OBJECTIVETo establish real-time PCR and loop-mediated isothermal amplification (LAMP) systems for detecting Cryptococcus neoformans CAP10 gene.

METHODSSpecific primers were designed targeting CAP10 gene of Cryptococcus neoformans, and the plasmid was constructed. After optimization of the reaction condition, the plasmid was quantitatively detected using real-time PCR and LAMP, and the detection sensitivity and specificity were evaluated. Clinical samples were also detected using the two methods.

RESULTSThe detection sensitivity of real-time PCR and LAMP was 6.8×10(1) and 6.8×10(3) copies, respectively. Real-time PCR yielded a higher positivity rate than LAMP. Both of the two methods showed a high detection specificity and produced negative results in the detection of Neisseria meningitidis, Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus niger and Escherichia coli.

CONCLUSIONReal-time PCR is highly sensitive and specific for detecting Cryptococcus neoformans CAP10 gene but requires sophisticated equipment. LAMP, though with a relatively lower sensitivity, is simple to operate without the need of special equipment, and the result can be conveniently observed. Both of the two methods are suitable for detecting Cryptococcus neoformans and evaluating the treatment outcomes.

Cryptococcus neoformans ; genetics ; Fungal Proteins ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Plasmids ; RNA, Fungal ; genetics ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Objective To explore the expression of TLR4/NF-κB pathway in cerebral injury resulting from DHCA ( deep hypothermia circulatory arrest ) as well as the effect of SACP ( selective antegrade cerebral perfusion). Methods Twelve pigs were randomly assigned to DHCA group (n = 6) or SACP group (n = 6) at 18 ℃ for 80 min. IL-6 was assayed by ELISA. Apoptosis and NF-κB proteins were detected by fluorescence TUNEL and Western blot, respectively. The level of TLR4 was determined through qRT-PCR and Western blot. Results Serum IL-6 level of SACP group was significantly lower at the end of circulation arrest and experiment and apoptotic index and NF-κB protein were apparently lower in SACP group (P < 0.05). The level of TLR4 protein and mRNA from SACP group decreased significantly (P < 0.05). Conclusions TLR4/NF-κB pathway plays a critical role in pathogenesis of DHCA cerebral injury and attenuating TLR4/NF-κB cytokines probably contributes to neuroprotection of SACP. TLR4/NF-κB pathway may be a novel target for DHCA.

Objective The primary culture of synovial fibroblasts is a convenient tool to study the pathology and physiology of synovial tissues .An improved method was constructed in this study by C57BL /6 mice to study the mechanism of rheumatoid ar-thritis(RA) .Methods The synovium around the hip joints were collected .Attention should be paid to eliminate the egg-yolk like yellow oval substance in the middle of the synovium .The synovium was transferred into a 1 .5 mL Eppendorf tube containing 0 .5%type Ⅳ collagenase and cut into 1 mm3 blocks or so .The Eppendorf tube was placed in 37 ℃ Constant temperature orbital shaker incubator for 60 min .After digestion ,the tube was placed on the Vortex for a high-speed oscillation for 1 .5 minutes to guarantee the separation of cells .Results Within about 1 week ,the first passage was performed by the trypsin digestion method .On day 10 , the number of synovial macrophages reached the maximum and then decreased gradually .After the third generation (day 15 to 20) , the synovial macrophages generally disappeared .Vimentin was suitable for the immunofluorescence cytochemical staining for the synovial fibroblasts .The cell purity was indicated as > 95% .The cytometric analysis indicated that purity of Vimentin and CD90 .2-labelled cells was over 95% ;the purity of CD54-labelled cells was 80% approximately .Conclusion It is a simple and effective method for primary culture of synovial fibroblasts in mice .

Objective To investigate the concentrations of indoor radon (222Rn) and its daughter products as well as indoor thoron (220Rn) in selected houses in Yuhang district and Sanmen county,Zhejiang province,and estimate their annual effective doses to the population.Methods Solid state nuclear track detectors were used in selected dwellings in Yuhang district and Sanmen county,and the detectors were placed in bedrooms or living rooms.Without changing the ventilation habits of residents,These detectors were continuously placed from March to September in 2009.Results Indoor 222 Rn and 220Rn concentrations in low-rise buildings were the highest among all types of houses.The indoor concentration of 222 Rn had no relation with the building age (F = 0.53,P ＞ 0.05),but that of 220 Rn was dependent on the building age (F = 3.56,P ＜ 0.05).Moreover,the investigation demonstrated indoor 220 Rn concentrations in houses with no decoration were higher than in the houses decorated (t = 2.33,P ＜0.05).The average indoor concentrations of 222Rn and 220Rn in Yuhang district were 32.5 Bq/m3 and 314.3 Bq/m3,respectively,and the annual effective doses were 0.88 mSy and 0.42 mSv respectively.The average indoor concentrations of 222Rn and 220Rn in Sanmen county were 26.8 Bq/m3 and 399.5 Bq/m3,and the annual effective doses were 0.72 mSy and 0.53 mSv respectively.Conclusion The concentrations of indoor 222 Rn in some areas of Zhejiang province are at natural background level,and the concentrations of indoor 220Rn in rural areas are relatively higher.The total annual effective dose from 220Rn and its progeny was larger than that from 222Rn and its progeny by 50 percents.

