This study was aimed at comparing performance differences among three metagenomic sequencing platforms,MGISEQ-2000,Illumina NextSeq 2000,and Ion GeneStudio S5 Plus,to optimize the sequencing process for trace samples.The three sequencing platforms were used to perform high-throughput sequencing on DNA standards and simulated samples.Through analysis of the quality of raw data and microbial detection capabilities,systematic differences among platforms were compared.The sequencing results were optimized for trace samples by incorporation of exogenous nucleic acids during the li-brary preparation process.In terms of data output per batch and base quality,MGISEQ-2000 surpassed the other two plat-forms.Illumina NextSeq 2000 had the lowest proportion of duplicate reads,whereas Ion GeneStudio S5 Plus had the highest proportion,and significant differences were observed across platforms(P＜0.001).In sequencing uniformity,MGISEQ-2000 and Illumina NextSeq 2000 were superior to Ion GeneStudio S5 Plus.MGISEQ-2000 provided a substantial advantage in microbial detection capability(P＜0.001),but the advantage diminished with decreasing bacterial fluid concentration.Ion GeneStudio S5 Plus had the shortest duration for single-batch sequencing.Moreo-ver,for trace samples with DNA content ≤0.05 ng,the experi-mental group(with added exogenous nucleic acids)achieved a higher number of reads than the control group(without exogenous nucleic acids),with a 11.09±8.03 fold increase.In conclu-sion,the different sequencing platforms each had advantages and disadvantages,thus allowing researchers to choose the appro-priate platform according to specific needs.Furthermore,the addition of exogenous nucleic acids improved the microorganism detection efficiency,and provided better support for subsequent diagnosis and evaluation of results.

OBJECTIVE: To explore the mechanisms underlying the proliferative inhibition of Chinese herbal medicine Kang-Ai injection (KAI) in gastric cancer cells. METHODS: Gastric cancer cell lines MGC803 and BGC823 were treated by 0, 0.3%, 1%, 3% and 10% KAI for 24, 48 and 72 h, respectively. The cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The apoptosis and cell cycle were evaluated by flow cytometry. Interleukin (IL)-6 mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA), respectively. The protein expression levels of cyclin A, cyclin E, cyclin B1, cyclin D1, p21, retinoblastoma (RB), protein kinase B (AKT), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT) 1 and STAT3 were detected by Western blot. RESULTS: KAI inhibited the proliferation of MGC803 and BGC823 gastric cancer cells in dose- and time-dependent manner. After treated with KAI for 48 h, the proportion of G1 phase was increased, expression level of cyclin D1 and phosphorylation-RB were down-regulated, whereas the expression of p21 was up-regulated (all P<0.01). Furthermore, 48-h treatment with KAI decreased the phosphorylation level of STAT3, inhibited the mRNA and protein expressions of IL-6 (all P<0.01). IL-6 at dose of 10 ng/mL significantly attenuated the proliferative effect of both 3% and 10% KAI, and recovered KAI-inhibited STAT3 phosphorylation and cyclin D1 expression level (all P<0.01). CONCLUSION KAI exerted an anti-proliferative function by inhibiting IL-6/STAT3 signaling pathway followed by the induction of G₁ phase arrest in gastric cancer cells.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1/pharmacology* ; Humans ; Interleukin-6/metabolism* ; RNA, Messenger/metabolism* ; STAT3 Transcription Factor/metabolism* ; Stomach Neoplasms/genetics*

【Objective】To evaluate the effects of methanol extract of Panax Notoginseng flower（PNFM）on platelet function in healthy human.【Methods】Platelet rich plasma were separated from venous blood of healthy volunteers and incubated with different concentrations（0，100，300 and 500 μg/mL）of PNFM for 20 min. After using ADP as agonist， granule-secretion were tested by CD62P expression and ATP release；integrin-αIIbβ3 activation was examined by PAC-1； Test platelet aggregation by turbidimetry ；Immunofluorescence examine platelet spreading on fibrinogen ；Changes in cytoplasmic calcium was studied using Fluo 3-AM，calcium ionophore. 【Results】After using ADP as agonist ，PNFM significantly inhibited platelet aggregation，compared to the control group（72.00±6.08），the 500μg/mL group decreased to 35.67±3.78（P<0.01）；Compared to the control group（30.05±6.48），PNFM reduced the CD62P expression on platelet surface，the 500 μg/mL group decreased to 2.66±0.90（P<0.001）；PNFM inhibited the expression of PAC-1 as a marker of the integrin- αIIbβ3 comformation，compared to the control group（33.37 ± 8.12），the 500 μg/mL group decreased to 11.89±6.12（P<0.01）；Compared to the control group（1.93±0.47），all dose groups attenuated platelet ATP release，the 500 μg/mL group decreased to 35.67±3.78（P<0.01）；Results demonstrated that 500 μg/mL PNFM markedly decreases the surface area of the spreading platelets（89.57±17.34 to 25.12±3.52，P<0.001），and all doses were affected；The Ca2 + mobilization was also reduced by all PNFM doses，compared to the control group（183.87 ± 11.59），the 500 μg/mL group was decreased to 71.25±5.33（P<0.001）.【Conclusions】PNFM attenuated platelet activation，spreading，and aggregation； Our results provided new ideas for prevention and treatment of cardiovascular and cerebrovascular diseases.

