Protective effect of liriodendrin against liver ischaemia/reperfusion injury in mice via modulating oxidative stress, inflammation and nuclear factor kappa B/toll-like receptor 4 pathway.

BACKGROUND: The aim of the present study was to investigate the protective effect and mechanism of liriodendrin (LDN) is a lignan diglucoside in hepatic ischaemia/ /reperfusion (I/R) injury. MATERIALS AND METHODS: The liver I/R was established in male C57BL/6 mice. The effect of LDN is initially investigated on hepatic I/R injury via estimating histopathology of liver. The level of metabolic enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) was studied along with apoptosis of mouse hepatocytes via TUNEL and flow cytometry analysis. The effect of LDN was investigated on oxidative stress biomarkers (glutathione [GSH] content, malondialdehyde [MDA] and superoxide dismutase [SOD] activities) and pro-inflammatory cytokines (tumour necrosis factor alpha [TNF-α], interleukin [IL]-1ß and IL-6). Western blot study was also conducted to elucidate the effect of LDN on toll-like receptor 4/nuclear factor kappa B (TLR4/NF-kB). RESULTS: Liriodendrin alleviates liver I/R injury, as manifested by decreased plasma ALT, AST and ALP with improvement in liver necrotic area. LDN also reduces apoptosis of mouse hepatocytes with reduction of oxidative stress and generation of pro-inflammatory cytokines. It significantly reduces the expression of TLR4 and NF-kB. CONCLUSIONS: The study demonstrated that LDN reduces liver injury and prevented apoptosis of hepatocytes following I/R injury. In addition, LDN also reduces oxidative stress, inflammation, and TLR4/NF-kB in I/R injured mice.

[Analytical performance verification protocols and performance specifications of platelet-dependent von Willebrand factor activity testing].

Objective: To investigate the analytical performance verification protocols and performance specifications of platelet-dependent von Willebrand factor (VWF) activity testing (VWF:GPIbM) for clinical laboratories. Methods: According to Clinical Laboratory Standards Institute (CLSI) documents and National Health Standard of China, the performance verification of VWF:GPIbM was designed and implemented using Sysmex CS-5100 instrument and its corresponding reagents. (1) Precision verification: Two commercial quality control samples (with normal and low activity levels) and three plasma pools (with activity range from 5.0% to 150.0%) were prepared. Each sample was tested five times daily for five consecutive days. The coefficient of variation (CV) of intra-and inter-run precisions were calculated, and the precision evaluation criterion was set according to package inserts. (2) Trueness verification: The calibrator was diluted to five reference materials with activity values of 5.2%, 31.2%, 62.4%, 104.0% and 138.7%, and each reference material was tested five times daily for five consecutive days. The bias between the measured value and the reference value was calculated, and the trueness evaluation criterion was set according to the total allowable error. (3) Linearity verification: Ten pooled plasmas with theoretical value range from 3.6% to 160.4% were prepared for the linearity verification of two calibration curves set by the manufacturer (i.e. low range and normal range calibration curve). Each pooled plasma was tested three times in a single run. The slope and R2 of linear regression of mean of measured value and theoretical value, as well as bias, were calculated, and the linearity evaluation criterion was set according to National Health Standard of China and package inserts. (4) Limit of quantitation verification: The calibrator was diluted to two reference materials with activity values of 3.3% and 2.7%, and each material was tested twelve times. The limit of quantitation evaluation criterion was set according to CLSI document. Results: The CVs of intra-and inter-run were 1.0%-2.5% and 1.1%-2.6%, respectively. The biases of trueness verification were -0.4%, 1.0%, -2.6%, 0.3% and -2.7%, respectively. The linearity verification results of low range (3.6%-31.8%) and normal range (28.4%-160.4%) showed that the slopes of regression equation were 1.021 7 and 0.996 2, respectively, R2 were 0.993 5 and 0.993 9, respectively, and the biases with 0-1.8% and -10.1%-0 of plasmas met the criterion. The biases ranged from -0.4% to 0.3% of test results in the verification of limit of quantitation met the criterion. Conclusion: The verification results of VWF:GPIbM assay for precision, trueness, linearity and limit of quantification meet the performance requirements indicated in the package inserts and the criteria set in this study, which can be taken as a reference of performance verification for the clinical laboratories.

Inhibition of A2AR gene methylation alleviates white matter lesions in chronic cerebral hypoperfusion rats.

