Rapid detection of Escherichia coli in dairy milk using static headspace-comprehensive two-dimensional gas chromatography.

A new approach is introduced for rapid and reliable bacteria detection in food. Namely, static headspace-comprehensive two-dimensional gas chromatography (HS-GC × GC) with backflushing. The introduced approach provides fast detection of Escherichia coli (E. coli) in enriched ultra-high-temperature processed (UHT) dairy milk. The presence of E. coli may be indicated by detecting microbial volatile organic compounds emanating from test solutions inoculated with E. coli. In the present investigation, HS-GC × GC analysis is preceded by conventional enrichment in nutrient broth and inoculated samples are clearly discernable from controls following as little as 15 h sample enrichment. Headspace equilibration for 28 min followed by an 8 min GC × GC analysis of enriched test solutions reduces time-to-response by approximately one full day compared to conventional culture-based methods. The presence of ethanol, 1-propanol, and acetaldehyde may be used as a putative marker of E. coli contamination in milk and the introduced approach is able to detect single-cell initial bacterial load. Faster, reliable detection of pathogens and/or spoilage microbes in food products is desirable for the food industry. The described approach has great potential to complement the conventional workflow and be utilised for rapid microbial screening of foodstuff.

Testosterone Deficiency Promotes Hypercholesteremia and Attenuates Cholesterol Liver Uptake via AR/PCSK9/LDLR Pathways.

Background: Testosterone deficiency is reportedly correlated with an elevation of cholesterol in plasma, but the mechanism remains unclear. Our objective was to investigate the effects of testosterone deficiency on cholesterol metabolism and the corresponding molecular changes in vivo and in vitro. Methods: SD rats were randomized into three groups: sham-operated (SHAM), subtotal orchiectomized (SO), and orchiectomized (ORX) and fed for 8 weeks. HepG2 cells were cultured with medium containing testosterone with the final concentrations of 0, 10, 30, and 300 nM. Method of isotope tracing and fluorescence labelling was adopted to investigate cholesterol metabolism. Several key molecules of cholesterol metabolism were also analyzed. Results: SO and ORX rats displayed dysfunctional liver uptake of cholesterol. HepG2 cells incubated with testosterone of lower and excessive level exhibited reduced capacity of cholesterol uptake. Further investigation revealed that lack of testosterone induced increased proprotein convertase subtilisin/kexin type 9 (PCSK9) and decreased low-density lipoprotein receptor (LDLR) both in vivo and in vitro. Moreover, the androgen receptor (AR) antagonist flutamide mimicked the effects of testosterone deficiency on PCSK9 and LDLR indicating the role of AR as a mediator in triggering attenuating liver cholesterol uptake in which testosterone instead of dihydrotestosterone (DHT) is the major functional form of androgen. Conclusion: Testosterone deficiency attenuated cholesterol liver uptake mediated by the PCSK9-LDLR pathway, in which AR and testosterone without transforming to DHT play important roles.

Analysis of influencing factors for arteriovenous graft dysfunction in hemodialysis patients / 中华肾脏病杂志