Background: Cervical cancer is the sixth most common cancer in Chinese women. A standard treatment modality for cervical cancer is the combination of surgery, chemotherapy, external-beam radiotherapy and intracavitary brachytherapy. The aim of this study was to retrospectively assess the long-term treatment outcomes of patients with cervical cancer who were treated with californium-252 neutron brachytherapy combined with external-beam radiotherapy plus concurrent chemotherapy.Methods: We retrospectively analyzed the medical records of 150 patients with primary stages IB-IVB cervical cancer who received neutron brachytherapy combined with external-beam radiotherapy concurrently with cisplatin chemotherapy.All patients were followed up. Using an actuarial analysis, patient outcomes and treatment-related adverse effects were evaluated and compared.Results: The median overall survival (OS) was 33.2 months. The 3-year progression-free survival rates for patients with stages Ⅰ—Ⅱ, Ⅲ, and Ⅳ diseases were 81.0% (68/84), 65.0% (39/60), and 0% (0/6), respectively; the 3-year OS rates were 90.5% (76/84), 85.0% (51/60), and 16.7% (1/6), respectively. Vaginal bleeding was controlled within the median time of 4.0 days. One month after treatment, 97.3% of patients achieved short-term local control. The local recurrence rates for patients with stages Ⅰ—Ⅱ, Ⅲ, and Ⅳ disease were 4.8% (4/84), 11.7% (7/60), and 33.3% (2/6), respectively, and the occurrence rates of distant metastasis were 16.7% (14/84), 25.0% (15/60), and 100.0% (6/6), respectively. Cancer stage,tumor size, and lymph node metastasis were identified as prognostic risk factors, but only lymph node metastasis was found to be an independent prognostic factor. The most common adverse effects during treatment were grades 1 and 2 irradiation-related proctitis and radiocystitis.Conclusion: For patients with cervical cancer, neutron brachytherapy combined with external-beam radiotherapy plus concurrent chemotherapy produces a rapid response and greatly improves local control and long-term survival rates with tolerable adverse effects.

BACKGROUNDAtrial fibrillation (AF) is the most frequent tachyarrhythmia in patients with a permanent pacemaker. Angiotensin II receptor antagonists have a protective effect against the occurrence of AF in patients with heart diseases. This study aimed to assess the effectiveness of olmesartan in the prevention of new-onset AF and AF burden in atrioventricular block (AVB) patients with dual-chamber (DDD) pacemaker implantation.

METHODSThis was a single-center, prospective, randomized, single-blind, controlled clinical study. A total of 116 AVB patients, who received DDD pacemakers implantation with the percentage of ventricular pacing (VP%) ≥40% from April 22, 2011 to December 24, 2012, were prospectively randomized to olmesartan group (20 mg per day; n = 57) or control group (n = 59). Patients were followed up using pacemaker programming, 12-lead electrocardiography in the intrinsic sinus rhythm, laboratory examinations, and transthoracic echocardiography at 24 months. Atrial high rate events (AHREs) were defined as 180 beats/min over a minimum of 5 min. AF burden was calculated by the number of hours with AHREs divided by the number of measurement hours.

RESULTSTen (17.5%) patients in the olmesartan group and 24 patients (40.7%) in the control group occurred new-onset AF, and the difference between two groups was statistically significant (P = 0.04). AF burden was lower in olmesartan group than that in control group (8.02 ± 3.10% vs. 13.66 ± 6.14%, P = 0.04). There were no significant differences in mean days to the first occurrence of AHREs and mean cumulative numbers of AHREs between two groups (P = 0.89 and P = 0.42, respectively). Moreover, olmesartan group had smaller values of maximal P-wave durations and P-wave dispersion (PD) after 24 months follow-up compared with the control group (109.5 ± 7.4 ms vs. 113.4 ± 7.1 ms, P = 0.00; and 40.6 ± 4.5 ms vs. 43.3 ± 4.4 ms, P = 0.02, respectively). Left ventricular end-diastolic diameter and left ventricular ejection fraction were not significantly different between two groups (both P > 0.05).

CONCLUSIONThis study suggested that 24-month of olmesartan therapy could reduce new-onset AF and AF burden in patients with DDD pacemakers.

CLINICAL TRIAL REGISTRATIONChiCTR-TRC-12004443; http://www.chictrdb.org.