OBJECTIVE: Chronic cerebral hypoperfusion (CCH) can cause ischemic cerebral white matter lesions (IWML). The aim of this study was to explore the roles of A2A receptors (A2AR) in IWML and the effect of methylation in A2AR gene. MATERIALS AND METHODS: SD rat model of CCH was constructed by bilateral common carotid artery occlusion (BCCAO) method. The rats were then treated with DNA methyltransferase (DNMT) inhibitor (decitabine), agonist (CGS21680) and A2AR inhibitor (SCH58261). Morris water maze and Kluver-Barrera staining were used to assess spatial learning and reference memory after IWML, respectively. Gene transcription and protein expression were measured by qRT-PCR, Enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. The concentration of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and DNMT were detected by assay kit. Methylation of A2AR gene promoter region was detected by bisulfite sequencing PCR (BSP). RESULTS: We found that the down-regulated expression of A2AR in corpus callosum under CCH was associated with IWML and cognitive impairment. We further showed that A2AR agonist can reduce the IWML under CCH, and A2AR inhibitor can aggravate the IWML under CCH. We also found that the expression level of DNMTs in corpus callosum and the methylation level in the promoter region of A2AR gene were increased under CCH. DNMT inhibitors could protect white matter by inhibiting the methylation of A2AR promoter and rescue the downregulation of A2AR under CCH. CONCLUSIONS: Our results demonstrate that the downregulation of A2AR mediates IWML in CCH, and A2AR downregulation is related to the increased methylation of A2AR gene promoter. DNMT inhibitors play a protective role in IWML.

[Effect of VDR gene silencing on proliferation and NF-κB activation of airway smooth muscle cells].

Objective: To investigate the effect of VDR gene silencing on proliferation of airway smooth muscle cells (ASMCs) and elucidate the role of NF-κB. Methods: A recombinant lentiviral vector specifically targeting VDR gene in rat was constructed by RNA interference. Rat ASMCs were divided into blank group, empty vector group and interference group. ASM cell line model stably silencing the VDR gene RNA expressing was selected by puromycin. Then MTT colorimetric assay and cell cycle analysis by flow cytometry were used to examine cell proliferation. The activation of nuclear factor-κB was determined by immunofluorescence double label method. Moreover, NF-κB-dependent transcription activity was tested through luciferase reporter gene assay. Western blotting was used for IκBα and phospho-IκBα protein levels and actinomycin D treatment was used to determine IκBα mRNA stability. All statistical analyses were performed using SPSS version 23.0 software. Differences between groups were analyzed using one-way ANOVA analysis. Multiple comparisons among groups were made by Student-Newman-Keuls test. Results: (1) As compared with those in the blank group and the empty vector group, the cell proliferation index (PI) and the percent of ASMCs at G2/M phase in the interference group were markedly increased (P<0.05), but their percent at G0/1 phase was decreased (P<0.05).(2) In the interference group, the nuclear translocation of NF-κB p65 in ASMCs was obviously induced. And its level of receptor gene NF-κB p65 (1.37±0.28) was significantly higher than that in the blank group (1.00±0.19,P=0.031) and in the empty vector group (0.96±0.18,P=0.027).(3) In the interference group, the IκBα protein level in ASMCs (0.13±0.04) was obviously less than that in the blank group (0.29±0.05, P=0.023) and in the empty vector group (0.32±0.07, P=0.014). Oppositely, the p-IκBα/IκBα level in the interference group (0.86±0.04) was much more than that in the blank control group (0.41±0.07, P=0.026) and in the empty vector group (0.37±0.05, P=0.017). (4) In the interference group, IκBα mRNA showed a shorter half-life, (171.31±9.67) min, compared to that in the blank group [(224.69±7.95) min,P=0.032] and in the empty vector group [(230.41±6.37) min,P=0.035]. Conclusion: VDR gene silencing could promote ASMC proliferation and the underlying mechanism may involve the activation of NF-κB signaling pathway.

[Comparative study ofmethylene blue tracer and double tracer containing nuclide in sentinel lymph node biopsy of breast cancer].