Objective:To explore the influencing factors of arteriovenous graft (AVG) dysfunction in hemodialysis (HD) patients, and provide a basis for predicting the risk of dysfunction and prolonging the service time.Methods:Retrospective analysis was performed on the clinical and follow-up data of patients who underwent AVG surgery in Department of Vascular and Interventional Surgery, Nanfang Hospital, Southern Medical University from January 2013 to September 2018. The factors of AVG dysfunction were determined by statistical methods.Results:A total of 139 patients were enrolled, including 58 males (41.7%); the median age was 57; in which 83 patients (59.7%) developed AVG dysfunction within 24 months. Kaplan-Meier survival analysis showed that the primary patency rates were 76.1%, 56.8%, and 38.5% at 6, 12, and 24 months after the establishment of AVG. The results of Kaplan-Meier survival analysis showed that at 24 months after surgery, the risk of AVG dysfunction in elderly patients (>65 years old) was significantly higher than that of patients≤65 years old (Log-rank χ2=7.632, P=0.006); the risk of AVG dysfunction in patients with mean platelet volume (MPV)>10.1 fl was significantly higher than that of patients with MPV≤10.1 fl (Log-rank χ2=19.910, P<0.001); the risk of AVG dysfunction in patients with platelet distribution width (PDW)>11.4 fl was significantly higher than that of patients with PDW≤11.4 fl (Log-rank χ2=35.410, P<0.001); the risk of AVG dysfunction in patients with platelet-larger cell ratio (P-LCR)>24.8% was significantly higher than that of patients with P-LCR≤24.8% (Log-rank χ2=7.181, P=0.007). Multivariate Cox proportional risk regression analysis showed that high MPV (MPV>10.1 fl, HR=6.501, 95% CI 1.916-22.054, P=0.003), high PDW (PDW>11.4 fl, HR=3.625, 95% CI 1.957-6.714, P<0.001) and low P-LCR (P-LCR>24.8%, HR=0.145, 95% CI 0.045-0.470, P=0.001) were independent influencing factors for AVG dysfunction. The establishment of a functional prediction equation based on the above factors had a certain value in predicting the risk of AVG dysfunction in HD patients (likelihood ratio test: χ2=49.360, P<0.001). Conclusions:There are multiple factors that affect AVG dysfunction in HD patients, among which MPV, PDW and P-LCR levels may be the influencing factors for AVG dysfunction. Preoperative examination or postoperative comprehensive review of these factors during the follow-up period has certain directive significance for the prevention of AVG dysfunction.

Mid-term follow-up results of 131 cases of arteriovenous graft patency / 中华普通外科杂志

Objective:To evaluate the mid-term follow-up results of arteriovenous graft(AVG) for patients on maintenance hemodialysis.Methods:The clinical data of 131 patients who implanted AVG from Jan 2014 to Dec 2016 at Department of Vascular Surgery, Nanfang Hospital, Southern Medical University were retrospectively analyzed.Results:The mean follow-up time was 22.8 months (ranging from 2 to 61 months). The average primary patency time after AVG was (22.20±1.97) months, and the primary patency rates at 1 year, 2 years, 3 years were 61.5%, 36.6% and 23.2%, respectively. The average secondary patency time after AVG was (38.30±2.30) months, and the secondary patency rates after 1 year, 2 years, 3 years were 85.6%, 68.6%, and 55.8%, respectively. Sixty five (49.6%) patients had thrombosis after operations, 50(38.2%) had access stenosis, 13(9.9%) had graft infections, and 2(1.5%) had pseudoaneurysm, 2(1.5%) had hemodialysis access-induced distal ischemia, 2(1.5%) had seroma.Conclusions:Though the primary patency rate of AVG is worse than that of arteriovenous fistula (AVF), a satisfactory secondary patency rate can be achieved through repair treatments. Regular follow-up, early detection of stenotic lesions and treatments are vital for long-term patency of AVG.

Effect of sex hormone levels on macrovascular complications in community-dwelling male patients with diabetes mellitus in Shanghai / 中华内分泌代谢杂志

Objective We aimed to investigate whether sex hormone levels including total testosterone, estradiol, estradiol/total testosterone, and dehydroepiandrosterone ( DHEA ) were associated with macrovascular complications among Shanghai community-dwelling diabetic men. Methods Relying on the Environmental Pollutant Exposure and Metabolic Diseases in Shanghai (METAL) study (ChiCTR1800017573, www.chictr.org.cn), 2147 male diabetic participants were recruited from 10 communities. Carotid plaques and common carotid artery ( CCA) diameters were detected by carotid ultrasound. Cardiovascular disease ( CVD) was defined as a self-reported diagnosis of CVD, including coronary heart disease, myocardial infarction, or stroke. Results ( 1) The prevalence of CVD in this study was 36. 0％, and patients with CVD had higher rates of hypertension and dyslipidemia than those without CVD. ( 2) After controlling for multiple factors, serum DHEA levels were negatively correlated with the prevalence of CVD while estradiol levels were positively correlated with both the prevalences of CVD and carotid plaque, estradiol/total testosterone ratio was also positively correlated with the prevalence of CVD. ( 3) In subgroup with unilateral/bilateral CCA plaque, and after controlling for multiple factors, total serum testosterone was negatively associated with the mean CCA diameter. Conclusion The incidence of macrovascular complications was lower in male diabetic patients with higher serum total testosterone and DHEA levels and lower estradiol levels, suggesting that sex hormone levels may be a window for the diagnosis and treatment of diabetic macrovascular complications.