OBJECTIVETo apply pyrosequencing technique in the detection of the common pathogens in sepsis.

METHODSThe primers for amplification and sequencing in pyrosequencing were designed according to alignment of the bacterial 16S rRNA sequence. Bacterial genomic DNA was extracted for pyrosequencing, and the pathogen species were determined according to the sequencing data obtained.

RESULTSPyrosequencing effectively yielded the sequencing data of the 28 bp sequences of the pathogens and clearly distinguished the pathogen species of Streptococcus pyogenes, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Neisseria meningitides, and Salmonella, but failed to distinguish Staphylococcus epidermidis from Staphylococcus aureus.

CONCLUSIONPyrosequencing technique can effectively distinguish the common pathogens in sepsis at the species level.

Bacteria ; classification ; isolation & purification ; DNA Primers ; DNA, Bacterial ; genetics ; Microbiological Techniques ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sepsis ; microbiology

OBJECTIVETo explore the effect of quercetin on the invasion, migration, proliferation and cell cycle of glioma U87 cells.

METHODSGlioma U87 cells were treated with 50, 100, or 150 µmol/L quercetin (Q(50), Q(100) and Q(150) groups, respectively) or with DMSO (Q(0) group). Transwell in vitro invasion and migration assays, Click-iT Edu test and flow cytometry were performed to evaluate the effect of quercetin on the invasion, migration, proliferation and cell cycle of U87 cells.

RESULTSAfter 36 h of quercetin treatment, the cells in Q(50), Q(100) and Q(150) groups showed invasive cell percentages (relative to Q(0) group) of 52.08%, 24.63%, and 13.13%, respectively (P<0.05). After quercetin treatment for 12 h, the migrating cell percentages (relative to Q(0) group) in Q(50), Q(100) and Q(150) groups were 49.46%, 26.78%, and 14.56%, respectively (P<0.05). After 24 h of quercetin treatment, the cell proliferation ratios in Q(0), Q(50), Q(100) and Q(150) groups were 25.21%, 18.38%, 16.74% and 15.24%; the cell percentages in phase G0/Gl were 71.14%, 72.71%, 69.29%, and 66.47%, phase S were 25.32%, 22.48%, 21.96%, and 23.32%, and phase G(2)/M were 3.53%, 4.80%, 8.75%, and 10.25% in the 4 groups, respectively, showing a significant difference between groups Q(100), Q(150) and group Q(0) in phase G(2)/M cell percentages (P<0.05).

CONCLUSIONSQuercetin can significantly inhibit the invasion, migration and proliferation of glioma U87 cells by blocking the cell cycle progression.

Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Glioma ; pathology ; Humans ; Quercetin ; pharmacology

OBJECTIVETo investigate the effect of quercetin on apoptosis and feedback regulation of MDM2-p53 in multiform glioblastoma U87 cells in vitro.

METHODSU87 cells exposed to different concentrations of quercetin (50, 100, and 150 µmol/L) were examined with flow cytometry, RT-PCR and Western blotting for detecting the cell apoptosis, MDM2 mRNA expression, and p53 and caspase-3 expressions.

RESULTSQuercetin induced obvious apoptosis in U87 cells in a concentration-dependent manner, with apoptosis rates of (12.40∓0.70)% at Q0, (22.53∓0.72)% at Q50, (29.06∓0.81)% at Q100, and (31.5∓0.45)% at Q150. Quercetin significantly increased the expressions of MDM2 mRNA and active caspase-3 protein but decreased the expression of p53 in the cells.

CONCLUSIONQuercetin promotes the apoptosis of multiform glioblastoma U87 cells mediated by caspase-3 and influences the feedback balance of MDM2-p53.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Glioma ; metabolism ; pathology ; Humans ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Quercetin ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism

Artificial intelligence (AI) is an emerging science and technology that studies and develops theories, methods, technologies, and application systems for simulating and expanding human intelligence. AI has made great breakthroughs in the field of intelligent medicine, and has shown great potential in the diagnosis and treatment of diabetic retinopathy (DR), retinopathy of prematurity, and other fundus diseases. A number of clinical trials on the application of AI technologies to DR screening have been carried out in the domestic and overseas, which not only have a high accuracy rate, but also save doctors' reading time and reduce the burden of society, medical work and patients. However, due to the lack of evaluation system for DR intelligent diagnosis technology, the accuracy of AI system still lacks of big data verification. Secondly, most of the color fundus photographs are taken in the posterior 45°, which only show the most vulnerable areas, making some lesions undetectable. In addition, the current DR screening system has not yet been applied to the clinic, most of which are in the stage of prospective research and trials. There are still many obstacles from the environment to the hospital or the clinic. Doctors cannot use real patient data to evaluate the AI system, so it is not popular in clinical practice. In the future, DR screening algorithms and diagnostic models can be further improved and established to make DR AI screening more accurate.