Objective: To compare the application effect of blue dye single tracer and blue dye combined with nuclide double tracer in sentinel lymph node biopsy (SLNB) of breast cancer surgery. Methods: A total of 92 breast cancer patients in Shandong Cancer Hospital and Institute from November 2017 to October 2019 underwent methyleneblue dye combined with (99)Tc(m) sulfur colloid nuclide double tracer in SLNB, while other 92 cases in Jining First People Hospital underwent blue dye single tracer. The number of SLN detection, detection rate, accuracy rate, sensitivity, and false negative rate of the two groups were compared. The impacts of age, menstruation, tumor location, tumor size, clinical stage, pathological type, and estrogen receptor (ER), progesterone receptor (PR), human epidermal receptor 2 (HER-2), molecular typing, dynamic enhanced magnetic resonance imaging (DCE-MRI)on the detection rate of SLN were analyzed. Results: The number of detection, detection rate, accuracy, sensitivity, and false negative rate of the blue dye single tracer group were 3.20±1.10, 90.22%, 93.48%, 95.24% and 4.76%, respectively; the double tracer group were 3.37±1.02, 92.39%, 95.65%, 95.65% and 4.35%, respectively, without significant difference (all P>0.05). In different age, menstrual condition, tumor location, clinical stage, pathological type, ER, PR, HER-2 expression and molecular typing, the detection rate of single tracer group and double tracer group had no significant difference (all P>0.05). However, in the tumor size of 2-5 cm and without DCE-MRI examination, the detection rate of single tracer group was significantly lower than that of double tracer group. Conclusion: The effect of blue dye single tracer in detecting SLN of breast cancer is equivalent to that of double tracer method, which is worthy of promotion and application in primary hospitals.

Lamin B1 deficiency promotes malignancy and predicts poor prognosis in gastric cancer.

Gastric cancer (GC) is a kind of global malignancy. However, the expression pattern and clinical relevance of lamin B1 in GC remain to be elucidated. We endeavored to investigate how GC is influenced by lamin B1 and the related mechanisms. The lamin B1 expression in GC tissues from 71 patients was assessed by using immunohistochemistry (IHC). The expression of lamin B1 was connected with the clinical stage, depth of invasion, and poorer overall survival. Colony formation assays and methyl thiazolyl tetrazolium (MTT) were used to assess cell viability. The migration ability of GC cells was determined by cell scratch assay and Transwell invasion assay. Moreover, we used two cell lines of GC to explore the underlying mechanism of lamin B1 in boosting the GC cells proliferation and invasion in vitro by assessing the effects on related signal transduction pathways. Our data demonstrated that the expression level of lamin B1 was downregulated in GC tissues, and low expression level of lamin B1 was significantly correlated with higher clinical stage, depth of invasion, nodal stage, and poor prognosis. Moreover, in vitro experiments demonstrated that lamin B1 knockdown promoted, whereas lamin B1 overexpression inhibited, gastric cancer cell proliferation and migration. We also observed that lamin B1 knockdown could promote the activity of the PI3K/PTEN/Akt and MAPK/ERK pathway with a decrease in the p53/p21WAF1/CIP1 expression, whereas lamin B1 overexpression contributed to the opposite results. In conclusion, our studies indicate that lamin B1 deficiency is crucial in GC progression. Furthermore, the results elucidating the biological mechanisms of lamin B1 may potentially contribute to current GC treatment modalities.

[High level of hemoglobin during the first trimester of pregnancy associated with the risk of gestational diabetes mellitus].

Objective: To explore the relationship between hemoglobin (Hb) level during the first trimester of pregnancy and gestational diabetes mellitus (GDM). Methods: A total of 1 276 participants, who underwent scheduled prenatal examination and normal singleton delivery at the Fifth People's Hospital of Shanghai and Hospital of Intergrated Chinese and Western Medicine in Minhang District, from January 2016 to May 2018 were included. There were 99 cases of GDM (GDM group) and 1 177 cases of normal (control group) pregnant women.Based on the serum Hb level during the first trimester of pregnancy, participants were divided into three groups, 236 cases of low Hb level group (Hb<110 g/L), 868 cases of normal Hb level group (110 g/L≤Hb<130 g/L), and 172 cases of high Hb level group (Hb≥130 g/L). Maternal clinical data were collected, including Hb level during the first trimester of pregnancy, three-point blood glucose (BG) of oral glucose tolerance test (OGTT) and fasting insulin during the second trimester of pregnancy. Homeostasis model assessment of insulin resistance index (HOMA-IR) and homeostasis model assessment of pancreatic ß cell function index (HOMA-ß) were used to evaluate insulin resistance and pancreatic ß cell function. Results: (1) Hb level during the first trimester of pregnancy in GDM group was significantly higher than that in control group [(123±10),(119±11) g/L, P<0.05]. There were no significant difference in gravidity, parity, index of liver and renal function (all P>0.05). (2) Pre-pregnancy body mass index (BMI), 1-hour BG and 2-hour BG of OGTT were significantly increased in the high Hb level group during the first trimester of pregnancy, which were (23±4) kg/m(2), (7.3±2.0) mmol/L, and (6.5±1.4) mmol/L (P<0.05), respectively. The pre-pregnancy BMI, 1-hour BG and 2-hour BG of the normal or low Hb level group were (22±3) kg/m(2), (6.7±1.6) mmol/L, (6.1±1.2) mmol/L; (22±3) kg/m(2), (6.5±1.5) mmol/L, (5.9±1.1) mmol/L, respectively. There were no statistically significant difference in levels of fasting blood glucose, fasting insulin, HOMA-IR and HOMA-ß within 3 groups (all P>0.05). (3) In the high Hb level group, prevalence of pregnancy overweight or obesity and GDM were the highest, which were 37.2%(64/172) and 15.1%(26/172), respectively; the differences were statistically significant (all P<0.05). (4) The serum Hb level in the first trimester was positively related with pre-pregnancy BMI (r=0.130, P<0.05), 1-hour BG (r=0.129, P<0.05), 2-hour BG (r=0.134, P<0.05), fasting insulin (r=0.096, P<0.05), and HOMA-IR (r=0.101, P<0.05).Logistic regression indicated that Hb≥130 g/L during the first trimester of pregnancy was an independent risk factor for GDM (OR=2.799, 95%CI: 1.186-6.604; P<0.05). Conclusion: The high level of Hb (Hb≥130 g/L) during the first trimester of pregnancy is associated with GDM.

