1.Chinese Medicine Regulates Hepatocellular Carcinoma-related Signaling Pathways: A Review
Chun YU ; Fen GAO ; Lanlan ZHENG ; Cai GUO ; Yanfang HE ; Jiaojiao XIE ; Xuan ZHANG ; Yanhua MA
Chinese Journal of Experimental Traditional Medical Formulae 2024;30(15):232-243
Hepatocellular carcinoma (HCC) is a common tumor in the digestive tract, the formation mechanism of which remains to be fully elucidated. Although surgery, radiation, chemotherapy, targeted therapy, and immunotherapy have achieved significant results in the treatment of HCC, these methods are accompanied by a considerable number of adverse reactions and complications. In recent years, Chinese medicine has shown remarkable efficacy in the treatment of HCC, and both basic experiments and clinical studies have confirmed the effectiveness of Chinese medicine, which exerts therapeutic effects via multiple components and multiple targets. However, the pathogenesis of HCC is exceptionally complex and not fully understood, which means that studies remain to be carried out regarding the specific mechanism of Chinese medicine in preventing and treating HCC. Network pharmacology and molecular biology can be employed to decipher the mechanism of Chinese medicine in the treatment of diseases. Studies have shown that Chinese medicine can regulate various pathways such as the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), Hedgehog, Wnt/β-catenin, nuclear factor-κB (NF-κB), Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3), and transforming growth factor-β (TGF-β)/Smad signaling pathways. Chinese medicine can exhibit its anti-HCC effects by inducing cell apoptosis, inhibiting cell proliferation and migration, and blocking the cell cycle via the above pathways. However, the specific mechanisms remain to be systematically studied. This study comprehensively reviews the regulatory effects of Chinese medicine on HCC-related signaling pathways to reveal the molecular mechanisms of Chinese medicine in the treatment of HCC. This view holds the promise of providing new targets, new perspectives, and new therapies for HCC treatment and advancing the modernization and development of Chinese medicine.
2.Expression and clinical significance of cell cycle protein-dependent kinase 1 and aurora kinase A in the serum of patients with hepatitis B virus-related hepatocellular carcinoma
Yanfang HE ; Jiaojiao XIE ; Lanlan ZHENG ; Cai GUO ; Yanhua MA
Journal of Clinical Hepatology 2024;40(7):1390-1396
Objective To investigate the value of serum cell cycle protein-dependent kinase 1(CDK1)and aurora kinase A(AURKA)in the diagnosis of patients with hepatitis B virus-related hepatocellular carcinoma(HBV-HCC).Methods A total of 50 HBV-HCC patients,50 patients with hepatitis B virus-related liver cirrhosis(HBV-LC),and 50 chronic hepatitis B(CHB)patients who were hospitalized in Department of Gastroenterology,Gansu Provincial Hospital,from June 2022 to December 2023 were enrolled,and 50 healthy individuals,matched for age and sex,who received physical examination at Physical Examination Center during the same period of time were enrolled as control group.Related data were recorded for all patients,including age,sex,complications,and the results of routine blood test,liver function,and coagulation for the first time after admission.ELISA was used to measure the serum levels of CDK1 and AURKA.A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups;the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups and the least significant difference Bonferroni test was used for further comparison between two groups;the chi-square test or the Fisher's exact test was used for comparison of categorical data between groups.The Spearman correlation analysis was used to investigate the correlation between CDK1 and AURKA,and the receiver operating characteristic(ROC)curve and the area under the ROC curve(AUC)were used to investigate the value of CDK1 and AURKA in the diagnosis of HBV-HCC.Results There were significant differences in liver function parameters between the HBV-HCC patients and the control group(all P<0.05);there were significant differences between the CHB group and the HBV-HCC group in albumin,Glb,direct bilirubin,aspartate aminotransferase(AST),gamma-glutamyl transpeptidase(GGT),and alkaline phosphatase(all P<0.05);there were significant differences between the HBV-LC group and the HBV-HCC group in Glb,AST,and GGT(all P<0.05).The