Objective:To investigate the effect of complex decongestive therapy(CDT) on elephantiasis and skin texture improvement.Methods:The clinical data of patients with elephantiasis of lower limbs who were admitted to the Department of Plastic Surgery, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from January 2016 to December 2018 were retrospectively analyzed. The patients with elephantiasis of different causes were treated with CDT for 1 course (4 weeks). The skin texture parameters (skin water content, transepidermal water loss, skin fibrosis), limb circumference and segmental edema were measured before and after treatment and analyzed by paired t test. Pearson correlation analysis was used to detect the correlation between segmental edema and skin texture parameters before and after treatment. The subjective feelings of patients were recorded during follow-up. A self-designed online questionnaire was used to follow up the patient’s disease control status, including the limb circumferences, changes in quality of life (life, work, social and emotion), the adaptability to bandages and elastic socks. Data were classified and analysed as excellent, good, medium and poor.Results:A total of 20 patients were included, including 16 females and 4 males. The average age was 50.45 years (ranged 9-70 years). 5 cases were primary lymphedema and 15 cases were secondary lymphedema. The duration of the disease ranged from 2 years to 27 years, with an average of 8.4 years. After 1 course of treatment, the skin texture was significantly improved. The skin water content decreased from 55.6%±7.4% before treatment to 42.1%±7.4% after treatment ( P< 0.001). The transepidermal water loss decreased from (14.981±5.699) g·m -2·h -1 before treatment to (9.312±2.590) g·m -2·h -1 after treatment. The skin fibrosis decreased from (0.087±0.042) N before treatment to (0.065±0.033) N after treatment ( P<0.001). The circumference of the affected limb decreased from (11.09±3.14) cm before treatment to (4.82±2.83)cm after treatment ( P<0.001). The segmental edema decreased from (4.00±2.14)L before treatment to (1.21±0.78) L after treatment. Segmental edema was positively correlated with skin water content, transepidermal water loss, and skin fibrosis ( r=0.447, r=0.429, r=0.751, P<0.05). The patients were followed up for 6 months after treatment. The circumference control was excellent in 13 cases, good in 5 cases and moderate in 2 cases. The quality-of-life changes were excellent in 16 cases, good in 3 cases, medium in 1 case. The adaptability of elastic material was excellent in 9 cases, good in 8 cases, medium in 2 cases and poor in 1 case. Conclusions:CDT can reduce subcutaneous edema, effectively reduce limb volume, decrease skin fibrosis, improve skin barrier function, improve skin texture, restore the appearance of the limb and improve patients’ quality of life.

Objective:To investigate the effect of complex decongestive therapy(CDT) on elephantiasis and skin texture improvement.Methods:The clinical data of patients with elephantiasis of lower limbs who were admitted to the Department of Plastic Surgery, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from January 2016 to December 2018 were retrospectively analyzed. The patients with elephantiasis of different causes were treated with CDT for 1 course (4 weeks). The skin texture parameters (skin water content, transepidermal water loss, skin fibrosis), limb circumference and segmental edema were measured before and after treatment and analyzed by paired t test. Pearson correlation analysis was used to detect the correlation between segmental edema and skin texture parameters before and after treatment. The subjective feelings of patients were recorded during follow-up. A self-designed online questionnaire was used to follow up the patient’s disease control status, including the limb circumferences, changes in quality of life (life, work, social and emotion), the adaptability to bandages and elastic socks. Data were classified and analysed as excellent, good, medium and poor.Results:A total of 20 patients were included, including 16 females and 4 males. The average age was 50.45 years (ranged 9-70 years). 5 cases were primary lymphedema and 15 cases were secondary lymphedema. The duration of the disease ranged from 2 years to 27 years, with an average of 8.4 years. After 1 course of treatment, the skin texture was significantly improved. The skin water content decreased from 55.6%±7.4% before treatment to 42.1%±7.4% after treatment ( P< 0.001). The transepidermal water loss decreased from (14.981±5.699) g·m -2·h -1 before treatment to (9.312±2.590) g·m -2·h -1 after treatment. The skin fibrosis decreased from (0.087±0.042) N before treatment to (0.065±0.033) N after treatment ( P<0.001). The circumference of the affected limb decreased from (11.09±3.14) cm before treatment to (4.82±2.83)cm after treatment ( P<0.001). The segmental edema decreased from (4.00±2.14)L before treatment to (1.21±0.78) L after treatment. Segmental edema was positively correlated with skin water content, transepidermal water loss, and skin fibrosis ( r=0.447, r=0.429, r=0.751, P<0.05). The patients were followed up for 6 months after treatment. The circumference control was excellent in 13 cases, good in 5 cases and moderate in 2 cases. The quality-of-life changes were excellent in 16 cases, good in 3 cases, medium in 1 case. The adaptability of elastic material was excellent in 9 cases, good in 8 cases, medium in 2 cases and poor in 1 case. Conclusions:CDT can reduce subcutaneous edema, effectively reduce limb volume, decrease skin fibrosis, improve skin barrier function, improve skin texture, restore the appearance of the limb and improve patients’ quality of life.