1.Carbon-friendly ecological cultivation mode of Dendrobium huoshanense based on greenhouse gas emission measurement.
Di TIAN ; Jun-Wei YANG ; Bing-Rui CHEN ; Xiu-Lian CHI ; Yan-Yan HU ; Sheng-Nan TANG ; Guang YANG ; Meng CHENG ; Ya-Feng DAI ; Shi-Wen WANG
China Journal of Chinese Materia Medica 2025;50(1):93-101
Ecological cultivation is an important way for the sustainable production of traditional Chinese medicine in the context of the carbon peaking and carbon neutrality goals. Facility cultivation and simulative habitat cultivation modes have been developed and applied to develop the endangered Dendrobium huoshanense on the basis of protection. However, the differences in the greenhouse gas emissions and global warming potential of these cultivation modes remain unexplored, which limits the accurate assessment of carbon-friendly ecological cultivation modes of D. huoshanense. Greenhouse gas emission flux monitoring based on the static chamber method provides an effective way to solve this problem. Therefore, this study conducted a field experiment in the facility cultivation and simulative habitat cultivation modes at a D. huoshanense cultivation base in Dabie Mountains, Anhui Province. From April 2023 to March 2024, samples of greenhouse gases were collected every month, and the concentrations of CO_2, CH_4, and N_2O of the samples were then detected by gas chromatography. The greenhouse gas emission fluxes, cumulative emissions, and global warming potential were further calculated, and the following results were obtained.(1)The two cultivation modes of D. huoshanense showed significant differences in greenhouse gas emission fluxes, especially the CO_2 emission flux, with a pattern of facility cultivation>simulative habitat cultivation [(35.60±11.70)mg·m~(-2)·h~(-1) vs(2.10±4.59)mg·m~(-2)·h~(-1)].(2) The annual cumulative CO_2 emission flux in the case of facility cultivation was significantly higher than that of simulative habitat cultivation[(3 077.00±842.00)kg·hm~(-2) vs(221.00±332.00)kg·hm~(-2)], while no significant difference was found in annual cumulative CH_4 and N_2O emission fluxes.(3) The facility cultivation mode had a significantly higher global warming potential than the simulative habitat cultivation mode [(3 053.00±847.00)kg·hm~(-2) vs(196.00±362.00)kg·hm~(-2)]. Overall, the simulative habitat cultivation of D. huoshanense has obvious carbon-friendly characteristics compared with facility cultivation, which is in line with the concept of ecological cultivation of medicinal plants. This study is of great reference significance for the implementation and promotion of the ecological cultivation mode of D. huoshanense under carbon peaking and carbon neutrality goals.
Dendrobium/chemistry*
;
Greenhouse Gases/metabolism*
;
Carbon/analysis*
;
Ecosystem
;
Carbon Dioxide/metabolism*
;
China
;
Global Warming
2.Phase changes and quantity-quality transfer of raw material, calcined decoction pieces, and standard decoction of Ostreae Concha (Ostrea rivularis).