Aged ; Angiotensin Receptor Antagonists ; therapeutic use ; Atrial Fibrillation ; drug therapy ; Atrioventricular Block ; drug therapy ; Female ; Humans ; Imidazoles ; therapeutic use ; Male ; Middle Aged ; Single-Blind Method ; Tetrazoles ; therapeutic use

Objective To discuss the clinical effect of two-dimensional oral care for patients with mechanical ventilation by orotracheal intubation,and improve the quality of oral care.Methods Seventy inpatients with mechanical ventilation by orotracheal intubation from Feb 2011 to Jul 2012 in ICU were selected and randomly divided into experimental group (n ＝ 35) and control group (n ＝ 35).Experimental group was given two-dimensional oral care (oral swab just one time before intubation,and brush teeth and suction three times a day after intubation).Control group was given traditional oral care three times a day.Throat swab specimens were gathered for bacterial colony counts before intubation,and in 4 h,12 h,24 h,48 h after intubation,respectively.Bacteria quantitative culture was performed in sputum specimens and in throat swab specimens (one time per three days) after intubation.Oropharyngeal bacteria change and the incidence rate of ventilator associated pneumonia (VAP) were observed.Results Among all 70 patients,61 cases were valid,15 cases had VAP,and 8 cases died.In the experimental group,31 were valid,4 had VAP and 3 died,while in the control group,30 were valid,11 had VAP and 5 died.The difference of the incidence rate of VAP was statistically significant (x2 ＝ 4.643,P ＝ 0.040) and the difference of the incidence rate of death was not statistically significant (x2 ＝ 0.654,P ＝ 0.473).The difference of oropharyngeal bacterial colony counts had no statistical significance between two groups before intubation (t ＝-0.563,P ＝ 0.589),while the difference of oropharyngeal bacterial colony counts had statistical significance between groups in 4 h,12 h,24 h,48 h after intubation (t＝1.957,-2.520,-3.560,-2.165,respectively; P＜0.05).Conclusions Two-dimensional oral care can effectively reduce oropharyngeal bacterial colony,and decrease the incidence rate of VAP for patients with mechanical ventilation by orotracheal intubation.Thus it is a better nursing method to improve oral care quality.

Oxaliplatin-based chemotherapy is used for treating gastric cancer. Autophagy has been extensively implicated in cancer cells; however, its function is not fully understood. Our study aimed to determine if oxaliplatin induce autophagy in gastric cancer MGC803 cells and to assess the effect of autophagy on apoptosis induced by oxaliplatin. MGC803 cells were cultured with oxaliplatin. Cell proliferation was measured using MTT assay, and apoptosis was determined by flow cytometry. Protein expression was detected by Western blot. Autophagy was observed using fluorescent microscopy. Our results showed that the rate of apoptosis was 9.73% and 16.36% when MGC803 cells were treated with 5 and 20 μg/mL oxaliplatin for 24 h, respectively. In addition, caspase activation and poly ADP-ribose polymerase (PARP) cleavage were detected. Furthermore, when MGC803 cells were treated with oxaliplatin for 24 h, an accumulation of punctate LC3 and an increase of LC3-II protein were also detected, indicating the activation of autophagy. Phosphorylation of Akt and mTOR were inhibited by oxaliplatin. Compared to oxaliplatin alone, the combination of autophagy inhibitor chlorochine and oxaliplatin significantly enhanced the inhibition of cell proliferation and the induction of cell apoptosis. In conclusion, oxaliplatin-induced protective autophagy partially prevents apoptosis in gastric cancer MGC803 cells. The combination of autophagy inhibitor and oxaliplatin may be a new therapeutic option for gastric cancer.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Autophagy ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Organoplatinum Compounds ; pharmacology ; Phosphatidylinositol 3-Kinase ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects ; Stomach Neoplasms ; metabolism ; pathology ; TOR Serine-Threonine Kinases ; metabolism

OBJECTIVEGastric cancer cells are insensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To sensitize gastric cancer cells to TRAIL, we treated gastric cancer MGC803 cells with TRAIL and cisplatin.

METHODSCell proliferation was measured using MTT assay. Cell apoptosis was determined by flow cytometry. Expression of proteins was analyzed by Western blot. The distribution of lipid rafts and death receptors was analyzed by immunofluorescence microscopy. MGC803 cells were pretreated with 50 mg/L nystatin for 1 h, and followed by the treatment of cisplatin and TRAIL.

