1.Research progress on the role of myeloid-derived suppressor cells in tuber-culosis
Jiaxue ZHANG ; Xiaoping WANG ; Li LIU ; Qinmei MA ; Guangcun DENG ; Xiaoling WU
Chinese Journal of Infection Control 2025;24(5):712-720
Tuberculosis,as a chronic infectious disease caused by Mycobacterium tuberculosis(MTB)infection,presenting a serious threat to human and animal health.When human body is infected by MTB,it quickly initiates an immune response,in which T lymphocyte-mediated cellular immunity plays a key role,as it can effectively recog-nize and eliminate MTB,serving as an important defense line for the body against MTB.However,during MTB in-fection process,myeloid-derived suppressor cells(MDSCs)undergo amplification and activation,which exhibit in-hibitory effect on the immune function of T lymphocytes and interfere the normal immune response of the body,making MTB easier to survive and reproduce in the body.This article discusses the role of MDSCs in the occu-rrence,development and prognosis of tuberculosis from two different perspectives,expects to provide new perspec-tives and ideas for the immunotherapy of tuberculosis as well as the research and development of targeted drugs,so as to open up a new way for the prevention and treatment of tuberculosis.
2.Consistency between MT-9000 and IOLMaster 700 in measuring ocular biometric parameters in cataract eyes
Guohuan WU ; Ying ZHANG ; Chaoyu DU ; Jingjing HUANG ; Liman WANG ; Qinmei WANG
Chinese Journal of Experimental Ophthalmology 2025;43(10):924-929
Objective:To analyze the consistency between MT-9000 and IOLMaster 700 in measuring preoperative ocular biometric parameters in patients with age-related cataracts.Methods:A cross-sectional study was conducted.A total of 536 patients (536 eyes) were consecutively included and scheduled for cataract extraction combined with intraocular lens implantation at Aier Eye Hospital of Wuhan University from June 20 to August 22, 2024.Two visualized sweep-frequency biometers, MT-9000 and IOLMaster 700, were used to measure the biometric parameters of all the patients.The parameters including axial length (AL), white to white (WTW), anterior chamber depth (ACD), lens thickness (LT), central corneal thickness (CCT), and mean keratometry (Km). The AL detection rates of the two instruments were compared, as were the differences in above parameters, and the correlation and consistency of the measurements were analyzed.Patients were divided into three groups based on AL measured by MT-9000: a short axis group (AL<22 mm) comprising 27 eyes, a normal axis group (22≤AL≤25 mm) comprising 392 eyes, and a long axis group (AL>25 mm) comprising 104 eyes.The differences in measured AL in different subgroups were compared.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Aier Eye Hospital, Wuhan University (No.2024IRB06017). Written informed consent was obtained from each subject.Results:The AL detection rate measured by MT-9000 was 99.3% (532/536), higher than 97.6% (523/536) measured by IOLMaster 700, with a statistically significant difference ( χ2=4.841, P=0.028). MT-9000 AL, LT and Km measurements were all higher than IOLMaster 700 measurements, showing statistically significant differences ( Z=6.418, t=4.971, 5.179; all P<0.001). WTW and CCT measurements from MT-9000 were both lower than those from IOLMaster 700, showing statistically significant differences ( Z=-3.017, t=16.322; both P<0.05). After grouping based on AL measurements from MT-9000, there was no statistically significant differences in AL measurements between MT-9000 and IOLMaster 700 in the short axis group and the normal axis group ( Z=-1.530, P=0.126; t=-0.880, P=0.380). In the long axis group, the AL measured by MT-9000 was longer than that by IOLMaster 700, with a statistically significant difference ( Z=7.254, P<0.001). The intra-class correlation coefficient values of AL, WTW, ACD, LT, CCT, and Km measured by MT-9000 and IOLMaster 700 were 1.000, 0.616, 0.997, 0.999, 0.988, and 0.995 (all P<0.001), respectively.The 95% limits of agreement (LoA) of AL, WTW, ACD, LT, CCT, and Km measured by Bland-Altman analysis were -0.06-+ 0.08 mm, -0.64-+ 0.57 mm, -0.06-+ 0.06 mm, -0.04-+ 0.05 mm, -13.6-+ 6.3 μm and -0.26-+ 0.32 D, respectively, which had 6.5%, 3.6%, 5.3%, 6.3%, 6.5% and 4.4% of the data points outside the 95% LoA. Conclusions:MT-9000 shows favorable agreement with IOLMaster 700 in the measurement of AL, ACD, LT, CCT and Km.MT-9000 is suitable for preoperative biometrics measurement in cataract patients.However, the agreement between the two devices is not desirable in the measurement of WTW, and interchangeable use of these two devices is not recommended in this regard.Additionally, MT-9000 outperforms IOLMaster 700 in terms of AL detection rate.
