Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.
Humans ; Animals ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; B7-H1 Antigen/metabolism* ; Ligands ; Liver Neoplasms/pathology* ; RNA, Small Interfering/metabolism* ; Neoplastic Stem Cells/physiology* ; Cell Line, Tumor ; Cell Proliferation

Lung cancers are the leading cause of cancer deaths worldwide and pose a grave threat to human life and health. Non-small cell lung cancer (NSCLC) is the most frequent malignancy occupying 80% of all lung cancer subtypes. Except for other mutations (

BACKGROUND: A patient's infectivity is determined by the presence of the virus in different body fluids, secretions, and excreta. The persistence and clearance of viral RNA from different specimens of patients with 2019 novel coronavirus disease (COVID-19) remain unclear. This study analyzed the clearance time and factors influencing 2019 novel coronavirus (2019-nCoV) RNA in different samples from patients with COVID-19, providing further evidence to improve the management of patients during convalescence. METHODS: The clinical data and laboratory test results of convalescent patients with COVID-19 who were admitted to from January 20, 2020 to February 10, 2020 were collected retrospectively. The reverse transcription polymerase chain reaction (RT-PCR) results for patients' oropharyngeal swab, stool, urine, and serum samples were collected and analyzed. Convalescent patients refer to recovered non-febrile patients without respiratory symptoms who had two successive (minimum 24 h sampling interval) negative RT-PCR results for viral RNA from oropharyngeal swabs. The effects of cluster of differentiation 4 (CD4)+ T lymphocytes, inflammatory indicators, and glucocorticoid treatment on viral nucleic acid clearance were analyzed. RESULTS: In the 292 confirmed cases, 66 patients recovered after treatment and were included in our study. In total, 28 (42.4%) women and 38 men (57.6%) with a median age of 44.0 (34.0-62.0) years were analyzed. After in-hospital treatment, patients' inflammatory indicators decreased with improved clinical condition. The median time from the onset of symptoms to first negative RT-PCR results for oropharyngeal swabs in convalescent patients was 9.5 (6.0-11.0) days. By February 10, 2020, 11 convalescent patients (16.7%) still tested positive for viral RNA from stool specimens and the other 55 patients' stool specimens were negative for 2019-nCoV following a median duration of 11.0 (9.0-16.0) days after symptom onset. Among these 55 patients, 43 had a longer duration until stool specimens were negative for viral RNA than for throat swabs, with a median delay of 2.0 (1.0-4.0) days. Results for only four (6.9%) urine samples were positive for viral nucleic acid out of 58 cases; viral RNA was still present in three patients' urine specimens after throat swabs were negative. Using a multiple linear regression model (F = 2.669, P = 0.044, and adjusted R = 0.122), the analysis showed that the CD4+ T lymphocyte count may help predict the duration of viral RNA detection in patients' stools (t = -2.699, P = 0.010). The duration of viral RNA detection from oropharyngeal swabs and fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (15 days vs. 8.0 days, respectively; t = 2.550, P = 0.013) and the duration of viral RNA detection in fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (20 days vs. 11 days, respectively; t = 4.631, P < 0.001). There was no statistically significant difference in inflammatory indicators between patients with positive fecal viral RNA test results and those with negative results (P > 0.05). CONCLUSIONS In brief, as the clearance of viral RNA in patients' stools was delayed compared to that in oropharyngeal swabs, it is important to identify viral RNA in feces during convalescence. Because of the delayed clearance of viral RNA in the glucocorticoid treatment group, glucocorticoids are not recommended in the treatment of COVID-19, especially for mild disease. The duration of RNA detection may relate to host cell immunity.
Adult ; Aged ; Betacoronavirus ; genetics ; Clinical Laboratory Techniques ; Coronavirus Infections ; diagnosis ; genetics ; rehabilitation ; Female ; Humans ; Male ; Middle Aged ; Pandemics ; Pneumonia, Viral ; genetics ; rehabilitation ; RNA, Viral ; genetics ; Real-Time Polymerase Chain Reaction ; Retrospective Studies