Hong-Yi ZHANG ; Jing-Wei ZHOU ; Jia-Wen LIU ; Wen-Bo FEI ; Shi-Ru HUANG ; Yu-Mei CHEN ; Chong-Yang LI ; Fei-Fei LI ; Qiao-Ling MA ; Fu WANG ; Yuan HU ; You-Ping LIU ; Shi-Lin CHEN ; Lin CHEN ; Hong-Ping CHEN
China Journal of Chinese Materia Medica 2025;50(5):1209-1223
The phase changes and quantity-quality transfer of 17 batches of Ostreae Concha(Ostrea rivularis) during the raw material-calcined decoction pieces-standard decoction process were analyzed. The content of calcium carbonate(CaCO_3), the main component, was determined by chemical titration, and the extract yield and transfer rate were calculated. The CaCO_3 content in the raw material, calcined decoction pieces, and standard decoction was 94.39%-98.80%, 95.03%-99.22%, and 84.58%-90.47%, respectively. The process of raw material to calcined decoction pieces showed the yield range of 96.85% to 98.55% and the CaCO_3 transfer rate range of 96.92% to 99.27%. The process of calcined decoction pieces to standard decoction showed the extract yield range of 2.86% to 5.48% and the CaCO_3 transfer rate range of 2.59% to 5.13%. The results of X-ray fluorescence(XRF) assay showed that the raw material, calcined decoction pieces, and standard decoction mainly contained Ca, Na, Mg, Si, Br, Cl, Al, Fe, Cr, Mn, and K. The chemometric results showed an increase in the relative content of Cr, Fe, and Si from raw material to calcined decoction pieces and an increase in the relative content of Mg, Al, Br, K, Cl, and Na from calcined decoction pieces to standard decoction. X-ray diffraction(XRD) was employed to establish XRD characteristic patterns of the raw material, calcined decoction pieces, and standard decoction. The XRD results showed that the main phase of all three was calcite, and no transformation of crystalline form or generation of new phase was observed. Fourier transform infrared spectroscopy(FTIR) was employed to establish the FTIR characteristic spectra of the raw material, calcined decoction pieces, and standard decoction. The FTIR results showed that the raw material had internal vibrations of O-H, C-H, C=O, C-O, and CO■ groups. Due to the loss of organic matter components after calcination, no information about the vibrations of C-H, C=O, and C-O groups was observed in the spectra of calcined decoction pieces and standard decoction. In summary, this study elucidated the quantity-quality transfer and phase changes in the raw material-calcined decoction pieces-standard decoction process by determining the CaCO_3 content, calculating the extract yield and transfer rate, and comparing the element changes, FTIR characteristic spectra, and XRD characteristic pattern. The results were reasonable and reliable, laying a foundation for the subsequent process research and quality control of the formula granules of calcined Ostreae Concha(O. rivularis Gould), and providing ideas and methods for the quality control of the whole process of raw material-decoction pieces-standard decoction-formula granules of Ostreae Concha and other testacean traditional Chinese medicine.
Drugs, Chinese Herbal/isolation & purification*
;
Calcium Carbonate/analysis*
;
Quality Control
3.Development of a Microstream End-Tidal Carbon Dioxide Monitoring System with Integrated Gas Circuit.
Yanan LIU ; Xuedong SONG ; Qi YIN ; Fuhao KANG ; Yan HANG ; Jilun YE ; Xu ZHANG
Chinese Journal of Medical Instrumentation 2025;49(2):204-211
End-tidal carbon dioxide monitoring is an important means of evaluating human lung function and is widely used in fields such as clinical emergency treatment and cardiopulmonary resuscitation. This paper develops a microstream end-tidal carbon dioxide monitoring system. It adopts an integrated gas circuit design to further reduce the size of the equipment. The system uses the method of calculating the root mean square (RMS) of differential pressure signals to regulate the gas circuit flow, enabling the system to stably operate at a flow state of 30 mL/min. In addition, by simultaneously detecting multiple environmental parameters such as temperature and pressure, the system realizes system state monitoring and gas parameter compensation. The test results show that various indicators of the system meet the requirements of relevant standards, laying a good foundation for subsequent engineering applications.
Carbon Dioxide/analysis*
;
Equipment Design
;
Monitoring, Physiologic/methods*
;
Humans
4.Effect of retinoic acid on delayed encephalopathy after acute carbon monoxide poisoning: Role of the lncRNA SNHG15/LINGO-1/BDNF/TrkB axis.
Fangling HUANG ; Su'e WANG ; Zhengrong PENG ; Xu HUANG ; Sufen BAI
Journal of Central South University(Medical Sciences) 2025;50(6):955-969
OBJECTIVES:
The neurotoxicity of carbon monoxide (CO) to the central nervous system is a key pathogenesis of delayed encephalopathy after acute carbon monoxide poisoning (DEACMP). Our previous study found that retinoic acid (RA) can suppress the neurotoxic effects of CO. This study further explores, in vivo and in vitro, the molecular mechanisms by which RA alleviates CO-induced central nervous system damage.
METHODS:
A cytotoxic model was established using the mouse hippocampal neuronal cell line HT22 and primary oligodendrocytes exposed to CO, and a DEACMP animal model was established in adult Kunming mice. Cell viability and apoptosis of hippocampal neurons and oligodendrocytes were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Annexin V/propidium iodide (PI) double staining. The transcriptional and protein expression of each gene was detected using real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. Long noncoding RNA (lncRNA) SNHG15 and LINGO-1 were knocked down or overexpressed to observe changes in neurons and oligodendrocytes. In DEACMP mice, SNHG15 or LINGO-1 were knocked down to assess changes in central nervous tissue and downstream protein expression.