RESULTS100 µg/L TRAIL resulted in (8.51 ± 3.45)% inhibition of cell proliferation and caused (3.26 ± 0.89)% cell apoptosis in MGC803 cells. Compared with the treatment with cisplatin alone, treatment with TRAIL (100 µg/L) and cisplatin (8.49 mg/L, IC(50) dose of 24 h) led to a dramatic increase in both inhibition of cell proliferation [(52.58 ± 4.57)% vs. (76.43 ± 5.35)%, P < 0.05] and cell apoptosis [(23.10 ± 3.41)% vs. (42.56 ± 4.11)%, P < 0.05]. Moreover, cleavage of caspase-8 and caspase-3 was detected. TRAIL (100 µg/L) did not induce obvious lipid rafts aggregation and death receptor 4 (DR4) clustering, while cisplatin (8.49 mg/L) significantly promoted the localization of DR4 in aggregated lipid rafts. Pretreatment with 50 mg/L nystatin, a cholesterol-sequestering agent, triggered (3.66 ± 0.52)% cell apoptosis after 24 h. Pretreatment with nystatin for 1 h before the addition of 8.49 mg/L cisplatin for 24 h caused a decreased tendency to cell apoptosis [(25.74 ± 3.28)% vs. (22.76 ± 2.97)%]. While, pretreatment with nystatin before the addition of cisplatin and TRAIL, the proportion of apoptotic cells decreased from (43.16 ± 4.26)% to (31.52 ± 3.99)% (P < 0.05).

CONCLUSIONCisplatin enhances TRAIL-induced apoptosis in gastric cancer MGC803 cells through clustering death receptors into lipid rafts.

Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Humans ; Membrane Microdomains ; metabolism ; Nystatin ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology

BACKGROUND AND OBJECTIVEc-Cbl and Cbl-b are two ubiquitous members of the Casitas B-lineage lymphoma (Cbl) family, which play important roles in the downregulation of epidermal growth factor receptor (EGFR) by acting as E3 ubiquitin ligases and multiadaptor proteins. This study investigated the expression of c-Cbl, Cbl-b, and EGFR in gastric carcinoma and its clinical significance.

METHODSThe expressions of c-Cbl, Cbl-b, and EGFR were detected by immunohistochemistry using tissue microarrays consisting of 124 specimens of gastric carcinoma and 16 specimens of normal gastric mucosa. The relationship between the expressions of c-Cbl, Cbl-b, and EGFR and clinicopathologic factors of gastric carcinoma were analyzed statistically.

RESULTSThe positive rates of c-Cbl, Cbl-b, and EGFR were higher in the gastric carcinoma group than in the normal group (71.0% vs. 18.0%, P<0.01; 82.3% vs. 25.0%, P<0.01; 56.5% vs. 12.5%, P<0.01, respectively). The expression of c-Cbl was positively correlated with depth of invasion (r=0.219, P=0.015), and TNM staging (r=0.266, P=0.003). The expression of Cbl-b was positively correlated with lymph node metastasis (r=0.190, P<0.034) and TNM staging (r=0.298, P<0.001). The expression of EGFR was positively correlated with depth of invasion (r=0.286, P<0.001) and TNM staging (r=0.362, P=0.000). The expression of both c-Cbl and Cbl-b was positively correlated with EGFR (r=0.241, P=0.007; r=0.183, P=0.042, respectively). Synchronous strong-positive expressions of c-Cbl, Cbl-b, and EGFR were observed in 27 specimens of gastric carcinoma, most of which were at advanced stage.

CONCLUSIONSOverexpressions of c-Cbl, Cbl-b, and EGFR are closely related to the invasion and progression of gastric carcinoma. c-Cbl and Cbl-b may serve as novel molecular markers for gastric carcinoma.

Adaptor Proteins, Signal Transducing ; metabolism ; Adenocarcinoma ; metabolism ; pathology ; Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; metabolism ; Disease Progression ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; Proto-Oncogene Proteins c-cbl ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Young Adult

The study was aimed to explore the mechanism of SYK and CBL family of ubiquitin ligases in Bufalin-induced HL-60 cells apoptosis. Cell viability was tested by trypan blue staining and apoptosis was detected by using flow cytometry. The expressions of CBL and CBL-b and the phosphorylation of SYK were detected by using immunoprecipitation and Western blot. The results showed that Bufalin inhibited HL-60 cell proliferation in time- and dose-dependent manners. IC(50) of suppressing cell viability at 24, 48 and 72 hours were about 26.3, 7.8 and 2.0 nmol/L respectively. The high dose of bufalin already induced apoptosis of HL-60 cells at 8 hours. SYK was quickly phosphorylated, and the expressions of CBL and CBL-b were down-regulated after treatment with Bufalin. It is concluded that SYK activation and CBL protein down-regulation may be involved in Bufalin-induced HL-60 cell apoptosis.
Apoptosis ; drug effects ; Bufanolides ; pharmacology ; Cell Proliferation ; drug effects ; Down-Regulation ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Proto-Oncogene Proteins c-cbl ; metabolism ; Signal Transduction ; Syk Kinase