3.Comparison and application of direct and indirect methods in establishing reference intervals for coagulation tests
Jing ZHENG ; Ping WANG ; Jiang LI ; Qinmei CAO ; Yongtong CAO
Chinese Journal of Preventive Medicine 2025;59(3):359-364
This study aimed to establish adult reference intervals for coagulation parameters using the CN-6000 fully automated coagulation analyzer. It also compared the applicability of direct and indirect methods in different populations. A cross-sectional and retrospective design was employed. The direct method included patients from the colorectal surgery and dermatology departments, as well as healthy participants from phase I clinical trials, admitted to the West Campus of China-Japan Friendship Hospital from April to December 2023. Participants were divided into two age groups: younger adults (18-64 years) and older adults (≥65 years). Coagulation parameters were tested, and reference intervals were determined. The indirect method applied the refineR algorithm to analyze coagulation data from non-ICU patients during the same period. The performance of both methods was evaluated in healthy participants, relatively healthy patients, and patients with high disease prevalence. The results showed that the direct method yielded similar reference intervals for APTT, PT, and TT in younger and older groups, with combined intervals of 23.7-31.2 seconds, 10.4-12.8 seconds, and 14.7-17.5 seconds, respectively. However, the Fib showed a significant difference between the two groups ( U=1 052 023, P<0.01), and separate reference intervals were established: 1.7-4.5 g/L for the younger group and 2.1-4.6 g/L for the older group. The reference intervals derived by the indirect method are similar to those from the direct method: APTT 23.8-31.4 seconds, PT 10.4-13.0 seconds, TT 14.5-17.4 seconds, and Fib 1.8-4.3 g/L (18-64 years) and 2.1-4.5 g/L (≥65 years). In relatively healthy patients, the reference intervals obtained by both methods were comparable, but significant differences were observed in patients with high disease prevalence. In conclusion, this study established localized reference intervals for the CN-6000 fully automated coagulation analyzer. The indirect method can serve as an alternative to the direct method in healthy and relatively healthy populations. However, its applicability in populations with high disease prevalence is limited and requires cautious interpretation in the context of clinical settings.
4.Consistency between MT-9000 and IOLMaster 700 in measuring ocular biometric parameters in cataract eyes
Guohuan WU ; Ying ZHANG ; Chaoyu DU ; Jingjing HUANG ; Liman WANG ; Qinmei WANG
Chinese Journal of Experimental Ophthalmology 2025;43(10):924-929
Objective:To analyze the consistency between MT-9000 and IOLMaster 700 in measuring preoperative ocular biometric parameters in patients with age-related cataracts.Methods:A cross-sectional study was conducted.A total of 536 patients (536 eyes) were consecutively included and scheduled for cataract extraction combined with intraocular lens implantation at Aier Eye Hospital of Wuhan University from June 20 to August 22, 2024.Two visualized sweep-frequency biometers, MT-9000 and IOLMaster 700, were used to measure the biometric parameters of all the patients.The parameters including axial length (AL), white to white (WTW), anterior chamber depth (ACD), lens thickness (LT), central corneal thickness (CCT), and mean keratometry (Km). The AL detection rates of the two instruments were compared, as were the differences in above parameters, and the correlation and consistency of the measurements were analyzed.Patients were divided into three groups based on AL measured by MT-9000: a short axis group (AL<22 mm) comprising 27 eyes, a normal axis group (22≤AL≤25 mm) comprising 392 eyes, and a long axis group (AL>25 mm) comprising 104 eyes.The differences in measured AL in different subgroups were compared.