RESULTS:
RA at 10 and 20 μmol/L significantly reversed CO-induced apoptosis of hippocampal neurons and oligodendrocytes, downregulation of SNHG15 and LINGO-1, and upregulation of brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB) (all P<0.05). Overexpression of SNHG15 or LINGO-1 weakened the protective effect of RA against CO-induced cytotoxicity (all P<0.05). Knockdown of SNHG15 or LINGO-1 alleviated CO-induced apoptosis of hippocampal neurons and oligodendrocytes and upregulated BDNF and TrkB expression levels (all P<0.05). Experiments in DEACMP model mice showed that knockdown of SNHG15 or LINGO-1 mitigated central nervous system injury in DEACMP (all P<0.05).
CONCLUSIONS
RA alleviates CO-induced apoptosis of hippocampal neurons and oligodendrocytes, thereby reducing central nervous system injury and exerting neuroprotective effects. LncRNA SNHG15 and LINGO-1 are key molecules mediating RA-induced inhibition of neuronal apoptosis and are associated with the BDNF/TrkB pathway. These findings provide a theoretical framework for optimizing the clinical treatment of DEACMP and lay an experimental foundation for elucidating its molecular mechanisms.
Animals
;
RNA, Long Noncoding/physiology*
;
Brain-Derived Neurotrophic Factor/genetics*
;
Carbon Monoxide Poisoning/complications*
;
Mice
;
Tretinoin/pharmacology*
;
Nerve Tissue Proteins/metabolism*
;
Membrane Proteins/metabolism*
;
Apoptosis/drug effects*
;
Hippocampus/cytology*
;
Receptor, trkB/metabolism*
;
Neurons/drug effects*
;
Male
;
Brain Diseases/etiology*
;
Oligodendroglia/drug effects*
;
Signal Transduction
;
Cell Line
5.Ferroptosis: a potential new therapeutic target for myocardial injury induced by acute carbon monoxide poisoning.
Anping LIU ; Xuheng JIANG ; Tianjing SUN ; Mo LI ; Haizhen DUAN ; Shuhong WANG ; Anyong YU
Chinese Critical Care Medicine 2025;37(4):407-412
Acute carbon monoxide poisoning (ACMP) is one of the most common gas poisonings in the emergency department, with tens of thousands of people seeking medical attention for carbon monoxide (CO) poisoning each year. The severity of poisoning is dependent upon environmental and human factors, with hypoxia and oxidative stress being important mechanisms of cardiac toxicity induced by CO. Myocardial involvement is common in moderate to severe ACMP, including myocardial injury, myocardial infarction, arrhythmia, and sudden death, which are associated with a high risk of death. Ferroptosis is a cell death mechanism caused by iron-dependent lipid peroxidation (LPO), although ferroptosis has been shown to play a critical role in various cardiovascular diseases, the potential mechanism by which it contributes to ACMP-induced myocardial injury is unclear. This review discusses the established link between ferroptosis and cardiovascular disease and summarizes the potential role of ferroptosis in ACMP-induced myocardial injury and the detrimental effects of ACMP on the heart. Elucidating these mechanisms could guide the development of novel therapeutic strategies that target ferroptosis to mitigate ACMP-induced myocardial injury. This review aims to provide a theoretical foundation for future research on the potential use of ferroptosis as a therapeutic target for ACMP-induced myocardial injury.
Humans
;
Carbon Monoxide Poisoning/complications*
;
Ferroptosis
;
Lipid Peroxidation
;
Myocardium/pathology*
;
Oxidative Stress
6.Optimization of promoter screening for heterologous expression of carbonic anhydrase and characterization of its enzymatic properties and carbon sequestration performance.