HBV-HCC group had significantly higher serum levels of CDK1 and AURKA than the HBV-LC group,the CHB group,and the control group(all P<0.05).There was a significant positive correlation between CDK1 and AURKA in the overall study population and the HBV-HCC patients(r=0.526 6 and 0.815 2,P<0.001).With the control group as reference,CDK1 had an AUC of 0.832 3 in the diagnosis of HBV-HCC,with a sensitivity of 92.86%and a specificity of 75%,and AURKA had an AUC of 0.886 6 in the diagnosis of HCC,with a sensitivity of 95.80%and a specificity of 74%.With the CHB group as reference,CDK1 had an AUC of 0.833 3 in the diagnosis of HBV-HCC,with a sensitivity of 93.75%and a specificity of 75%,and AURKA had an AUC of 0.972 7 in the diagnosis of HBV-HCC,with a sensitivity of 95.83%and a specificity of 91.67%.With the HBV-LC group as reference,CDK1 had an AUC of 0.608 5 in the diagnosis of HBV-HCC,with a sensitivity of 66.67%and a specificity of 54.17%,and AURKA had an AUC of 0.762 2 in the diagnosis of HBV-HCC,with a sensitivity of 95.83%and a specificity of 47.92%.Conclusion The serum levels of CDK1 and AURKA increase with the progression of hepatitis B-associated chronic liver disease,and significant increases in serum CDK1 and AURKA have a certain value in the diagnosis of HBV-HCC.
3.Blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle
Huiling MA ; Xiuxiu CAI ; Jiaojiao ZHANG
Chinese Journal of Plastic Surgery 2024;40(5):537-544
Objective:To modify the blepharoplasty through palpebral margin incision with preservaton of the pretarsal orbicularis oculi muscle, and to discuss the clinical application effects.Methods:The clinical data of patients who underwent double-eyelid blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle were retrospectively analyzed from August 2021 to August 2023 in Changsha Aist Medical Cosmetology Hospital. After the skin peeling rang was designed, the skin was removed, tissue was separated under the orbicularis oculi muscle. The pretarsal orbicularis oculi muscle was thinned-shaped at the upper edge of tarsus as wedge-shaped, and was detached from the upper edge of tarsus. The pretarsal fascia or levator aponeurosis was fixed internally with the orbicularis oculi muscle at the position of the double-eyelid crease. After confirming the shape and symmetry of the double-eyelid, the skin was sutured. Incision healing, eyelid swelling and complications were observed, and double-eyelid morphology was followed up. Six months after surgery, the patients’ satisfaction with the surgical effect (satisfied, somewhat satisfied, dissatisfied) was investigated, and the upper eyelid scar status (including pigmentation, thickness, vascularity, and pliability) was scored using the Vancouver scar scale (VSS). The higher the score, the more serious the scar was.Results:A total of 78 patients with single eyelid or subtle double eyelid were included, including 6 males and 72 females, aged 18-40 years old. All the patients underwent modified double-eyelid blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle, 29 patients underwent ptosis correction simultaneously, and 47 patients underwent epicanthus correction simultaneously. The incision healed by first intention after the operation. 78 cases were followed up for 1 to 6 months, and slight upper eyelid swelling was eliminated in 71 cases at 10-15 days and in 7 cases at 25-30 days. Among them, 61 cases were followed up for 6 months, the creases of double-eyelid was natural and smooth, bilateral basic symmetry, and there were no adverse manifestations such as double-eyelid shallowing and pretarsal tissue pilling, and no complications such as incompleted eye-closure. 57 cases were satisfied with the results and 4 cases were somewhat satisfied. The incision scar was not obvious when the eyes were closed in 61 patients. The VSS score was close to 0, and the average score of pigmentation, thickness, vascularity, and pliability were 0.29, 0, 0.31 and 0 points, respectively.Conclusion:This operation method preserves almost all the pretarsal orbicularis oculi muscle to promote the healling of lymph circulation. After the orbicularis oculi muscle is choosed for internal fixation, this more simple operation method can not only guarantee the efficacy of double-eyelid blepharoplasty through palpebral margin incision, but also accelerate the healling of the eyelid after surgery and reduce the incidence of incompleted eye-closure.