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Aier Eye Hospital, Wuhan University (No.2024IRB06017). Written informed consent was obtained from each subject.Results:The AL detection rate measured by MT-9000 was 99.3% (532/536), higher than 97.6% (523/536) measured by IOLMaster 700, with a statistically significant difference ( χ2=4.841, P=0.028). MT-9000 AL, LT and Km measurements were all higher than IOLMaster 700 measurements, showing statistically significant differences ( Z=6.418, t=4.971, 5.179; all P<0.001). WTW and CCT measurements from MT-9000 were both lower than those from IOLMaster 700, showing statistically significant differences ( Z=-3.017, t=16.322; both P<0.05). After grouping based on AL measurements from MT-9000, there was no statistically significant differences in AL measurements between MT-9000 and IOLMaster 700 in the short axis group and the normal axis group ( Z=-1.530, P=0.126; t=-0.880, P=0.380). In the long axis group, the AL measured by MT-9000 was longer than that by IOLMaster 700, with a statistically significant difference ( Z=7.254, P<0.001). The intra-class correlation coefficient values of AL, WTW, ACD, LT, CCT, and Km measured by MT-9000 and IOLMaster 700 were 1.000, 0.616, 0.997, 0.999, 0.988, and 0.995 (all P<0.001), respectively.The 95% limits of agreement (LoA) of AL, WTW, ACD, LT, CCT, and Km measured by Bland-Altman analysis were -0.06-+ 0.08 mm, -0.64-+ 0.57 mm, -0.06-+ 0.06 mm, -0.04-+ 0.05 mm, -13.6-+ 6.3 μm and -0.26-+ 0.32 D, respectively, which had 6.5%, 3.6%, 5.3%, 6.3%, 6.5% and 4.4% of the data points outside the 95% LoA. Conclusions:MT-9000 shows favorable agreement with IOLMaster 700 in the measurement of AL, ACD, LT, CCT and Km.MT-9000 is suitable for preoperative biometrics measurement in cataract patients.However, the agreement between the two devices is not desirable in the measurement of WTW, and interchangeable use of these two devices is not recommended in this regard.Additionally, MT-9000 outperforms IOLMaster 700 in terms of AL detection rate.
5.Research progress on the role of myeloid-derived suppressor cells in tuber-culosis
Jiaxue ZHANG ; Xiaoping WANG ; Li LIU ; Qinmei MA ; Guangcun DENG ; Xiaoling WU
Chinese Journal of Infection Control 2025;24(5):712-720
Tuberculosis,as a chronic infectious disease caused by Mycobacterium tuberculosis(MTB)infection,presenting a serious threat to human and animal health.When human body is infected by MTB,it quickly initiates an immune response,in which T lymphocyte-mediated cellular immunity plays a key role,as it can effectively recog-nize and eliminate MTB,serving as an important defense line for the body against MTB.However,during MTB in-fection process,myeloid-derived suppressor cells(MDSCs)undergo amplification and activation,which exhibit in-hibitory effect on the immune function of T lymphocytes and interfere the normal immune response of the body,making MTB easier to survive and reproduce in the body.This article discusses the role of MDSCs in the occu-rrence,development and prognosis of tuberculosis from two different perspectives,expects to provide new perspec-tives and ideas for the immunotherapy of tuberculosis as well as the research and development of targeted drugs,so as to open up a new way for the prevention and treatment of tuberculosis.