Background: A patient’s infectivity is determined by the presence of the virus in different body fluids, secretions, and excreta. The persistence and clearance of viral RNA from different specimens of patients with 2019 novel coronavirus disease (COVID-19) remain unclear. This study analyzed the clearance time and factors influencing 2019 novel coronavirus (2019-nCoV) RNA in different samples from patients with COVID-19, providing further evidence to improve the management of patients during convalescence. Methods: The clinical data and laboratory test results of convalescent patients with COVID-19 who were admitted to from January 20, 2020 to February 10, 2020 were collected retrospectively. The reverse transcription polymerase chain reaction (RT-PCR) results for patients’ oropharyngeal swab, stool, urine, and serum samples were collected and analyzed. Convalescent patients refer to recovered non-febrile patients without respiratory symptoms who had two successive (minimum 24 h sampling interval) negative RT-PCR results for viral RNA from oropharyngeal swabs. The effects of cluster of differentiation 4 (CD4)+ T lymphocytes, inflammatory indicators, and glucocorticoid treatment on viral nucleic acid clearance were analyzed. Results: In the 292 confirmed cases, 66 patients recovered after treatment and were included in our study. In total, 28 (42.4%) women and 38 men (57.6%) with a median age of 44.0 (34.0–62.0) years were analyzed. After in-hospital treatment, patients’ inflammatory indicators decreased with improved clinical condition. The median time from the onset of symptoms to first negative RT-PCR results for oropharyngeal swabs in convalescent patients was 9.5 (6.0–11.0) days. By February 10, 2020, 11 convalescent patients (16.7%) still tested positive for viral RNA from stool specimens and the other 55 patients’ stool specimens were negative for 2019-nCoV following a median duration of 11.0 (9.0–16.0) days after symptom onset. Among these 55 patients, 43 had a longer duration until stool specimens were negative for viral RNA than for throat swabs, with a median delay of 2.0 (1.0–4.0) days. Results for only four (6.9%) urine samples were positive for viral nucleic acid out of 58 cases; viral RNA was still present in three patients’ urine specimens after throat swabs were negative. Using a multiple linear regression model (F=2.669, P=0.044, and adjusted R²=0.122), the analysis showed that the CD4+ T lymphocyte count may help predict the duration of viral RNA detection in patients’ stools (t=-2.699, P=0.010). The duration of viral RNA detection from oropharyngeal swabs and fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (15 days vs 8.0 days, respectively; t=2.550, P=0.013) and the duration of viral RNA detection in fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (20 days vs 11 days, respectively; t=4.631, P <0.001). There was no statistically significant difference in inflammatory indicators between patients with positive fecal viral RNA test results and those with negative results (P >0.05). Conclusions In brief, as the clearance of viral RNA in patients’ stools was delayed compared to that in oropharyngeal swabs, it is important to identify viral RNA in feces during convalescence. Because of the delayed clearance of viral RNA in the glucocorticoid treatment group, glucocorticoids are not recommended in the treatment of COVID-19, especially for mild disease. The duration of RNA detection may relate to host cell immunity.

Background: Brain death is the irreversible cessation of the function of the brain including the brainstem. In 2013, the Brain Injury Evaluation Quality Control Centre (BQCC) of the National Health and Family Planning Commission issued criteria and practical guidelines for the determination of brain death. This study aimed to evaluate whether the institutions have adopted these guidelines and to make suggestions for the improvement of the current criteria and practical guidelines for brain death determination in China. Methods: Consecutive brain death cases from 44 hospitals were evaluated for summary statistics for the following data: the performance of BQCC criteria and practical guidelines, clinical examination, apnea testing, ancillary testing, and the number of examinations as well as the waiting periods between examinations and details of who determined brain death. Data analysis was conducted from January 2013 to December 2017. Results: A total of 550 cases were obtained. All patients were determined to have deep coma and met the prerequisites for clinical testing. The performance rates of four brainstem reflex examinations (except cough reflex) ranged from 97.5% to 98.0%, and the completion rate as well as the coincidence rate were both 100.0%. The 238 cases (50.7%) completed apnea testing, and 231 cases (42.0%) had to stop apnea testing during the examination because of instability. The performance rates of the three ancillary tests, including electroencephalogram, short-latency somatosensory evoked potential, and transcranial Doppler, were 89.5%, 67.5%, and 79.5%, respectively; furthermore, the coincidence rates were 98.6%, 96.5%, and 99.5%, respectively. The combination of two ancillary tests was more accurate than one single ancillary test. A total of 401 (72.9%) cases successfully underwent two separate examinations to determine brain death with at least a 12-h waiting period. All brain death cases were determined by at least two qualified physicians. Conclusion This study might provide suggestions for brain death determination in China.
Brain Death ; diagnosis ; physiopathology ; Electroencephalography ; Evoked Potentials, Somatosensory ; Humans ; Ultrasonography, Doppler, Transcranial