RNA-seq reveals transcriptome changes of the embryonic lens cells in Prox1 tissue specific knockout mice.

OBJECTIVE: Prox1 is expressed in both lens epithelial cells and fiber cells and is essential for lens fiber cell elongation. This study aimed to explore the molecular mechanisms of how Prox1 mutations influence lens fiber cells development. MATERIALS AND METHODS: Comparative transcriptomes analysis of Prox1 conditional knockout (cKO) lens and wild-type (WT) lens were performed using the data GSE69940 downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were determined by the R package "edgeR" of Trinity software. GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes databases) enrichment analysis were performed using the cluster Profiler R package. Then, the protein-protein interaction (PPI) network was predicted using Cytoscape, and the Module analysis of the PPI network was analyzed through the Cytoscape MCODE plugin. Moreover, MotifDb package in R was used to predict the transcription factors binding to Prox1 promoter regions. RESULTS: In total, 2263 differentially expressed genes were identified between the two groups. GO and KEGG analysis showed that the down-regulated genes were enriched in camera-type eye term, nucleosome assembly, lens fiber cell differentiation, and cell modified and amino acid metabolism. The KEGG pathway of up-regulated genes was associated with lens development, including Hedgehog signaling pathway and MAPK signaling pathway. GO terms of up-regulated DEGs were mainly relevant to bone morphological development, muscle development, and sensory organ morphological development. Next, the PPI network of DEGs was constructed, and 4 modules were analyzed. Moreover, 30 transcription factors were predicted, which are likely to be downstream targets of Prox1 with potential roles in lens development in mice. CONCLUSIONS: This study provides insights into the unique transcriptome profile of lens cells in Prox1 conditional knockout mice, which is a valuable resource for further study of mouse lens genomics.

Dexmedetomidine enhances ropivacaine-induced sciatic nerve injury in diabetic rats.

BACKGROUND: Previous studies suggest that dexmedetomidine has a protective effect against local anaesthetic-induced nerve injury in regional nerve blocks. Whether this potentially protective effect exists in the context of diabetes mellitus is unknown. METHODS: A diabetic state was established in adult male Sprague-Dawley rats with intraperitoneal injection of streptozotocin. Injections of ropivacaine 0.5%, dexmedetomidine 20 µg kg-1 (alone and in combination), or normal saline (all in 0.2 ml) were made around the sciatic nerve in control and diabetic rats (n=8 per group). The duration of sensory and motor nerve block and the motor nerve conduction velocity (MNCV) were determined. Sciatic nerves were harvested at post-injection day 7 and assessed with light and electron microscopy or used for pro-inflammatory cytokine measurements. RESULTS: Ropivacaine and dexmedetomidine alone or in combination did not produce nerve fibre damage in control non-diabetic rats. In diabetic rats, ropivacaine induced significant nerve fibre damage, which was enhanced by dexmedetomidine. This manifested with slowed MNCV, decreased axon density, and decreased ratio of inner to outer diameter of the myelin sheath (G ratio). Demyelination, axon disappearance, and empty vacuoles were also found using electron microscopy. An associated increase in nerve interleukin-1ß and tumour necrosis factor-α was also seen. CONCLUSIONS: Ropivacaine 0.5% causes significant sciatic nerve injury in diabetic rats that is greatly potentiated by high-dose dexmedetomidine. Although the dose of dexmedetomidine used in this study is considerably higher than that used in clinical practice, our data suggest that further studies to assess ropivacaine (alone and in combination with dexmedetomidine) use for peripheral nerve blockade in diabetic patients are warranted.