6.Comparison and application of direct and indirect methods in establishing reference intervals for coagulation tests
Jing ZHENG ; Ping WANG ; Jiang LI ; Qinmei CAO ; Yongtong CAO
Chinese Journal of Preventive Medicine 2025;59(3):359-364
This study aimed to establish adult reference intervals for coagulation parameters using the CN-6000 fully automated coagulation analyzer. It also compared the applicability of direct and indirect methods in different populations. A cross-sectional and retrospective design was employed. The direct method included patients from the colorectal surgery and dermatology departments, as well as healthy participants from phase I clinical trials, admitted to the West Campus of China-Japan Friendship Hospital from April to December 2023. Participants were divided into two age groups: younger adults (18-64 years) and older adults (≥65 years). Coagulation parameters were tested, and reference intervals were determined. The indirect method applied the refineR algorithm to analyze coagulation data from non-ICU patients during the same period. The performance of both methods was evaluated in healthy participants, relatively healthy patients, and patients with high disease prevalence. The results showed that the direct method yielded similar reference intervals for APTT, PT, and TT in younger and older groups, with combined intervals of 23.7-31.2 seconds, 10.4-12.8 seconds, and 14.7-17.5 seconds, respectively. However, the Fib showed a significant difference between the two groups ( U=1 052 023, P<0.01), and separate reference intervals were established: 1.7-4.5 g/L for the younger group and 2.1-4.6 g/L for the older group. The reference intervals derived by the indirect method are similar to those from the direct method: APTT 23.8-31.4 seconds, PT 10.4-13.0 seconds, TT 14.5-17.4 seconds, and Fib 1.8-4.3 g/L (18-64 years) and 2.1-4.5 g/L (≥65 years). In relatively healthy patients, the reference intervals obtained by both methods were comparable, but significant differences were observed in patients with high disease prevalence. In conclusion, this study established localized reference intervals for the CN-6000 fully automated coagulation analyzer. The indirect method can serve as an alternative to the direct method in healthy and relatively healthy populations. However, its applicability in populations with high disease prevalence is limited and requires cautious interpretation in the context of clinical settings.
7.Identification of osteoid and chondroid matrix mineralization in primary bone tumors using a deep learning fusion model based on CT and clinical features: a multi-center retrospective study.
Caolin LIU ; Qingqing ZOU ; Menghong WANG ; Qinmei YANG ; Liwen SONG ; Zixiao LU ; Qianjin FENG ; Yinghua ZHAO
Journal of Southern Medical University 2024;44(12):2412-2420
METHODS:
We retrospectively collected CT scan data from 276 patients with pathologically confirmed primary bone tumors from 4 medical centers in Guangdong Province between January, 2010 and August, 2021. A convolutional neural network (CNN) was employed as the deep learning architecture. The optimal baseline deep learning model (R-Net) was determined through transfer learning, and an optimized model (S-Net) was obtained through algorithmic improvements. Multivariate logistic regression analysis was used to screen the clinical features such as sex, age, mineralization location, and pathological fractures, which were then connected with the imaging features to construct the deep learning fusion model (SC-Net). The diagnostic performance of the SC-Net model and machine learning models were compared with radiologists' diagnoses, and their classification performance was evaluated using the area under the receiver operating characteristic curve (AUC) and F1 score.
RESULTS:
In the external test set, the fusion model (SC-Net) achieved the best performance with an AUC of 0.901 (95% CI: 0.803-1.00), an accuracy of 83.7% (95% CI: 69.3%-93.2%) and an F1 score of 0.857, and outperformed the S-Net model with an AUC of 0.818 (95% CI: 0.694-0.942), an accuracy of 76.7% (95% CI: 61.4%-88.2%), and an F1 score of 0.828. The overall classification performance of the fusion model (SC-Net) exceeded that of radiologists' diagnoses.
CONCLUSIONS
The deep learning fusion model based on multi-center CT images and clinical features is capable of accurate classification of osseous and chondroid matrix mineralization and may potentially improve the accuracy of clinical diagnoses of osteogenic versus chondrogenic primary bone tumors.