Carotid artery stenosis treated with modified carotid endarterectomy: report of two cases / 南方医科大学学报

Based on standard carotid endarterectomy, we performed modified carotid endarterectomy in two cases of carotid artery stenosis by changing the direction of the carotid artery incision to avoid restenosis of the internal carotid artery without using a patch. The two patients recovered smoothly without any complications. Compared with eversion or patch endarterectomy, this modified carotid endarterectomy avoids restenosis of the carotid artery and shortens operation time.

Prevalence of chronic kidney disease and its risk factors in subjects with different glucose metabolism status / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the prevalence of chronic kidney disease (CKD) in subjects with different glucose metabolism status.</p><p><b>METHODS</b>Between January, 2015 and October, 2015, a total of 934 subjects without a previous diagnosis of diabetes visiting the Department of Endocrinology or Health Examination Center underwent oral glucose tolerance test (OGTT), which identified 266 subjects with normal glucose tolerance (NGT group), 243 pre-diabetic subjects, and 425 patients with diabetes mellitus group. The baseline characteristics and laboratory test data of the subjects were collected. The diagnosis of CKD was established for an eGFR <60 mL/min/1.73 m(2) or a ACR≥30 mg/g, and the prevalence of CKD were compared among the 3 groups. Logistic regression model was used to analyze the OR value of the risk factors of CKD.</p><p><b>RESULTS</b>The prevalences of CKD in NGT, pre-diabetic and diabetic groups were 10.2%, 26.3% and 32.5%, respectively. Pairwise comparisons showed that the prevalence of CKD was significantly higher in pre-diabetic group (P<0.001, OR=3.17, 95% CI 1.94-5.17) and diabetic group (P<0.001, OR=4.27, 95% CI 2.72-6.65) than in NGT group, and was comparable between the pre-diabetic and diabetic groups (P=0.115, OR=1.35, 95% CI 0.95-1.91). Logistic regression analysis, after adjustment for age, gender, blood pressure, hypertension, blood lipids and uric acid, showed that pre-diabetes (OR=2.03, P=0.044) and diabetes mellitus (OR=2.22, P=0.016) were independently associated with CKD.</p><p><b>CONCLUSION</b>Glucose metabolism status has a significant independent impact on the incidence of CKD, suggesting the importance of early detection of pre-diabetes and timely interventions in pre-diabetic subjects in prevention CKD.</p>

