation indicators,respectively,which can reflect changes in clinical status.
4.Spatial Distribution Characteristics and Influencing Factors of Suitable Habitats of Codonopsis pilosula(Franch.)Nannf.Based on Maximum Entropy Modeling and Geographical Detector
Shaoyang XI ; Gonghan TU ; Huaqian GONG ; Fei CHEN ; Xudong GUO ; Ling JIN
Chinese Journal of Information on Traditional Chinese Medicine 2025;32(5):1-6
Objective To investigate the spatial distribution pattern of the suitable habitats for Codonopsis pilosula(Franch.)Nannf.and the factors influencing the spatial differentiation of its habitat under current climatic conditions in China.Methods Based on maximum entropy(MaxEnt)model,a species distribution model was established with filtered species occurrence data and environmental variable data.The geographical detector was applied to quantify the factors influencing the spatial differentiation of the suitable areas,employing both factor detection and interaction detection functionalities.Additionally,an overlay analysis with land cover types was conducted to quantify the distribution characteristics of arable land and forest land within the potential suitable areas for Codonopsis pilosula(Franch.)Nannf..Results Based on the contribution rate of environmental factors and the Jackknife method,average solar radiation intensity in December,precipitation in July,and highest temperature in December were identified as key environmental factors affecting the suitability of Codonopsis pilosula(Franch.)Nannf.habitats,which were further validated by the geographical detector.The total suitable area for Codonopsis pilosula(Franch.)Nannf.in China under current climatic conditions was approximately 4.46×106 km2,with the areas of high,moderate,and low suitability being 1.08×106 km2,1.70×106 km2,and 1.68×106 km2,respectively.After overlaying with arable and forest lands,the potentially suitable total forest area for Codonopsis pilosula(Franch.)Nannf.was 1.55×106 km2,and the arable land area was 1.63×106 km2.Conclusion This study provides reference for the optimization of cultivation areas for Codonopsis pilosula(Franch.)Nannf.and the conservation of its wild resources.
5.Human tissue Organoids:A New Frontier of Antiviral Research in Traditional Chinese Medicine
Ling GAO ; Changshu QU ; Zhiwen XU ; Shibo LIU ; Ruoshi TU ; Yunzhong NIE
Journal of Nanjing University of Traditional Chinese Medicine 2025;41(11):1441-1451
Viruses,with high adaptability and immune evasion capabilities,are responsible for a wide spectrum of diseases from lo-calized infections to global pandemics,continuously posing threats to global public health.In recent years,traditional Chinese medi-cine(TCM)has demonstrated significant potential in antiviral therapy.With its multi-component,multi-target synergistic mechanism,TCM offers unique advantages in the intervention of viral diseases.However,progress in elucidating the active antiviral constituents of TCM and identifying their precise molecular targets remains limited,primarily due to the lack of humanized models that can faithfully recapitulate the natural course of viral infection.The emergence of organoid technology has introduced new opportunities for antiviral research in TCM.Human organoids,which recapitulate the three-dimensional architecture and physiological functions of human tis-sues,provide an advanced platform for modeling host-virus interactions in a human-relevant context.This article highlights the latest advances in applications of human tissue organoids,including lung,liver,and brain organoids,in the study of respiratory viruses(SARS-CoV-2,influenza virus,respiratory syncytial virus),hepatitis viruses(HBV,HCV,HEV),and neurotropic viruses(ZIKV,pri-ons,HCMV);illustrates how organoid models have been leveraged to evaluate both the efficacy and safety of TCM-based antiviral inter-ventions.Although human tissue organoids still face technical challenges in insufficient vascularization and incomplete immune micro-environment,they represent a powerful tool for dissecting the mechanism of TCM antiviral action and for guiding the development of personalized antiviral strategies.Looking ahead,human tissue organoid technology is expected to become key enabling platform for pro-moting the clinical transformation of TCM in the field of antiviral medicine.