Dandan YAO ; Yunhui LI ; Xingjia FU ; Hui WANG ; Yun LIU
Chinese Journal of Biotechnology 2025;41(4):1588-1604
In this study, high-throughput promoter screening was employed to optimize the heterologous expression of Mesorhizobium loti carbonic anhydrase (MlCA) in order to reduce the costs associated with carbon capture and storage (CCS). To simplify the complexity of traditional vectors, a fusion protein expression system was constructed using superfolder green fluorescent protein (sfGFP) and MlCA. The synthetic promoter library in Escherichia coli was utilized for efficient one-step screening. Based on fluorescence intensity on agar plates, a total of 143 monoclonal colonies were identified, forming a library with varying expression levels. The top four recombinants with the highest fluorescence intensity were selected, among which MlCA driven by the promoter 342042/+ exhibited the highest enzymatic activity, with a specific activity of the 34.6 Wilbur-Anderson units (WAU)/mg. Optimization experiments revealed that MlCA exhibited the best performance when cultured for 4 days under pH 7.0 and 40 ℃ conditions. The Michaelis constant (Km·hdy) and maximum reaction rate (Vmax·hdy) for CO2 hydration were determined to be 62.46 mmol/L and 0.164 mmol/(s·L), respectively. For esterase hydrolysis, MlCA showed the Km and Vmax of 639.8 mmol/L and 0.035 mmol/(s·L), respectively. MlCA accelerated the CO2 hydration process, promoting CO2 mineralized into CaCO3 within 9 min at low pH and room temperature conditions. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses confirmed that the precipitated product was calcite. This study provides a low-cost and environmentally friendly alternative for future CCS applications.
Carbonic Anhydrases/biosynthesis*
;
Promoter Regions, Genetic/genetics*
;
Escherichia coli/metabolism*
;
Carbon Sequestration
;
Carbon Dioxide/metabolism*
;
Green Fluorescent Proteins/metabolism*
7.Preparation of decellularized bone graft material with supercritical carbon dioxide extraction technique.
Feng HAO ; Kaifeng PAN ; Liuyun HUANG ; Xuhong CHEN ; Haikun WEI ; Xianhua CHEN ; Jianfeng ZHANG
Journal of Zhejiang University. Medical sciences 2024;53(6):772-778
OBJECTIVES:
To evaluate the immunogenicity and osteogenic ability of animal-derived bone graft material decellularized with supercritical carbon dioxide.
METHODS:
Porcine femurs were randomly divided into two groups after preliminary treatment, and decellularized with conventional method (control group) or supercritical carbon dioxide (experimental group). Allogenic demineralized bone matrix was used as positive control. Clearance rate of galactose-α-1, 3-galactose (α-Gal) antigen was determined by enzyme-linked immunosorbent assay and residual DNA was detected by a fluorescence method. Nine SPF-grade male athymic nude mice of 6 weeks old were randomly divided into experimental, control and positive control groups. Samples were implanted over biceps femoris muscle of athymic nude mice. The explants were collected 4 weeks post implantation. Hematoxylin and eosin (HE) staining and immunohistochemistry were applied to determine the osteogenic ability and bone tissue-associated protein expressions of the implants.
RESULTS:
The clearance rates of α-Gal antigen in the experimental group and the control group were (99.09±0.26)% and (30.18±2.02)%, respectively (t=58.67, P<0.01). The residual DNA of the experimental, control and positive control groups were (13.49±0.07), (15.20±0.21) and (14.70±0.17) ng/mg. The residual DNA in the experimental group was significantly lower than that in the control group (t=-13.41, P<0.01) and positive control group (t=-11.30, P<0.01). HE staining results showed that multiple bone formation centers with active osteogenesis and rich bone marrow were observed in experimental group 4 weeks after implantation, but only a small number of bone formation centers were observed in the control and positive control groups, with no obvious osteoblasts present. Immunohistochemistry results indicated that the expressions of alkaline phosphatase, Runt-related transcription factor 2, collagen typeⅠand osteocalcin in the experimental group showed an increasing trend compared with those in the control and positive control groups.
CONCLUSIONS
Compared with clinically used allogenic demineralized bone matrix and bone graft material decellularized with conventional method, bone graft material decellularized with supercritical carbon dioxide exhibits lower immunogenicity and better osteogenic ability.
Animals
;
Mice
;
Swine
;
Male
;
Bone Transplantation/methods*
;
Mice, Nude
;
Carbon Dioxide
;
Osteogenesis/drug effects*
;
Femur
;
Bone Substitutes
;
Tissue Engineering/methods*
8.Effect of extra corporeal reducing pre-load on pulmonary mechanical power in patients with acute respiratory distress syndrome.