Role of Treg/Th17 cells and related cytokines in Graves' ophthalmopathy / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of CD4⁺ CD25⁺ Foxp3⁺ Treg/CD4⁺ IL-17A(+)Th17 cells and the related cytokines in Graves' ophthalmopathy.</p><p><b>METHODS</b>Based on clinical activity scores (CAS), we divided patients with untreated Graves' ophthal- mopathy into active group (AGO group with CAS ≥ 3 (15 cases) and non-active group (NGO group) with CAS<3 (15 cases), with another 15 patients with untreated Graves' disease free of eye symptoms (GD group) and 15 normal subjects as controls. Peripheral venous blood Treg/Th17 cell ratio was determined using flow cytometry. RT-PCR was used to detect the mRNA expression levels of Treg-specific transcription factor Foxp3 and Th17-specific transcription factor RORγt. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of Th17 cell-related cytokines (IL-17A, IL-23, and IL-6) and Treg-related cytokines (TGF-β, IL-10, and IL-35).</p><p><b>RESULTS</b>Compared with the normal subjects, the patients in GD, NGO, AGO groups all showed significantly increased Th17 cell count (P<0.05), which was the highest in AGO group. RT-PCR results revealed significantly increased RORγt in GD, NGO, and AGO groups, also the highest in AGO group. Serum IL-17A, IL-23, and IL-6 levels all showed significant increments in GD, NGO, and AGO groups (P<0.05), especially in AGO group. Among the Treg-related cytokines, TGF-β and IL-35 levels decreased (P<0.05) but IL-10 increased significantly (P<0.05) in GD, NGO, AGO groups.</p><p><b>CONCLUSION</b>Decreased immunosuppressive capacity of Treg cells can be an important factor in the pathogenesis of Graves' ophthalmopathy. Th17 cells may also participate in the occurrence and progression of Graves' ophthalmopathy and can serve along with related cytokines as novel indicators of the disease activity. Impaired Treg/Th17 balance may importantly contribute to the occurrence of Graves' ophthalmopathy.</p>

Application of case-based learning in training vascular surgery resident doctors / 中华医学教育探索杂志

Objective To explore the application and effect of case-based learning(CBL)in vas-cular surgery clinical teaching. Methods Totally 37 resident doctors were randomly divided into 2 groups respectively: CBL teaching group (n=21)and traditional teaching group (n=16). CBL teaching was con-ducted through the following procedures:selecting typical cases-establishing and applying typical case library-autonomous learning-holding regular seminars. Traditional teaching was conducted through the following procedures: basic theory studying-participating in clinical practice-participating in case discus-sion. Evaluation was conducted based on test socre (written test and clinical operational skill test)and res-idents' feedback of teaching effect. Data were statistically described and independent sample t test was performed. Results Theoretical exam score and clinical skill test score were high in CBL group than in traditional group ((thoretical score:(85.53 ±1.75) vs. (79.94 ±2.29);clinical skill test score:(85.10±1.64)vs.(80.31±1.82)). CBL teaching group had advantages in improving learning efficiency, cultivat-ing clinical thinking,promoting mastery and application of knowledge,broadening knowledge, promoting communication and expression ability and improving study enthusiasm ,et al . Conclusion CBL teaching can effectively improve the teaching quality and obtain higher evaluation. Typical case li-brary should be constantly improved and education of vascular surgical basic theory should be strength-ened to promote CBL.

Experience with endovenous laser treatment combined with high ligation and Muller's phlebectomy for C3-6 grade primary superficial varicose in the lower limbs / 南方医科大学学报

<p><b>OBJECTIVE</b>To summarize the experience with endovenous laser treatment(EVLT) combined with high ligation and Muller's phlebectomy for primary superficial varicose in the lower limbs.</p><p><b>METHODS</b>In 95 patients with C3-6 grade primary superficial varicose in 146 lower limbs, the extent of varicose was accurately marked, the guiding wires were manipulated precisely, and the proximal great saphenous veins (GSV) were ligated after exsanguinations. The stems of the GSV were ablated with laser with the lower limbs lift up and pressed hard along the stems, and Muller's incisions were carefully planned.</p><p><b>RESULTS</b>All the operations were completed successfully. The guiding wires entered into the deep veins through the communicating branches in 2 limbs, 1 patient experienced capillary hemorrhage from Muller's incisions, 8 had thrombotic phlebitis of the GSV, 7 sustained heat-related injury of the saphenous nerves, 1 experienced skin heat-related lesion, 2 developed hematoma in the inguinal region, 2 had pitting edema in the dorsum of the foot, 1 had fat liquefaction of the Muller incision, and 1 showed rejection of the thrum. After conservative treatment, all the patients recovered and were discharged. Part of the superficial varicose remained after the operation in 6 limbs.</p><p><b>CONCLUSION</b>It is necessary to standardize the routine procedure of EVLT combined with high ligation and Muller's phlebectomy to reduce the complications.</p>