This study was aimed to explore differential effects of various sections of the velvet antler on promoting cell proliferation in vitro. The NRK-49F cell line from rat kidney fibroblasts was used as the cell model. The cell proliferation rates of the water extracts from the upper, middle and lower section of fresh velvet antler were measured by the MTT method. BCA method was used in the detection of protein concentration. The SDS-PAGE method was used in the analysis of difference composition of the sample protein. The results showed that soluble protein content of the upper, middle and lower section were 17.89, 16.04 and 6.89 mg·mL-1, respectively. From the top to the base, the soluble protein content of velvet antler was decreased. After 24 h treatment, when the protein concentration of the upper and middle section samples of the velvet antler were 800 μg·mL-1 and 600 μg·mL-1, the cell proliferation promoting rates reached the maximum, which were 66.76% and 64.36%, respectively. And when the lower section sample of the velvet antler was 1 000 μg·mL-1, the cell proliferation promoting rates reached the maximum, which was 58.87%. After 48 h treatment, when the upper and middle section samples of the velvet antler were 800μg·mL-1, the cell proliferation promoting rates reached the maximum, which were 219.56% and 215.86%, respectively. And when the lower section sample of the velvet antler was 1 000 μg·mL-1, the cell proliferation promoting rates reached the maximum, which was 169.20%. The velvet antler on the proliferation of cells was much better than the 10% fetal bovine serum. The figure of SDS-PAGE showed the slight difference in the protein composition of three part of the velvet antler. It was concluded that all samples had promoting effects on cell proliferation with concentration-depen-dent, and the main protein in different part of the velvet antler had minor differences.

Objective To evaluate the therapeutic efficacy of stereotactic body radiotherapy (SBRT) with gamma knife on stage Ⅰ-Ⅱ non-small-cell lung cancer(NSCLC)and the quality of life of the patients undergoing this therapy.Methods Twenty NSCLC patients with the median age of 76,10 at stage Ⅰ and 10 at stage Ⅱ who were unable or unwilling to undergo surgery were given SBRT with gamma knife at the doses of 3-6 Gy in 8-15 fractions,finished within 2 to 3 weeks.The prescription isodose line was 50％,the marginal dose was 39-56 Gy,the central dose was 78-112 Gy,and the total biologically effective dose was 51-83 Gy.The patients were observed after admission and followed up by chest CT 1,3,6,and 12 months after treatment until progressive disease or death.EORTC QLQ-LC43 questionnaire was used to investigate the changes in quality of life.Results The 20 patients were followed up for 24 (12-46) months.At six months after the treatment,the overall response rate was 80％,and the complete response rate was 35％.The 1,2 and 3-year local control rates were 100％,95％ and 95％,respectively.The 1,2 and 3-year overall survival rates were 95％,80％ and 50％ respectively; The 1,2,and 3-year progression free survival rates were 85％,64％ and 33％,respectively.The failure rate was 20％ and the rate of progress within the planning target volume was 5％.No acute toxicity at grade 3 and over occurred in any patient during the treatment.15％ of the patients developed grade 1-2 radiation pneumonia.Age,gender,pathologic index or not were weakly correlated with the overall survival.The emotional function was improved significantly after treatment (P ＜ 0.05),dyspnea and cough were improved at different degrees,however,not significantly.There were no significant changes in the physical function and symptoms,such as fatigue,lack of appetite,insomnia,etc.Conclusions Significantly improving the motional function and maintaining the quality of life,SBRT with gamma knife is effective for elderly NSCLC patients with high local control rate fair overall survival rate and few side effects.

Objective To analyze the efficacy and prognosis of stereotactic radiotherapy (SRT) and whole-brain radiotherapy (WBRT) in treatment of brain metastases,and to observe the influence of temozolomide (TMZ) on survival rate during the period of radiotherapy.Methods A total of 52 patients with brain metastases were divided into two groups according to treatment methods,including 35 patients treated with WBRT plus SRT and 17 patients treated with SRT alone.WBRT dose was 1.8 - 3.0 Gy per fraction,one fraction a day,five fractions per week,with total dose of 30 - 40 Gy.After WBRT,gamma knife was performed with prescription isodose line of 45％ -70％ surrounding the planned target volume in WBRT + SRT group.The marginal dose was 12 - 15 Gy and the center dose was 20-30 Gy.In SRT group,the prescription isodosc line was 45％ - 70％ and the marginal dose was 36 - 40 Gy while the center up to 70 - 80 Gy.The follow up time was 1 - 2 years.Besides 20 patients in this study took temozolomide capsule during and after radiotherapy.The schedule of concomitant chemotherapy was temozolomide of 75 mg/m2 by oral administration every day until radiotherapy was over,and then temozolomide of 150 mg/m2 was taken for 3 -6 months after radiotherapy.Results The efficiency during 1 -3 months after treatment was 84.62％ in this study.In the WBRT + SRT group,the efficiency was 88.57％ and declined to 76.47％ in the SRT group.The six month-and one year-local control rate were 92.10％ and 85.20％,respectively.The average survival time of WBRT + SRT was 13.2 months and median survival time was 11 months.Six month-,one year-and eighteen months-survival rate were 71.40％,54.30％ and 14.30％,respectively.In the SRT group,the average survival time was 10.2 months and median survival time was 9 months.Six month-,one year- and eighteen month-survival rate were 41.20％,23.50％ and 5.88％,respectively,while those for RT + TMZ group were 80.00％,60.00％ and 10.00％.In comparison,those in RT group were 56.30％,37.50％ and 12.50％,respectively.Conclusions Effect of gamma knife stereotactic radiotherapy combined with WBRT is better than GK stereotactic radiotherapy alone in treatment of brain metastases.Compared with radiotherapy alone,concomitant temozolomide chemotherapy could improve the survival rate of the patients with brain metastases without increasirg adverse reactions significantly.