4.Blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle
Huiling MA ; Xiuxiu CAI ; Jiaojiao ZHANG
Chinese Journal of Plastic Surgery 2024;40(5):537-544
Objective:To modify the blepharoplasty through palpebral margin incision with preservaton of the pretarsal orbicularis oculi muscle, and to discuss the clinical application effects.Methods:The clinical data of patients who underwent double-eyelid blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle were retrospectively analyzed from August 2021 to August 2023 in Changsha Aist Medical Cosmetology Hospital. After the skin peeling rang was designed, the skin was removed, tissue was separated under the orbicularis oculi muscle. The pretarsal orbicularis oculi muscle was thinned-shaped at the upper edge of tarsus as wedge-shaped, and was detached from the upper edge of tarsus. The pretarsal fascia or levator aponeurosis was fixed internally with the orbicularis oculi muscle at the position of the double-eyelid crease. After confirming the shape and symmetry of the double-eyelid, the skin was sutured. Incision healing, eyelid swelling and complications were observed, and double-eyelid morphology was followed up. Six months after surgery, the patients’ satisfaction with the surgical effect (satisfied, somewhat satisfied, dissatisfied) was investigated, and the upper eyelid scar status (including pigmentation, thickness, vascularity, and pliability) was scored using the Vancouver scar scale (VSS). The higher the score, the more serious the scar was.Results:A total of 78 patients with single eyelid or subtle double eyelid were included, including 6 males and 72 females, aged 18-40 years old. All the patients underwent modified double-eyelid blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle, 29 patients underwent ptosis correction simultaneously, and 47 patients underwent epicanthus correction simultaneously. The incision healed by first intention after the operation. 78 cases were followed up for 1 to 6 months, and slight upper eyelid swelling was eliminated in 71 cases at 10-15 days and in 7 cases at 25-30 days. Among them, 61 cases were followed up for 6 months, the creases of double-eyelid was natural and smooth, bilateral basic symmetry, and there were no adverse manifestations such as double-eyelid shallowing and pretarsal tissue pilling, and no complications such as incompleted eye-closure. 57 cases were satisfied with the results and 4 cases were somewhat satisfied. The incision scar was not obvious when the eyes were closed in 61 patients. The VSS score was close to 0, and the average score of pigmentation, thickness, vascularity, and pliability were 0.29, 0, 0.31 and 0 points, respectively.Conclusion:This operation method preserves almost all the pretarsal orbicularis oculi muscle to promote the healling of lymph circulation. After the orbicularis oculi muscle is choosed for internal fixation, this more simple operation method can not only guarantee the efficacy of double-eyelid blepharoplasty through palpebral margin incision, but also accelerate the healling of the eyelid after surgery and reduce the incidence of incompleted eye-closure.
5.Effects of calcitriol on PI3K/AKT signaling pathway and wound healing in diabetic foot ulcer rats
Yanhong LUO ; Yu TIAN ; Yunfeng WU ; Jiaojiao LI ; Shuxiang YANG ; Lingrui WANG ; Tingting XIE ; Yan YANG ; Yulan CAI
Chinese Journal of Diabetes 2024;32(7):532-539
Objective To investigate the effects of calcitriol intervention on PI3K/AKT signaling pathway and wound healing in rats with diabetic foot ulcer(DFU).Methods Thirty-two male SD rats were divided into normal control(Con)group,DFU group,calcitriol low dose(L)group and calcitriol high dose(H)group.A circular wound of 5 mm in diameter and deep to the fascia was made on the dorsum of the left foot of rats in each group.HE staining was used to evaluate the histopathological changes of the wounds.Immunohistochemical method was selected to compare the distribution of CD34-positive cells and the expression of p-PI3K and p-AKT in traumatic tissues of each group.ELISA was adopted in the detection of serum IL-6,IFN-γ,TNF-α and IL-7.RT-PCR was used to detect the mRNA expression of PI3K and AKT in each group,and western blot was used to detect the protein expression of PI3K,p-PI3K,AKT,p-AKT and VEGF.Results Compared with Con group,the expressions of IL-6,IFN-γ,TNF-α,IL-7,CD34,PI3K mRNA,AKT mRNA,p-AKT protein,p-PI3K protein,p-PI3K/PI3K,p-AKT/AKT increased,while PI3K protein expression decreased in DFU,L and H groups(P<0.05),VEGF and AKT protein expression decreased in DFV and L groups(P<0.05).Compared with DFU group,the expressions of VEGF,AKT and PI3K protein increased(P<0.05),while the expressions of p-PI3K/PI3K,p-AKT/AKT decreased in L and H groups(P<0.05),IL-6 decreased in L group(P<0.05),and CD34 expression increased in H group(P<0.05),while IL-6,IFN-γ,TNF-α and IL-7,PI3K mRNA,AKT mRNA,p-AKT protein and p-PI3K protein expression decreased(P<0.05).Compared with L group,the expressions of CD34,VEGF,AKT and PI3K protein increased(P<0.05),while IL-6,PI3K mRNA,AKT mRNA,p-AKT protein,p-PI3K protein,p-PI3K/PI3K and p-AKT/AKT decreased in H group(P<0.05).Conclusions Calcitriol intervention may reduce the activity of the PI3K/AKT signaling pathway,inhibit inflammation,promote neovascularization,and facilitate wound healing in rats with DFU.