6.Safe and effective concentration of ropivacaine in interscalene brachial plexus block for children aged 6 to 10 years:a sequential allocation study
Shangyingying LI ; Li YANG ; Fei YANG ; Yaqiong TIAN ; Ling LIU ; Wen GAO ; Yuan SHI ; Shengfen TU
Journal of Army Medical University 2025;47(21):2698-2705
Objective To determine the median effective concentration(EC50)of ropivacaine at the volume of 0.5 mL/kg for ultrasound-guided interscalene brachial plexus block in children aged 6 to 10 years.Methods A prospective dose-finding trial was conducted based a sequential Dixon up-and-down allocation.We recruited children aged 6 to 10 years who were scheduled for unilateral surgery on upper extremity regions in our hospital from April to December 2022.All of them were subjected to general intravenous anaesthesia combined with ultrasound-guided interscalene brachial plexus block.The ropivacaine volume for each patient was 0.5 mL/kg.The concentration of 0.2%for the first patient,subsequent concentrations were adjusted based on the block effect of previous patient,increase or decrease by 0.02%.The trial was stopped when there were 7 turning points.Isotonic regression and Bootstrap were used to calculate the values of EC50 and 95%effective concentration(EC95),along with their 95%confidence intervals(CI).General data,incidence of adverse events,and score of postoperative pain were recorded.Results A total of 26 children aged 6 to 10 years were included.The EC50 of ultrasound-guided interscalene brachial plexus block was determined to be 0.091%(95%CI:0.077%~0.105%),and the EC95 was estimated to be 0.117%(95%CI:0.110%~0.118%).Successful blockade was achieved in 16 cases(61.5%),while 10 cases(38.5%)failed.No statistical differences existed between successful and failed cases regarding sex,age,body weight,surgical site,surgical laterality,operative time,or anesthesia duration.None of the patients experienced adverse events such as pneumothorax,vascular injury,or Horner's syndrome,and the score of postoperative pain were all<6 by modified Children's Hospital of Eastern Ontario Pain Scale.Conclusion In children aged 6 to 10 years,the EC50 of ropivacaine is 0.091%at a volume of 0.5 mL/kg for ultrasound-guided interscalene brachial plexus block,and this low dose of regional anesthetic can reduce the risks such as systemic toxicity and direct neurotoxicity.
7.Clinical analysis of pediatric renal abscess
Jinshan SUN ; Chaoying CHEN ; Juan TU ; Haiyun GENG ; Huarong LI ; Ling WAN ; Yuchun YAN
Chinese Journal of Pediatrics 2025;63(5):524-528
Objective:To analyze the clinical characteristics of renal abscess in children and provide suggestions for early diagnosis and treatment.Methods:The clinical data including general information, laboratory data, imaging results, treatment and prognosis of 20 pediatric patients with renal abscess admitted to the Department of Nephrology, Capital Center for Children's Health Capital Medical University were analyzed retrospectively.Results:A total of 8 males and 12 females were enrolled. The age of onset was 3.0 (0.8, 9.0) years. All cases had fever. Six cases presented with abdominal pain, 6 cases had poor appetite, 5 cases had vomiting and 5 cases urinary tract irritation symptoms. Laboratory data showed elevated white blood cells 20.4 (17.4,26.3)×10 9/L, C-reactive protein 126 (77, 154)mg/L, erythrocyte sedimentation rate 60 (41,73) mm/1 h in 20 cases and procalcitonin 4.7 (1.2,33.5)μg/L in 10 cases. Totally 18 cases had pyuria. Urine culture was positive in 8 cases. Enterococcus faecium and Pseudomonas aeruginosa was detected in 2 cases. Klebsiella pneumoniae was found in 1 case after performing blood culture. Renal abscess was confirmed in all cases by doing contrast-enhanced CT scan, while only 9 cases with abscesses were identified by using renal ultrasound. There were 14 cases with renal abscess formation confirmed at onset by performing magnetic resonance imaging. Nine cases were accompanied with congenital anomalies of the kidney and urinary tract. All cases received conservative medical treatment. Intravenous broad-spectrum antibiotics were administered for 23 (14, 39) d initially, while the medication in 11 cases were upgraded to meropenem or imipenem. Oral antibiotics were continued for 23 (14, 28) d after discharge in all cases. Within 1 year of follow-up, except for 1 case of recurrence, the others had a favorable prognosis. Conclusions:Renal abscess should be suspected for children presenting with unexplained fever, vomiting, abdominal pain, elevated white blood cell count, C-reactive protein, erythrocyte sedimentation rate and pyuria. Ultrasonography is suitable for screening and follow-up, while CT or magnetic resonance imaging can be used to confirm the diagnosis. Conservative management with broad-spectrum antibiotics is effective and can be considered the first-line therapy for pediatric renal abscess.