OBJECTIVEDuring 2003-2005, an outbreak of meningitis due to Neisseria meningitidis serogroup C occurred in China. With the aim to find strain clues result in the final epidemics, the ancestral strain 053442, a clinical isolate, and a carrier strain 053426 with different gene type were analyzed.

METHODSClinical strain 053442 and carrier strain 053426 were cultured on GC agar plates under the same condition. Two-dimensional electrophoresis was performed using the pH 3-10 nonlinear IPG strips of 24 cm length, and all the protein spots were identified by matrix-assisted laser desorption/ionization time of flight spectrometry.

RESULTS502 and 380 protein spots were identified in 053426 and 053442 respectively, relating to 266 and 202 different genes covering a wide range of cellular functions. The express volume and number of proteins involved in energy metabolism, protein synthesis and amino acid biosynthesis in 053426 were higher than in 053442. Virulence factor Opa, Opc and a series of proteins involved in pilus assembly and retraction were identified in 053442, which appear to be of primary importance in colonization and invasion of human cells. Compared to 053442, virulence protein species were less in 053426, with lower express volumes too. No Opa and Opc were detected in 053426.

CONCLUSIONSThe different protein expression profiles of the clinical strain 053442 and carrier strain 053426 in the present study provide some clues of the different pathogenicity of the two strains, which may account for result in the final epidemics.

Bacterial Proteins ; analysis ; Bacterial Typing Techniques ; China ; epidemiology ; Disease Outbreaks ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Meningitis, Meningococcal ; cerebrospinal fluid ; epidemiology ; microbiology ; Neisseria meningitidis, Serogroup C ; classification ; isolation & purification ; Proteome ; analysis ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Objective To investigate the epidemiological characteristics of non-toxic goiter and non-toxic thyroid nodules in the regions with different iodine intakes and the factors influencing the occurrence, development and outcome of goiter and thyroid nodules. Methods 3 385 subjects, who had taken part in the previous survey in 1999 with the ultrasonic examination of thyroid, were composed of individuals in Panshan with chronic mild iodine deficiency,in Zhangwu with more than adequate iodine "after iodine supplementation and in Huanghua with excessive iodine. These 3 groups of subjects were followed up in 2004. Results (1) The cumulative incidences of diffuse goiter in Panshan ,Zhangwu and Huanghua were 7.1% ,4.4% and 6.9% ,respectively ,being the lowest in Zhangwu (P<0.01) and those of nodular goiter were 5.0% ,2. 4% and 0.8%, respectively, being the highest in Panshan (P<0.01). (2) The incidences of single nodule were 4.0% ,5.7% and 5.6%, respectively, and those of multiple nodules 0.4%, 1.2% and 1.0%, respectively. (3)The result of logistic analysis showed that iodine deficiency,iodine excess and positive thyroid autoantibodies (TAA) were the independent risk factors for the occurrence of goiter. (4)In Zhangwu ,the incidence of non-toxic goiter in the group with positive TAA was higher than that in the group with negative TAA(P<0.01) ,while there were no such differences in Panshan and Huanghua. (5)In these three regions, the rates of positive TAA in the individuals with diffuse non-toxic goiter were higher than those in the healthy subjects (P<0.05). And in Huanghua,the rate of positive TAA in subjects with non-toxic nodular goiter was also higher than that in the healthy individuals (P<0.05). Conclusion Iodine deficiency and iodine excess may both induce the raising incidence of goiter. Nodular goiter is prevalent in iodine deficient district and diffuse goiter is the predominant form in places with iodine excess. Thyroid autoimmunity is associated with occurrence and maintenance of goiter, and this phenomenon is more obvious in the community with previous iodine deficiency followed then by treatment with more than adequate iodine.