6.Ferroptosis in Treatment of Liver Fibrosis with Chinese Medicine: A Review
Lanlan ZHENG ; Cai GUO ; Yanfang HE ; Jiaojiao XIE ; Fen GAO ; Chun YU ; Yanhua MA
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(23):235-244
Hepatic fibrosis characterized by various chronic liver injuries can lead to abnormal activation of hepatic stellate cells, unbalanced production and degradation of extracellular matrix proteins, and excessive deposition that destroys the normal structure of the liver. The aggravated liver fibrosis can cause irreversible cirrhosis and hepatocellular carcinoma, becoming a great challenge to the global health. Ferroptosis is a new form of iron-dependent cell death discovered in recent years, which mainly involves abnormal iron metabolism, lipid peroxide accumulation, and weakening of the antioxidant defense system. A number of studies have reported that inducing ferroptosis in hepatic stellate cells or alleviating ferroptosis in the liver can ameliorate liver fibrosis and reduce liver injury. Chinese medicine widely applied in the treatment of chronic liver diseases has demonstrated good safety, wide therapeutic effects, and easy access compared with Western medicine. Therefore, The intervention of hepatic stellate cells or hepatic ferroptosis by Chinese medicine may be a new direction for the prevention and treatment of liver fibrosis in the future. This paper summarized the various regulatory mechanisms of ferroptosis and expounded how ferroptosis affected the progression of liver fibrosis, providing theoretical support for the prevention and treatment of liver fibrosis with Chinese medicine in the future.
7.Multicenter study on the etiology characteristics of neonatal purulent meningitis
Yanli LIU ; Jiaojiao CAI ; Xiaoyi ZHANG ; Minli ZHU ; Zhenlang LIN ; Yicong PAN ; Junhu ZHENG ; Yiwei ZHAO ; Xiang WANG ; Hongping LU ; Meifang LIN ; Ji WANG ; Haihong GU ; Lizhen WANG ; Keping CHENG ; Yuxuan DAI ; Yuan GAO ; Junsheng LI ; Hongxia FANG ; Na SUN ; Lihua LI ; Xiaoquan LI ; Ying LIU ; Yingyu LI ; Wa GAO ; Minxia LI
Chinese Journal of Infectious Diseases 2023;41(6):393-400
Objective:To study the distribution and antibiotics resistance of the main pathogens of neonatal purulent meningitis in different regions of China.Methods:A retrospective descriptive clinical epidemiological study was conducted in children with neonatal purulent meningitis which admitted to 18 tertiary hospitals in different regions of China between January 2015 to December 2019. The test results of blood and cerebrospinal fluid, and drug sensitivity test results of the main pathogens were collected. The distributions of pathogenic bacteria in children with neonatal purulent meningitis in preterm and term infants, early and late onset infants, in Zhejiang Province and other regions outside Zhejiang Province, and in Wenzhou region and other regions of Zhejiang Province were analyzed. The chi-square test was used for statistical analysis.Results:A total of 210 neonatal purulent meningitis cases were collected. The common pathogens were Escherichia coli ( E. coli)(41.4%(87/210)) and Streptococcus agalactiae ( S. agalactiae)(27.1%(57/210)). The proportion of Gram-negative bacteria in preterm infants (77.6%(45/58)) with neonatal purulent meningitis was higher than that in term infants (47.4%(72/152)), and the difference was statistically significant ( χ2=15.54, P=0.001). There were no significant differences in the constituent ratios of E. coli (36.5%(31/85) vs 44.8%(56/125)) and S. agalactiae (24.7%(21/85) vs 28.8%(36/125)) between early onset and late onset cases (both P>0.05). The most common pathogen was E. coli in different regions, with 46.7%(64/137) in Zhejiang Province and 31.5%(23/73) in other regions outside Zhejiang Province. In Zhejiang Province, S. agalactiae