Humans
;
Deep Learning
;
Bone Neoplasms/diagnostic imaging*
;
Retrospective Studies
;
Tomography, X-Ray Computed/methods*
;
Neural Networks, Computer
;
Male
;
Female
;
ROC Curve
;
Algorithms
8.Correlation study on the changes of retinal artery angle in idiopathic epiretinal membrane and its correlation with visual acuity and optical coherence tomography classification
Ziyi XIANG ; Jianbo MAO ; Qinmei WANG ; Zhengxi ZHANG ; Yijing CHEN ; Shian ZHANG ; Xiaoya ZHANG ; Jing ZHONG ; Lijun SHEN
Chinese Journal of Ocular Fundus Diseases 2024;40(3):190-195
Objective:To observe the change of retinal artery angle in eyes with idiopathic epiretinal membrane (ERM) and to analyze the relationship between retinal artery angle, ERM classification based on optical coherence tomography (OCT), and visual acuity.Methods:A retrospective cross-sectional clinical study. A total of 187 eyes in 187 patients diagnosed with monocular idiopathic ERM (IERM group) in Department of Ophthalmology of Zhejiang Provincial People's Hospital and the Affiliated Eye Hospital of Wenzhou Medical University at Hangzhou from November 2018 to January 2023 were included in the study. The contralateral healthy eyes were included as the control group. All patients underwent best corrected visual acuity (BCVA), fundus photography, spectral-domain OCT, OCT angiography (OCTA) and axial length (AL) measurement. BCVA examination was performed using the standard logarithmic visual acuity chart, which was converted to the logarithm of the minimum angle of resolution (logMAR) visual acuity. The foveal avascular zone (FAZ) area was measured by OCTA. The central macular thickness (CMT) was measured by spectral domain OCTaccording to the grading criteria of ectopic inner foveal layer (EIFL) was divided into stages 1 to 4 with 42, 45, 62, and 38 eyes, and the IERM group was subdivided into stage 1, stage 2, stage 3, and stage 4 groups accordingly. Image J was used to measure the retinal artery angle and the 1/2 retinal artery angle on fundus images. Multiple linear regression analysis was used to analyze the correlation between BCVA and artery angle, 1/2 artery Angle, CMT, FAZ area and AL.Results:Compared with the control group, eyes in IERM group had worse BCVA ( t=9.727), thicker CMT ( t=12.452), smaller FAZ area ( t=-14.329), smaller artery angle ( t=-9.165) and smaller 1/2 artery angle ( t=-9.549). The differences were statistically significant ( P<0.001). With the increase of IERM stage, the artery angle and 1/2 artery angle decreased significantly ( F=21.763, 12.515; P<0.001). There was no significant difference in artery angle and 1/2 artery angle between stage 1 group and stage 2 group, and 1/2 arterial angle between stage 2 group and stage 3 group ( P>0.05). There were significant differences in artery angle and 1/2 artery angle between the other groups ( P<0.05). There were significant differences in CMT and logMAR BCVA among different classification subgroups in IERM groups ( P<0.05). There was no significant difference in FAZ area between grade 3 group and grade 4 group ( P>0.05). There were significant differences in FAZ area between the other groups ( P<0.05). Correlation analysis showed that decreased artery angle ( P=0.013) and increased CMT ( P<0.001) were associated with decreased BCVA. Conclusions:Compared with healthy eyes, the artery angle decreases significantly with the increase of ERM stage. Decreased retinal artery angle is associated with decreased visual acuity in IERM eyes.
9.Effect of NOD-like receptor family pyrin domain containing 3 knockdown on a mouse model of nonalcoholic steatohepatitis induced by high-fat high-carbohydrate diet
Qian HUANG ; Zhuoyuan WANG ; Ziming AN ; Xin XIN ; Qinmei SUN ; Xiaojun GOU ; Yiyang HU ; Qin FENG
Journal of Clinical Hepatology 2024;40(5):952-960
Objective To investigate the effect of NOD-like receptor family pyrin domain containing 3(NLRP3)knockdown on a mouse model of nonalcoholic steatohepatitis(NASH)induced by high-fat high-carbohydrate(HFHC)diet.Methods A total of 44 mice were randomly divided into normal diet group(CON group)with 20 mice and HFHC group with 24 mice.At the end of week 14 of modeling,4 mice were randomly selected from the HFHC group for the pre-experiment of adeno-associated virus(AAV)by tail vein injection,and NLRP3 knockdown was verified after 4 weeks.After NLRP3 knockdown was verified at the end of week 18,the remaining 40 mice were given a single tail vein injection of AAV,and then they were divided into CON+NLRP3 knockdown negative control group(CON+NLRP3-NC group),CON+NLRP3 knockdown group(CON+NLRP3-KD group),HFHC+NLRP3-NC group,and HFHC+NLRP3-KD group,with 10 mice in each group.At the end of week 24,the activation of NLRP3 inflammasome was observed;related indicators were measured,including body weight,liver weight,liver index,and glucose metabolism(fasting blood glucose,fasting insulin,and Homeostasis Model Assessment of Insulin Resistance[HOMA-IR]index);the indicators of liver lipid content(liver triglyceride[TG]and oil red O staining),liver inflammation(serum alanine aminotransferase[ALT]activity,HE staining,and inflammation-related genes),and liver fibrosis(Sirius Red staining and fibrosis-related genes)were measured.