Cerebellar Schistosomiasis: A Case Report with Clinical Analysis

The authors report here a rare case of cerebellar schistosomiasis identified by pathological diagnosis, lacking extracranial involvement. The clinical symptoms included headache, dizziness, and nausea. Studies in blood were normal and no parasite eggs were detected in stool. Computed tomography of brains showed hypodense signal, and magnetic resonance imaging showed isointense signal on T1-weighted images, hyperintense signal on T2-weighted images, and intensely enhancing nodules in the right cerebellum after intravenous administration of gadolinium. A high-grade glioma was suspected, and an operation was performed. The pathologic examination of the biopsy specimen revealed schistosomal granulomas scattered within the parenchyma of the cerebellum. The definitive diagnosis was cerebellar schistosomiasis japonica. A standard use of praziquantel and corticosteroid drugs was applied, and the prognosis was good. When the pattern of imaging examinations is present as mentioned above, a diagnosis of brain schistosomiasis should be considered.

EGFR mutations are more frequent in well-differentiated than in poor-differentiated lung adenocarcinomas.

Somatic mutations in epidermal growth factor receptor (EGFR) tyrosine kinase domain, particularly deletions in exon 19 and point mutation in exon 21, are associated with clinical outcome in patients with lung adenocarcinoma, suggesting that EGFR mutation would have an important role in clinical decision making. DNA was extracted from the excised specimens of 60 lung adenocarcinoma patients with phenol-chloroform and ethanol precipitation. Exon 19 and 21 were amplified by PCR, and direct sequenced from both sense and antisense directions. EGFR somatic mutations were present in 13 of 60 patients (21.67%), including seven cases of in-frame deletion in exon 19 around codon 746 and six cases of amino acid substitution in exon 21. Exon 21 mutation is more frequent in adenocarcinomas with bronchi-alveolar component than exon 19 deletions. Mutations were more prevalent in well-differentiated adenocarcinomas (9/27, 33.33%) than in moderate to poor-differentiated adenocarcinomas (4/33, 12.12%) (P < 0.05). Adenocarcinomas with bronchi-alveolar components had higher mutation frequency (8/22,36. 36%) than those without bronchi-alveolar components (5/38, 13.16%) (P < 0.05). In this study, female patients had more mutation rate than male patients. This trend was also observed in the patients with pathologic stage I-II compared with stage III-IV, but neither of them was statistically significant. Patients with cisplatin-based adjuvant chemotherapy had no significantly prolonged survival compared with single radical resection. But patients with EGFR mutation had relative longer survival. In conclusion, our study suggest that EGFR mutations may be a valuable prognostic factor for disease free survival of surgically treated lung adenocarcinoma patients independently from adjuvant chemotherapy.

[P2Y purinergic receptor activated PI-3K/Akt signaling pathway in regulation of growth and invasion of prostatic cancer].

OBJECTIVE: To investigate P2Y purinergic receptor activated PI-3K/Akt signaling pathway in the regulation of growth and invasion of prostate cancer in vitro. METHODS: Western blot was used to detect phosphorylation of Akt (a downstream target molecule of PI-3K) by P2Y receptor agonist in 1E8 cells (a highly metastatic subclone derived from PC-3 prostatic cancer cell line). Cell counts, flow cytometry, Matrigel invasion assay, wound healing assay and gelatin zymography were used to detect changes of biological behaviors of 1E8 cells after P2Y receptor activation. RESULTS: AMP-PNP, one non-hydrolysis ATP analogue and P2Y receptor agonist, induced significant phosphorylation of Akt in a time- and dose-dependent manner in IE8 cells. LY294002, a specific inhibitor of PI-3K, effectively blocked Akt phosphorylation induced by AMP-PNP. Continuous exposure to AMP-PNP induced significant growth inhibition of 1E8 cells (inhibition rate at 50.2% at the 8th day), and this inhibition was mainly due to an arrest at S phase of the cell cycle (the S phase fraction of AMP-PNP treated cells was 22.3% higher than that of the control). Application of LY294002 did not reverse the growth inhibition effect of AMP-PNP. Matrigel invasion assay showed that AMP-PNP stimulation increased invasive ability of 1E8 cells, and this effect was effectively blocked by LY294002. No significant changes in the activation of MMP-2 and MMP-9 were detected by gelatin zymography, although wound healing assay showed 21.2% increase in cell migration after AMP-PNP treatment. CONCLUSIONS: PI-3K/Akt signaling pathway participates in P2Y receptor-stimulated prostate cancer invasion by enhancing cell motility, rather than up-regulating MMP-2 and MMP-9 activities. PI-3K signaling pathway plays an important role in prostate cancer proliferation, but is not involved in P2Y receptor mediated growth inhibition.