was detected in 49 out of 137 cases (35.8%), which was significantly higher than other regions outside Zhejiang Province (11.0%(8/73)). The proportions of Klebsiella pneumoniae, and coagulase-negative Staphylococcus in other regions outside Zhejiang Province (17.8%(13/73) and 16.4%(12/73)) were both higher than those in Zhejiang Province (2.9%(4/137) and 5.1%(7/137)). The differences were all statistically significant ( χ2=14.82, 12.26 and 7.43, respectively, all P<0.05). The proportion of Gram-positive bacteria in Wenzhou City (60.8%(31/51)) was higher than that in other regions in Zhejiang Province (38.4%(33/86)), and the difference was statistically significant ( χ2=6.46, P=0.011). E. coli was sensitive to meropenem (0/45), and 74.4%(32/43) of them were resistant to ampicillin. E. coli had different degrees of resistance to other common cephalosporins, among which, cefotaxime had the highest resistance rate of 41.8%(23/55), followed by ceftriaxone (32.4%(23/71)). S. agalactiae was sensitive to penicillin, vancomycin and linezolid. Conclusions:The composition ratios of pathogenic bacteria of neonatal purulent meningitis are different in different regions of China. The most common pathogen is E. coli, which is sensitive to meropenem, while it has different degrees of resistance to other common cephalosporins, especially to cefotaxime.
8.Analysis of efficacy of ultrasound-guided percutaneous polidocanol sclerotherapy for thyroglossal duct cysts in children
Xiao ZHANG ; Wenjia CAI ; Shengcai WANG ; Jiabin WAN ; Jiaojiao DING ; Yuanhu LIU ; Xin NI ; Wenyuan SHI
Chinese Journal of Ultrasonography 2023;32(4):348-353
Objective:To evaluate the effectiveness and safety of ultrasound-guided percutaneous polidocanol sclerotherapy of the thyroglossal duct cysts in children.Methods:A retrospective analysis of 26 children who were treated with ultrasound-guided percutaneous puncture sclerosis for thyroglossal duct cysts in Beijing Children′s Hospital, Capital Medical University from October 2019 to April 2022, the size of the cyst before treatment was recorded, and the cyst volume in accordance with V(ml) =1/6 πabc cyst volume was calculated. The follow-up time was the 1st, 3rd, 6th, and 12th months after sclerotherapy. And the cyst volume and volume reduction ratio at different time points was calculated. According to changes in the cystic volume, the treatment was considered effective if the cyst volume reduction rate was≥50%. Cosmetic grading scores (CGS) were performed pre-treatment and at the last follow-up after sclerotherapy using the WHO grading system. Complications were recorded during the follow-up period.Results:The cyst volume before treatment was 2.67 (3.78)ml, and the cyst volume at the 1st, 3rd, 6th and 12th month after treatment were 0.66(1.83), 0.45(0.87), 0.40(0.70), 0.38 (0.63)ml, respectively, there were significant differences between pre-treatment and each time point after-treatment (all P<0.001); the volume reduction rate at the last follow-up was (81.48±14.57)%. The reduction rate of cyst volume was more than 50% at the last follow-up in 22 children, the treatment efficiency was 84.62% (22/26). The cosmetic grading scores at the last follow-up after sclerotherapy was 1.74(1.50), and it decreased significantly compared with the pre-treatment score 3.85(0)( P<0.001). There was no skin pigmentation, pain in the injection area, local urticaria and blistering after sclerotherapy, no adverse reactions such as cyst bleeding and drunkenness-like reaction, and no serious complications occurred. There were 2 cases of cysts that presented infection with fever, and no serious complications occurred. Conclusions:Ultrasound-guided percutaneous sclerotherapy is a safe and effective minimally invasive treatment for thyroglossal duct cysts in children.