Wenwen ZHANG ; Xin'gang HU ; Lixia YUE ; Jie ZHANG ; Zhida LIU ; Shuai GAO ; Zhigang ZHAO ; Xinliang LIANG
Chinese Critical Care Medicine 2024;36(12):1244-1248
OBJECTIVE:
To explore the effects of veno-venous extra corporeal carbon dioxide removal (V-V ECCO2R) on local mechanical power and gas distribution in the lungs of patients with mild to moderate acute respiratory distress syndrome (ARDS) receiving non-invasive ventilation.
METHODS:
Retrospective research methods were conducted. Sixty patients with mild to moderate ARDS complicated with renal insufficiency who were transferred to the respiratory intensive care unit (RICU) through the 96195 platform critical care transport green channel from January 2018 to January 2020 at the collaborative hospitals of Henan Provincial People's Hospital were enrolled. According to different treatment methods, they were divided into a conventional treatment group and an ECCO2R group, with 30 patients in each group. Both groups received standard treatments including primary disease treatment, airway management, and non-invasive ventilation. The conventional treatment group received bedside continuous renal replacement therapy (CRRT), and the ECCO2R group received V-V ECCO2R treatment. General information of patient such as gender, age, cause of disease, and acute physiology and chronic health evaluation II (APACHE II) were recorded; arterial blood gas analysis was performed before treatment and at 12 hours and 24 hours during treatment, recording arterial partial pressure of oxygen (PaO2), arterial partial pressure of carbon dioxide (PaCO2), and oxygenation index (PaO2/FiO2). Respiratory mechanics parameters [tidal volume, respiratory rate, maximal inspiratory pressure (MIP), and maximal expiratory pressure (MEP)] were recorded, and the rapid shallow breathing index (RSBI) was calculated; electrical impedance tomography (EIT) was used to measure regional of interest (ROI) values in different lung areas at 12 hours and 24 hours of treatment, and the pulmonary mechanical energy was calculated.
RESULTS:
The arterial blood gas analysis indicators, respiratory mechanics parameters, and pulmonary mechanical energy of patients in the conventional treatment group and ECCO2R group improved significantly after 24 hours of treatment compared to 12 hours of treatment (all P < 0.05). The levels of PaCO2, RSBI, total mechanical power, and non-dependent zone mechanical power in the ECCO2R group were significantly lower than those in the conventional treatment group at both 12 hours and 24 hours during the treatment [PaCO2 (mmHg, 1 mmHg ≈ 0.133 kPa): 44.03±2.96 vs. 49.96±2.50 at 12 hours, 41.65±3.21 vs. 48.53±2.33 at 24 hours; RSBI (times×min-1×L-1): 88.67±4.05 vs. 92.35±4.03 at 12 hours, 77.66±4.64 vs. 90.98±4.21 at 24 hours; total mechanical power (mJ): 10.40±1.15 vs. 12.93±1.68 at 12 hours, 11.13±1.18 vs. 14.05±1.69 at 24 hours; non-dependent zone mechanical power (mJ): 7.15±0.84 vs. 7.98±0.75 at 12 hours, 7.77±0.93 vs. 9.13±1.10 at 24 hours], and MEP and MIP in the ECCO2R group were significantly higher than those in the conventional treatment group at both 12 hours and 24 hours during the treatment [MEP (cmH2O, 1 cmH2O ≈ 0.098 kPa): 89.88±5.04 vs. 86.09±5.57 at 12 hours, 96.57±2.59 vs. 88.66±2.98 at 24 hours; MIP (cmH2O): 47.64±2.82 vs. 41.93±2.44 at 12 hours, 60.11±6.53 vs. 43.63±2.80 at 24 hours], the differences were statistically significant (all P < 0.05).
CONCLUSIONS
V-V ECCO2R combined with non-invasive ventilation can effectively reduce the regional tidal volume, mechanical power, and respiratory rate in the non-gravitational dependent zones of patients with mild to moderate ARDS, and improve respiratory distress and oxygenation status.