8.Prediction Study on the Potential Suitable Habitats of Gastrodiae Rhizoma Based on MaxEnt Model and Geodetector
Shaoyang XI ; Huaqian GONG ; Gonghan TU ; Fei CHEN ; Xudong GUO ; Ling JIN
Chinese Journal of Information on Traditional Chinese Medicine 2025;32(4):1-6
Objective To analyze the spatial distribution pattern of potential suitable habitats and the influencing factors of habitat spatial differentiation of Gastrodiae Rhizoma under current climate conditions.Methods Based on 137 distribution records of Gastrodiae Rhizoma sourced from the Global Biodiversity Information Facility and 104 environmental variables acquired from the WorldClim dataset,a predictive model for the potential suitable habitat of Gastrodiae Rhizoma was developed using the MaxEnt model.The geodetector was applied to quantitatively analyze the environmental factors influencing the spatial heterogeneity of the suitable habitats.Subsequently,an overlay analysis with land cover types was conducted to obtain the distribution characteristics of forest land and cultivated land in the potential suitable habitats.Results Under current climate conditions,key environmental factors affecting the distribution of Gastrodiae Rhizoma included precipitation in July,average temperature in the coldest quarter,precipitation in the warmest quarter,water vapor pressure in September,altitude,and solar radiation in December.The potential geographical distribution range of Gastrodiae Rhizoma was 1.64×106 km2.Considering land cover types,the actual potential suitable area for Gastrodiae Rhizoma was 1.33×106 km2,of which the forest land area was 8.56×105 km2 and the cultivated land area was 4.74×105 km2.The highly suitable forest areas were mainly located within the provinces of Guizhou,Sichuan,Shaanxi,Hubei,Hunan and Gansu.The cultivated land suitable areas are mainly located within the provinces of Guizhou,Sichuan,Yunnan,Shaanxi and Hubei.Conclusion The highly suitable habitats of Gastrodiae Rhizoma are mainly located in provinces such as Guizhou,Sichuan,Shaanxi and Hubei,and are affected by factors such as solar radiation,water vapor pressure,temperature and precipitation.The research results can provide reference for the protection of wild Gastrodiae Rhizoma resources,artificial nurturing and optimal selection of ecological planting areas.
9.Diagnosis and treatment of special type of laryngeal foreign body with granuloma in children.
Dezhen TU ; Zongtong LIN ; Ling SHEN ; Deping CHEN ; Zhongjie YANG ; Qiaoyu LIAO
Journal of Clinical Otorhinolaryngology Head and Neck Surgery 2025;39(2):163-167
Objective:This article explores the diagnosis and treatment of a special type of laryngeal foreign body with granuloma in children, providing a clinical reference for standardized diagnosis and treatment. Methods:This article retrospectively analyzes one case admitted to our department and five other cases reported in relevant literature involving a special type of laryngeal foreign body with granuloma in children. It summarizes the clinical characteristics of this disease and explores its diagnosis and treatment. Results:①A total of 6 cases were misdiagnosed and had a long course of illness. Five cases had no clear history of foreign body at the first visit. One case had foreign body history but no foreign body was found. ②The clinical symptoms were diverse, which could be hoarseness, cough, wheezing, dyspnea, or no symptoms. ③A total of 3 cases were diagnosed by laryngoscopy, and 3 cases were diagnosed by laryngoscopy and imaging examination. ④Foreign bodies that were not wrapped in granulomas, were removed first and then the granulomas were treated; foreign bodies wrapped in granulomas were removed after the removal of granulomas. Conclusion:A special type of laryngeal foreign body with granuloma may lack of accurate foreign body history, resulting in long-term retention of foreign bodies, leading to granuloma formation and misdiagnosis. Electronic laryngoscopy combined with CT examination contribute to early diagnosis. Low temperature plasma under endoscope and anesthetic laryngoscope is conducive to the exposure and precise removal of foreign bodies and avoids tracheotomy to a certain extent.