9.Development of an approach for simultaneous determination ofmulti-mycotoxins in fresh fruits
Jiaojiao XU ; Zengxuan CAI ; Junlin WANG ; Meijia XU ; Baifen HUANG
Journal of Preventive Medicine 2022;34(6):555-560
Objective:
To develop an approach for simultaneous detection of multi-mycotoxins in fresh fruits, so as to provide technical supports for mycotoxins surveillance in fresh fruits.
Methods:
Fresh fruits were collected from markets and homogenized. Then, 2 g of fresh fruits were added with 10 mL of 0.1% formic acid ( 99∶1, v/v ) in acetonitrile and wortexed for 10 min. Following extraction with 1 g of sodium chloride and 4 g of anhydrous sodium sulfate, samples were centrifuged and 5 mL of the supernatant was cleaned up with 25 mg C18. Following centrifugation, the supernatant was dried under nitrogen. The residue was dissolved in 300 μL of methanol-acetonitrile mixture solution ( 1∶1, v/v ), and mixed evenly in 700 μL of the distilled water. Samples were then eluted in gradient series of 0.1% formic acid and 5 mmol ammonium formate and methanol-acetonitrile mixture solution ( 1∶1, v/v ). The 15 mycotoxins were determined using liquid chromatography-tandem mass spectrometry ( LC-MS/MS ) with electrospray ion source (ESI+/ESI-) under multiple reaction monitoring. In addition, a matrix-matched standard curve was employed for quantitative analysis.
Results:
There was a good linear relationship for 15 mycotoxins at concentrations of 0.25 to 10 ng/mL ( R2>0.992 ), the LC-MS/MS method showed the detection limits of 0.1-1.0 μg/kg, the spiked recovery rates of 71.68%-117.50%, and the relative standard deviations ( RSDs ) of 0.01%-13.60%. The detection rate of mycotoxins was 27.09% in 203 fresh fruits sold in markets.
Conclusions
The optimized LC-MS/MS method can be used for simultaneous determination of multi-mycotoxins in fresh fruits.
10.Determination of ibotenic acid and muscimol in wild mushroom by dansyl chloride derivatization-liquid chromatography-tandem mass spectrometry
XU Xiaomin ; CAI Zengxuan ; XU Jiaojiao ; ZHENG Yibin ; CHEN Qing ; HAN Jianlong
Journal of Preventive Medicine 2021;33(12):1223-1227
Objective :
To develop an analytical method of ibotenic acid (IBA) and muscimol (MUS) in wild mushroom by dansyl chloride (DNSCl) derivatization-liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to provide technical support for etiological identification of mushroom poisoning events.
Methods :
The sample was extracted with hydrochloric acid solution, derived by bimolecular DNSCl, diluted and inorganic salts precipitated with acetonitrile. The extract was separated by a waters XBridgeTM BEH C18 column and measured by LC-MS/MS.
Results :
The limits of detection for IBA and MUS in wild mushroom were 0.15 mg/kg and 0.1 mg/kg, respectively. Good linear relationship was obtained for IBA and MUS at the range of 0.5-250 mg/kg with the correlation coefficient of 0.997 and 0.999, respectively. The average recoveries at three spiking levels were 84.5%-102.0% with relative standard deviations (RSDs, n=6) of 4.7%-8.6% for IBA. The average recoveries were 88.6%-95.4% with RSDs (n=6) of 4.9%-7.5% for MUS.
Conclusion
The optimized sample extraction and bimolecular DNSCl derivatization conditions can achieve rapid and accurate analysis of IBA and MUS in wild mushroom poisoning sample.


Result Analysis
Print
Save
E-mail