Study on the relationship between passive smoking and blood lipids, fibrinogen and viscosity among women who never smoke / 中华流行病学杂志

<p><b>OBJECTIVE</b>To determine whether blood lipids profile, fibrinogen and viscosity were associated with passive smoking (i. e. environmental tobacco smoke, ETS) in Chinese women who never smoke.</p><p><b>METHODS</b>In Xi'an, China, a case-control study was carried out on 115 cases of coronary heart disease (CHD) defined by coronary arteriography (CAG) and 208 non-CHD controls confirmed by CAG and/or exercise electrocardiography. Data on exposure to ETS, defined as exposure from cigarettes smoking husband or co-workers or both for at least 5 years, was obtained through standardized interviews. Standard laboratory methods were used and the lipid measurements were under US CDC quality control programs.</p><p><b>RESULTS</b>In the subjects defined by CAG, the levels of high density lipoprotein cholesterol (HDL-C), HDL2C, apolipoprotein (apo) A1 among passive smokers appeared lower than those in non-passive smokers,but the low density lipoprotein cholesterol (LDL-C), apoB, apoB/A1, fibrinogen, plasma and whole blood viscosity were higher than that in non-passive smokers. There were positive associations of the numbers of coronary arteriosclerosis with the levels of blood lipids,fibrinogen and viscosity. In the non-CHD controls, 81 subjects were not exposed and 127 were exposed to ETS. The P values of t-test for the adjusted (for age, body mass index, present diseases history) means between two groups were listed below: 0.06 (total cholesterol), 0.30 (triglyceride), 0.004 (HDL-C), <0.001 (HDL2-C), < 0.001 (apoA1), 0.009 (apoB), <0.001 (apoB/apoA1), <0.001 (fibrinogen), <0.001 (plasma viscosity), <0.001 and 0.004 [two measures (5.75/s and 230/s) of whole blood viscosity]. The correlation coefficients between cumulative exposure of passive smoking and HDL-C,HDL2-C,apoA1, apoB, apoB/apoA1, fibrinogen, plasma viscosity, and two measures of whole blood viscosity were -0.25, -0.27, -0.30, 0.24, 0.31, 0.32, 0.43, 0.51 and 0.36 (all P<0.01), respectively.</p><p><b>CONCLUSION</b>Passive smoking could affect blood lipid metabolism, fibrinogen and viscosity in the never smoking women which might contribute to the causation of coronary heart disease.</p>

Novel polymeric microspheres containing norcantharidin for chemoembolization.

Chemoembolization has been found to be a potentially effective method of treating certain types of cancer. It involves arterial embolization of a tumor, in combination with simultaneous or subsequent local delivery of chemotherapeutic agents. In this study, PLGA-alginate microspheres were evaluated for their potential application in chemoembolization. Norcantharidin, which possesses anti-tumor properties, was used to investigate the application of drug-containing microspheres for chemoembolization. The release profiles of alginate, PLGA and PLGA-alginate microspheres were markedly different in phosphate buffered saline, with the composite microspheres showing the most appropriate release rate for chemoembolization. Burst effect decreased while particle size increased with increasing proportion of alginate in the PLGA-alginate microspheres. PLGA-alginate microspheres containing norcantharidin were effective in destroying the cancer cells used in this study. The growth inhibitory effect was concentration and time dependent. These microspheres also exhibited excellent embolization and therapeutic effects on rats with transplanted tumors.