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the CON+NLRP3-NC group based on the results of Western Blot,the HFHC+NLRP3-NC group had significant increases in the protein expression levels of NLRP3,pro-Caspase1,Caspase1,ASC,and IL-1β,while the HFHC+NLRP3-KD group had significant reductions in these levels(all P<0.05).The HFHC+NLRP3-NC group showed varying degrees of increase in body weight,liver weight,liver index,and glucose metabolism indicators,while the HFHC+NLRP3-KD group showed significant improvements in these indicators(all P<0.05).As for hepatic fat deposition,compared with the CON+NLRP3-NC group,the HFHC+NLRP3-NC group had a significant increase in liver TG,with a large number of red lipid droplets shown by oil red O staining,and the HFHC+NLRP3-KD group had significant reductions in liver TG and the number of lipid droplets in the liver(all P<0.01).In terms of liver inflammation,compared with the CON+NLRP3-NC group,the HFHC+NLRP3-NC group had significant increases in serum ALT,NAFLD activity score,and inflammation-related genes,while the HFHC+NLRP3-KD group had significant reductions in these indicators(all P<0.01).As for liver fibrosis,compared with the CON+NLRP3-NC group,the HFHC+NLRP3-NC group had significant increases in collagen fiber area and fibrosis-related genes,and the HFHC+NLRP3-KD group had significant reductions in fibrosis-related genes(all P<0.05)and a tendency of reduction in collagen fiber area(P>0.05).Conclusion NLRP3 knockdown can significantly improve hepatic fat deposition and inflammation in a mouse model of HFHC-induced NASH.
10.Attraction of different concentrations of glucose solution to Aedes albopictus, Culex pipiens pallens and their egg-laying behaviors
WU Yuyan ; CHEN Enfu ; LIU Qinmei ; LI Tianqi ; WANG Jinna ; LUO Mingyu ; GONG Zhenyu
Journal of Preventive Medicine 2024;36(6):543-547
Objective:
To explore the effects of different concentrations of glucose solution on the survival of Aedes albopictus and Culex pipiens pallens larvae, the attraction to mosquitoes and egg-laying behaviors, so as to provide the reference for developing mosquito control technology based on sugar bait.
Methods:
White porcelain bowls were filled with 100 mL of 3%, 5%, 8%, 10% and 15% glucose solutions. Ten of fourth instar larvae of Aedes albopictus or Culex pipiens pallens were added to each bowl, and the survival of larvae was recorded after 2, 4, 6, 24, 48 and 72 hours. Egg-laying cups containing 5%, 8% and 15% glucose solution were put in mosquito cages containing fully blooded female mosquitoes of Aedes albopictus and Culex pipiens pallens (50 mosquitoes each), and the total number of eggs laid in 72 hours was observed. The analogous site room was filled with fully blooded and starved female mosquitoes of Aedes albopictus and Culex pipiens pallens (100 mosquitoes each), and simple mosquito control buckets containing 5% and 8% glucose solution and black sticky insect plates. The number of mosquitoes and eggs was observed after 6 days. All the above experiments were repeated 3 times using dechlorinated water as the control.
Results:
The 72 hour corrected mortality rates of Aedes albopictus and Culex pipiens pallens larvae gradually increased with the increase of glucose concentration. The glucose solution with 5% and higher concentrations was not suitable for mosquito larvae to survive. The attraction of egg-laying behaviors to Aedes albopictus and Culex pipiens pallens gradually decreased with the increase of glucose concentration. The effects were similar between 5% and 8% glucose solution, with the averages of 686.67 and 682.33 eggs for Aedes albopictus, and 3.00 and 2.33 egg rafts for Culex pipiens pallens. In analogous site room, there were 93.33, 105.00 and 130.33 adult mosquitoes captured on average in the control group, 5% and 8% glucose solution groups, respectively, with 8% glucose solution group more attractive to adult mosquitoes than the control group (F=3.283, P=0.030); there were 70.33, 55.33 and 63.00 Aedes albopictus eggs (eggs counts+larvae counts) on average, respectively, with statistically significant differences among the three groups (H=6.761, P=0.034).
Conclusion
Glucose solution with concentration of 5% or higher can effectively inhibit the survival of Aedes albopictus and Culex pipiens pallens larvae, and attractive to adult mosquitoes and egg-laying behavoirs.


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