Child
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Child, Preschool
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Female
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Humans
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Infant
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Male
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Foreign Bodies/surgery*
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Granuloma, Foreign-Body/therapy*
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Larynx/pathology*
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Retrospective Studies
10.Diabetic vascular calcification inhibited by soluble epoxide hydrolase gene deletion via regressing NID2-mediated IGF2-ERK1/2 signaling pathway.
Yueting CAI ; Shuiqing HU ; Jingrui LIU ; Jinlan LUO ; Wenhua LI ; Jiaxin TANG ; Siyang LIU ; Ruolan DONG ; Yan YANG ; Ling TU ; Xizhen XU
Chinese Medical Journal 2025;138(20):2657-2668
BACKGROUND:
Epoxyeicosatrienoic acids (EETs), which are metabolites of arachidonic acid catalyzed by cytochrome P450 epoxygenase, are degraded into inactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). Many studies have revealed that sEH gene deletion exerts protective effects against diabetes. Vascular calcification is a common complication of diabetes, but the potential effects of sEH on diabetic vascular calcification are still unknown.
METHODS:
The level of aortic calcification in wild-type and Ephx2-/- C57BL/6 diabetic mice induced with streptozotocin was evaluated by measuring the aortic calcium content through alizarin red staining, immunohistochemistry staining, and immunofluorescence staining. Mouse vascular smooth muscle cell lines (MOVAS cells) treated with β-glycerol phosphate (0.01 mol/L) plus advanced glycation end products (50 mg/L) were used to investigate the effects of sEH inhibitors or sEH knockdown and EETs on the calcification of vascular smooth muscle cells, which was detected by Western blotting, alizarin red staining, and Von Kossa staining.
RESULTS:
sEH gene deletion significantly inhibited diabetic vascular calcification by increasing levels of EETs in the aortas of mice. EETs (especially 11,12-EET and 14,15-EET) efficiently prevented the osteogenic transdifferentiation of MOVAS cells by decreasing nidogen-2 (NID2) expression. Interestingly, suppressing sEH activity by small interfering ribonucleic acid or specific inhibitors did not block osteogenic transdifferentiation of MOVAS cells induced by β-glycerol phosphate and advanced glycation end products. NID2 overexpression significantly abolished the inhibitory effect of sEH gene deletion on diabetic vascular calcification. Moreover, NID2 overexpression mediated by adeno-associated virus 9 vectors markedly increased insulin-like growth factor 2 (IGF2) and phospho-ERK1/2 expression in MOVAS cells. Overall, sEH gene knockout inhibited diabetic vascular calcification by decreasing aortic NID2 expression and, then, inactivating the downstream IGF2-ERK1/2 signaling pathway.
CONCLUSIONS
sEH gene deletion markedly inhibited diabetic vascular calcification through repressed osteogenic transdifferentiation of vascular smooth muscle cells mediated by increased aortic EET levels, which was associated with decreased NID2 expression and inactivation of the downstream IGF2-ERK1/2 signaling pathway.
Animals
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Mice
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Vascular Calcification/metabolism*
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Mice, Inbred C57BL
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Epoxide Hydrolases/metabolism*
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Diabetes Mellitus, Experimental/genetics*
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Male
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Gene Deletion
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MAP Kinase Signaling System/genetics*
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Cell Line
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Immunohistochemistry
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Muscle, Smooth, Vascular/metabolism*
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Signal Transduction/genetics*
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Mice, Knockout

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