[Clinicopathological features of second primary lung cancer and pulmonary metastasisin patients with breast cancer].

Objective: To compare the clinicopathological characteristics of second primary lung cancer following breast cancer and lung metastases from breast cancer, and then to analyze the risk factors in breast cancer patients with second primary lung tumor. Methods: Clinical data of 55 breast cancer patients with second primary lung tumor and 205 breast cancer patients with solitary pulmonary metastasis in Shandong Cancer Hospital from January 2006 to January 2017 were retrospectively analyzed. The risk factors of primary lung cancer following breast cancer were analyzed using logistic regression model. Results: Second primary lung cancer in patients with first breast cancer accounted for approximately 21.2%(55/260) of pulmonary malignant solitary nodules, and 0.84%(55/6 580) of all breast cancer patients. The median intervals between the diagnosis of second primary lung cancer or lung metastasis and first breast cancer were 52 months and 42 months, respectively. These two groups showed significant difference between age, time interval between diagnoses, breast tumor size, axillary lymph node metastasis, estrogen receptor, molecular subtype (luminal B and triple-negative) and history of radiotherapy (P<0.05 for all). A multivariate logistic regression model confirmed that age (OR=1.088, P<0.001), breast tumor size(OR=0.480, P<0.001), and radiotherapy history (OR=3.460, P=0.004) were all independent factors for second primary lung cancer. Conclusions: For isolated pulmonary nodules in patients with breast cancer, especially for those with elder age, larger tumor size and radiotherapy history, we should distinguish the second primary lung cancer from pulmonary metastasis. The treatment regimen for lung metastasis and primary lung cancer in patients with breast cancer are entirely distinct. The timely histopathology examinations for pulmonary nodes in patients with breast cancer are recommended.

[Comparative study on the efficacy and safety between pegfilgrastim (PEG-rhG-CSF) and recombinant human granulocyte colony-stimulating factor in promoting hematopoietic recovery after allogeneic hematopoietic stem cell transplantation after hematological malignancy].

Objective: To observe the efficacy and safety between Pegfilgrastim (PEG-rhG-CSF) and Recombinant human granulocyte colony stimulating factor (rhG-CSF) in hematological malignancy after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: 157 patients after allo-HSCT were enrolled in this study from June 2015 to November 2016. Two agents of G-CSF were used to stimulate hematopoietic recovery after transplantation. There were 65 cases in PEG-rhG-CSF and 92 cases in rhG-CSF groups. Patients in PEG-rhG-CSF group were given a single subcutaneous dose of 6 mg on the first day and +8 d, while cases in rhG-CSF group were given in dose of 5 µg·kg(-1)·d(-1) by subcutaneous injection from +1 d continuing to neutrophils more than 1.5×10(9)/L, and then the indicators and survival rates in two groups after transplantation were compared. Results: ①There were no significant differences of the neutrophil implantation time[13.5 (8-12) d vs 13 (9-24) d, P=0.393] and platelet implantation time [14 (9-160) d vs 14 (9-92) d, P=0.094] between PEG-rhG-CSF and rhG-CSF groups respectively. There were no significant differences in terms of neutropenia period (P=0.435) , number of cases who got fever during neutropenia (P=0.622) , and the median time of fever in neutropenia period (P=0.460) , respectively between the two groups. There were no significant differences of erythrocyte and platelet transfusions (P=0.074, P=0.059) within 1 month after transplantation. ②There were no significant differences with regard to the incidences of acute GVHD[23.1% (15/65) vs 34.8% (32/92) , P=0.115], chronic GVHD[20.0% (13/65) vs 32.6% (32/92) , P=0.081], Ⅱ-Ⅳdegree of acute GVHD[30.0% (13/65) vs 30.4% (30/92) , P=0.287] and extensive chronic GVHD[9.2% (6/65) vs 20.7% (19/92) , P=0.135] between PEG-rhG-CSF and rhG-CSF groups. ③There were no significant differences in terms of disease free survival (DFS) (62.5% vs 61.4%, P=0.478) and overall survival (OS) (67.4% vs 67.3%, P=0.718) between PEG-rhG-CSF and rhG-CSF groups. ④There was no significant difference of the non-relapse mortality (NRM) between PEG-rhG-CSF and rhG-CSF groups[20.5% (95%CI 11.4%-37.0%) vs 32.6% (95%CI 22.2%-47.9%) , P=0.141]. The relapse rate was not statistically significant[14.9% (95%CI 7.4%-29.8%) vs 10.0% (95%CI 5.0%-20.0%) , P=0.299]. Conclusion: Compared with rhG-CSF, PEG-rhG-CSF could reduce the times of injection. There were no differences in terms of hematopoietic recovery, the incidence of GVHD, relapse rate, DFS and OS rates after allo-HSCT between two groups.