Humans
;
Respiratory Distress Syndrome/physiopathology*
;
Retrospective Studies
;
Carbon Dioxide
;
Blood Gas Analysis
;
Lung/physiopathology*
;
Intensive Care Units
;
Male
;
Female
;
Noninvasive Ventilation/methods*
;
Continuous Renal Replacement Therapy/methods*
;
APACHE
;
Middle Aged
9.Research progress on the application of end-tidal carbon dioxide monitoring in prehospital emergency care.
Jingtao MA ; Renbao LI ; Qin LI ; Wei HAN
Chinese Critical Care Medicine 2024;36(12):1340-1344
Prehospital emergency care is the primary stage in the treatment of critically ill patients, where efficient and accurate monitoring methods are crucial for patient survival and prognosis. End-tidal carbon dioxide (EtCO2) monitoring is a real-time, non-invasive method that can sensitively capture the status of respiratory, circulatory, and metabolic functions, particularly in the urgent and complex pre-hospital environment, a immediate detection and non-invasive method, can sensitively capture the respiratory, circulatory, and metabolic status of patients. It provides valuable guidance for rapid decision-making and precise interventions. This is particularly valuable in the complex and urgent prehospital environment, providing critical data for rapid decision-making and precise intervention. This paper systematically reviews the advancements in the application of EtCO2 monitoring across various fields, including sepsis identification, trauma assessment, cardiac arrest, respiratory critical care, endotracheal intubation confirmation, and management of metabolic diseases, aiming to explore its application value and prospects in pre-hospital emergency care.
Humans
;
Emergency Medical Services/methods*
;
Carbon Dioxide/analysis*
;
Monitoring, Physiologic/methods*
;
Critical Illness
;
Capnography/methods*
10.Effect of transcutaneous acupoint electrical stimulation at Neiguan (PC 6) on general anesthesia under preserving spontaneous breathing in thoracoscopic lobectomy.
Yi DING ; Sheng-Yong SU ; Ya-Li LIN ; Yi-Tong WEI ; Jun-Dan SHI ; Si-Li GAO ; Jin-Yi ZHUO ; Yuan-Chun CAI
Chinese Acupuncture & Moxibustion 2023;43(3):282-286
OBJECTIVE:
To evaluate the effect of transcutaneous acupoint electrical stimulation (TEAS) at Neiguan (PC 6) on general anesthesia under preserving spontaneous breathing in thoracoscopic lobectomy.
METHODS:
A total of 66 patients of primary lung cancer undergoing thoracoscopic lobectomy were divided to an observation group (33 cases, 1 case discontinued) and a control group (33 cases). In the observation group, TEAS at Neiguan (PC 6) was used 30 min before anesthesia induction till the end of surgery. The surgery time, maximum value of partial pressure of end-tidal carbon dioxide (PETCO2) and minimum value of oxygen saturation (SpO2) of the two groups were recorded. The dosage of propofol, sufentanil, remifentanil and dexmedetomidine were analyzed. Separately, before induction (T0), at the start of surgery (T1), thoracic exploration (T2) and lobectomy (T3), as well as 30 min (T4) and 60 min (T5) after lobectomy, the mean arterial pressure (MAP), heart rate (HR), respiratory rate (RR), serum cortisol (Cor) and norepinephrine (NE) were measured. The time of post anesthesia care unit (PACU) stay, ambulation, flatus, chest drainage and the incidence of nausea and vomiting were compared between the two groups.
RESULTS:
The maximum value of PETCO2, the dosage of propofol and remifentanil in the observation group were lower than those in the control group (P < 0.05, P < 0.01), the minimum value of SpO2 in the observation group was higher than that of the control group (P < 0.01). At T1-T5, the MAP, HR, serum Cor and NE levels in the observation group were all lower than those in the control group (P < 0.05). The ambulation time, the time for the flatus, chest drainage time, and the incidence of nausea and vomiting in the observation group were all lower than those in the control group (P<0.001, P < 0.01).
CONCLUSION
For the general anesthesia under preserving spontaneous breathing in thoracoscopic surgery, TEAS at Neiguan (PC 6) relieves stress response, reduces opioids dosage and promotes postoperative recovery.
Humans
;
Acupuncture Points
;
Carbon Dioxide
;
Flatulence
;
Propofol
;
Remifentanil
;
Anesthesia, General
;
Nausea
;
Norepinephrine
;
Electric Stimulation

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