[Clinical efficacy of recombinant activated factor â ¦ a for 16 hematonosis with moderate or severe bleeding].

Objective: To analyze the efficacy of recombinant activated factor Ⅶ a (rF Ⅶ a) on hematonosis with moderate or severe bleeding signs. Methods: Of total 16 cases with rF Ⅶ a treatment from May 2013 to May 2016, 8 cases received allogeneic hematopoietic stem cells transplantation (allo-HSCT) and the other were non-transplantation patients. In two groups, there was no significant difference on rF Ⅶ a usage and dosage. 15 patients with acute graft-versus-host disease (aGVHD) after allo-HSCT were control group (without rF Ⅶ a) . Results: ①The total response rate was 75.0% (6/8) in non-transplantation group and 37.5% (3/8) in transplantation group, respectively. Median interval for hemorrhage stop was 38.5 hours in non-transplantation group and 63.0 hours in transplantation group. The median overall survival (OS) was 201.0 and 29.0 days for non-transplantation group and transplantation group, respectively, and the OS rate was 50.0% (4/8) and 25.0% (2/8) , respectively. The bleeding-related mortality rate was 50.0% (2/4) and 83.3% (5/6) , respectively. ②Of the 16 cases, 9 showed response to rF Ⅶ a treatment and the other 7 cases'bleeding signs did not alleviate. The median OS was 268.0 in 9 cases with response and 24.0 days in 7 cases without response, respectively. ③In patients with intestinal aGVHD complicated with intestinal hemorrhage, the median OS of observation group (n=6) and control group (n=15) were 25.5 days and 20.0 days, respectively. Conclusion: Patients with hematological diseases, especially patients after allo-HSCT, had high bleeding-related mortality, and rFⅦa therapy had a obvious hemostatic efficacy. The survival rate of patients with response was higher than that of cases without response. The causes of poor hemostasis efficacy of rF Ⅶ a therapy were associated with unsatisfactory control of complications in patients with intestinal bleeding after allo-HSCT.

Outbreak of Occupational Brucellosis at a Pharmaceutical Factory in Southeast China.

Brucellosis is an occupational disease affecting workers in butcher shops, the milking and dairy product industry, causing more than 500 000 new cases around the world. As a national statutory B infectious disease in China, morbidity of brucellosis is rapidly increasing in recent years. We report an occupational outbreak of brucellosis infection in a pharmaceutical factory. Exposure was a result of manual operation in the process line, close contact with sheep placentas, insufficient disinfection and repeated using of protective suits and infected by aerosol dissemination. Improved preventive methods, appropriate public health measures and spread of health education would be helpful to prevent the occupational outbreak of brucellosis in future.

[Effect of autophagy inhibitor combined with EGFR inhibitor on triple-negative breast cancer MDA-MB-468 and MDA-MB-231 cells].

OBJECTIVE: To investigate the effect of combined administration of autophagy inhibitor 3-methyladenine/bafilomycin A1 and EGFR inhibitor gefitinib on triple-negative breast cancer MDA-MB-468, MDA-MB-231 cells and estrogen receptor-positive MCF-7 cells. METHODS: All the cells were treated with 3-methyladenine/bafilomycin A1 and/or gefitinib. The effect of autophagy inhibitor and gefitinib on the cell growth was evaluated by MTT assay. Cell apoptosis was detected by flow cytometry. Western blot analysis was used to determine the alteration of autophagy-related protein (such as LC3) and apoptosis-related proteins (such as caspase-3 and caspase-9). RESULTS: MTT assay showed that the IC50 in the GE+ 3-MA and GE+ BAF groups were (4.1±0.2) µmol/L and (3.8±0.3) µmol/L, significantly lower than that of the gefitinib alone group [(7.0±0.2) µmol/L] in MDA-MB-468 cells (P<0.05). Similarly, the IC50 in the GE+ 3-MA and GE+ BAF groups were (9.7±0.1) µmol/L and (7.7±0.2) µmol/L, significantly lower than that of the gefitinib alone group [(14.7±0.1) µmol/L]in MDA-MB231 cells (P<0.05). The flow cytometry assay revealed that the apoptosis rates of MDA-MB-468 cells in GE, GE+ 3-MA and GE+ BAF groups were (12.43±3.18)%, (23.37±2.71)% and (18.71±2.81)%, respectively. The apoptosis rates of MDA-MB-231 cells of the GE, GE+ 3-MA and GE+ BAF groups were (12.15±1.82)%, (16.94±2.19)% and (33.83±5.92) %, significantly higher than that of the gefitinib alone group (All P<0.05). The apoptosis rates of the MCF-7 cells were not changed significantly among the three groups (P>0.05). Western blot data showed that the expression levels of LC3 and p-Akt were decreased in the combined groups than that of the gefitinib alone group, while the p-PTEN, caspase-3 and caspase-9 were increased. CONCLUSIONS: Autophagy inhibitor may enhance the sensitivity to gefitinib in MDA-MB-468 and MDA-MB-231 cells by activation of the PTEN/P13K/Akt pathway. Apoptosis in MDA-MB-468 and MDA-MB-231 cells might be enhanced by the combination treatment through caspase cascade.

Chlorpyrifos-induced hormesis in insecticide-resistant and -susceptible Plutella xylostella under normal and high temperatures.

Hormesis induced by insecticides at the dosage lower than what ostensibly directly causes death on insects was studied. This paper reports the effects of the in vivo application of varied concentrations of chlorpyrifos (CPF) on Plutella xylostella (DBM). The insecticide concentrations applied included 0.000025-2.5 mg l-1, which are far lower than LC1 (7.2 mg l-1), for the CPF-susceptable (Si) DBM, and 250 mg l-1 which is far below LC1 (1286 mg l-1), for the CPF-resistant (Rc) DBM, as well as LC10- and LC50-doses for both strains. Significant hormesis was found with the 'hermetic-CPFs', i.e., 0.0025 mg l-1 for Si DBM and 2.5 mg l-1 for Rc DBM, at the normal or high temperature either in a 24 h or under a long-term treatment. These doses of CPF significantly stimulated the development and increased the fecundity of Si and Rc DBM at 25°C with approximately 23.5-29.8% activity increase on acetylcholinesterase (AChE) and 30.5-91.3% increase on glutathione S-transferases (GSTs) at 25 or 38°C in 4-24 h. The enzymatic activities were significantly reduced by LC50-CPF at 25°C in vivo, but the inhibition was relieved significantly, if the insects were first subjected to a hormetic-CPF pretreatment. It was remarkable that the average rates of enzymatic activity increase were 67.5-76.6% for AChE and 366-546% for GSTs. Consequently, it was concluded that the hormesis on Si and Rc DBM could be induced by CPF doses far below LC1 at normal or high temperature in short- or long-term treatment. These findings might help to improve the current insect control practices in the field.

Heterologous expression of the Hsp24 from Trichoderma asperellum improves antifungal ability of Populus transformant Pdpap-Hsp24 s to Cytospora chrysosperma and Alternaria alternate.

The tolerance of plants to biotic and abiotic stresses could be improved by transforming with fungal resistance-related genes. In this study, the cDNA sequence (GenBank Acc. No. KP337939) of the resistance-related gene Hsp24 encoding the 24 kD heat shock protein was obtained from the biocontrol fungus Trichoderma asperellum ACCC30536. The promoter region of Hsp24 contained many cis-regulators related to stresses response, such as "GCN4" and "GCR1" etc. Hsp24 transcription in T. asperellum was up-regulated under six different environmental stresses, compared with the control. Furthermore, following heterologous transformation into Populus davidiana × P. alba var. Pyramidalis (Pdpap), Hsp24 was successfully transcribed in transformant Pdpap-Hsp24s. Pathogen-related genes (PRs) in four Pdpap-Hsp24s were up-regulated compared with those in the control Pdpap (Pdpap-Con). After co-culture of Pdpap-Hsp24s with the weak parasite Cytospora chrysosperma, the transcription of genes related to hormone signal pathway (JA and SA) were up-regulated in Pdpap-Hsp24s, and ethidium bromide (EtBr) and Nitro-blue tetrazolium (NBT) staining assays indicated that the cell membrane permeability and the active oxygen content of Pdpap-Hsp24s leaves were lower than that of the control Pdpap-Con. And when the Pdpap-Hsp24s were under the Alternaria alternate stress, the activities of superoxide dismutase (SOD) and peroxidase (POD) got higher in Pdpap-Hsp24s than that in Pdpap-Con, and the disease spots in Pdpap-Con leaves were obviously larger than those in Pdpap-Hsp24s leaves. In summary, Hsp24 of T. asperellum ACCC30536 is an important defense response gene, and its heterologous expression improved the resistance of transformant Pdpap-Hsp24s to C. chrysosperma and A. alternate.

Rare Variants in LAMA5 Gene associated with FLT4 and FOXC2 Mutations in Primary Lymphedema May Contribute to Severity.

Mutations in the Fms-related tyrosine kinase 4 (FLT4) and forkhead box protein C2 (FOXC2) genes cause Milroy disease (MD) and lymphedema-distichiasis syndrome (LDS), respectively, but the mechanism underlying disease pathology remains unclear. Applying whole-exome sequencing to two families with MD, one LDS family, and one sporadic LDS case, we identified four rare variants in the laminin subunit alpha-5 gene (LAMA5) in subjects carrying novel and known missense FLT4 mutations and a 7-bp duplication and 1-bp insertion in FOXC2. Phenotyping was expanded in some individuals using magnetic resonance lymphangiography, indiocyanine green fluorescence lymphography, and immunofluorescent lymphatic staining of skin tissue. Skin lymphatic staining showed the existence of dermal lymphatic vasculature in the MD case. Significant lymphatic dysfunction was observed in both MD and LDS patients. In the MD patient, tortuous lymphatics in the dorsum of the foot were slowly enhanced on indocyanine green fluorescent lymphography (ICG) imaging. Dilated lymph collectors with disruption and lymph leakage were observed in the familial LDS case on magnetic resonance lymphangiography (MRL). Numerous tortuous lymph collectors were visualized along the entire length of affected lower limbs on MRL imaging, and retrograde lymph flow was observed in the lymph collectors during ICG lymphography in the isolated LDS case. The finding of rare LAMA5 variants together with FLT4 and FOXC2 mutations suggests that these mutations may be co-responsible for these disorders and most likely interfere with the function of lymphatics. Further, larger studies are needed to confirm these results.

Correlation between p53 and epidermal growth factor receptor expression in breast cancer classification.

This study aimed to explore new opportunities for developing targeted therapy for triple-negative breast cancer (TNBC) by analyzing the significance and association between p53 and epidermal growth factor receptor (EGFR) expression in different molecular subtypes of breast cancer. The clinical and pathological data of 264 patients with breast cancer receiving surgery in our hospital from January 2012 to August 2013 were retrospectively analyzed. According to the expression of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER2), Ki-67, CK5/6, p53, and EGFR detected by immunohistochemical methods, breast cancer was divided into four molecular subtypes. Then, the expression of p53 and EGFR as well as their correlation in the different subtypes were determined. Among the four subtypes, luminal B breast cancer was the most common type. TNBC and HER2-enriched breast cancer had larger tumor sizes with higher expression of Ki-67 as compared with the luminal types. TNBC had a lower lymph node metastasis rate but higher CK5/6 and EGFR expression than the other three types. The expression of p53 was higher in luminal B, HER2-enriched, and triple-negative breast cancers, and this was positively correlated with the expression of EGFR in TNBC but not in the other subtypes. p53 and EGFR expression was positively correlated in TNBC, which enables us to explore the molecular biological characteristics of TNBC, so as to provide new ideas for the treatment of TNBC.

Cloning of an anthocyanidin synthase gene homolog from blackcurrant (Ribes nigrum L.) and its expression at different fruit stages.

Anthocyanidin synthase (ANS), a 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase, catalyzes the penultimate step in anthocyanin biosynthesis, from leucoanthocyanidins to anthocyanidins, the first colored compound in the anthocyanin pathway. In this study, a full-length, 1427-bp long cDNA named RnANS1, which is homologous to the anthocyanidin synthase gene, was cloned from blackcurrant using a homologous cloning strategy. RnANS1 is highly homologous to other plant ANS genes at both the nucleotide and amino acid sequence levels. The deduced protein contains domains conserved in the 2OG and Fe(II)-dependent oxygenase, and is phylogenetically closely related to Paeonia suffruticosa and Paeonia lactiflora. The expression of RnANS1 was upregulated during fruit maturation, and correlated with the accumulation of anthocyanins and soluble carbohydrates in the fruit. Further characterization of the structure and expression patterns of RnANS1 will clarify our understanding of anthocyanin biosynthesis in blackcurrant, and support the development of molecular approaches to manipulate anthocyanin production in this plant.