[Status and clinical implication of c-kit and PDGFRA mutations in 165 cases of gastrointestinal stromal tumor (GIST)].

OBJECTIVE: To investigate the status of c-kit and PDGFRA mutations of GIST in a the large sample of Chinese patients. METHOD: One hundred and sixty-five cases were evaluated for the presence of c-kit and PDGFRA mutations. Exon 9, 11, 13, 17 of c-kit and exon 12, 18 of PDGFRA were analyzed by PCR amplification and direct sequencing. RESULTS: Immunohistochemical demonstrations of KIT (CD117) were seen in 94% of the cases (155/165). Overall, c-kit mutations were identified in 76.1% (118/155) of CD117 positive cases: 67.1% (104/155) involving exon 11, 7.1% (11/155) involving exon 9, 1.3% (2/155) involving exon 13 and 0.6% (1/155) involving exon 17. The c-kit exon 11 mutations were mostly heterogeneous and clustered in the classic "hot spot" at the 5' end of the exon, including in-frame deletion and point mutation. The second "hot spots" were internal tandem duplications (ITD) at the 3' end of the exon, which were associated with female patient, older age, stomach location and low mitotic counts. The exon 9 mutations correlated with a distinct subset of GISTs involving the small bowel of young male patients. A new point mutation of L641P was identified in exon 13. PDGFRA mutations were present in 50% (5/10) of CD117-negative GISTs, all involving exon 18 with the majority of mutations being D842V. One novel in-frame deletion of IMHD mutation at codon 843 - 846 with S847T was identified. GISTs with PDGFRA mutations were often larger tumors arising from the omentum/mesentery of young male patients with high risk of aggressive behavior. CONCLUSIONS: The vast majority of GISTs in this study harbored c-kit and PDGFRA mutations, there were non-random relations between the gene mutation patterns and the locations of GISTs. It appears that Chinese GIST patients have some unique mutation patterns. It is necessary to evaluate the gene mutations status of GISTs to guide target therapy.

[Detection of Mycobacterium tuberculosis DNA in sarcoidosis samples using real-time fluorescence polymerase chain reaction].

OBJECTIVE: To study the role of Mycobacterium tuberculosis in the pathogenesis of sarcoidosis. METHODS: Archival material of 22 patients with a histologic diagnosis of sarcoidosis were retrieved. Real-time fluorescent polymerase chain reaction (PCR) was used to detect DNA fragments of the complex-specific insertion sequence IS6110 of Mycobacterium tuberculosis in formalin-fixed and paraffin-embedded biopsy samples. RESULTS: Among the 22 samples studied, Mycobacterium tuberculosis DNA was detected in 11 cases. The sequence of PCR amplified IS6110 DNA fragments completely matched with the related sequence in Mycobacterium tuberculosis gene. CONCLUSIONS: Mycobacterium tuberculosis DNA is identified in a certain proportion of patients with a clinicopathologic diagnosis of sarcoidosis. Mycobacterium tuberculosis may be an important etiologic agent, at least in some of these patients.

[SHP2 and MKP5 in P2Y purinergic receptor-mediated prostate cancer invasion].

OBJECTIVE: To investigate the effects of protein tyrosine phosphatase-SHP2 and dual-specificity MAPK phosphatase-MKP5 on the activation of MAPKs and cell invasion induced by P2Y purinergic receptor in human prostate cancer cell lines with different metastatic potentials. METHODS: The wide type (-wt) SHP2, mutant type (-cs) SHP2 and wide type (-wt) MKP5 cDNA expression vectors were constructed and stably transfected into 1E8 cells (highly metastatic) and/or 2B4 cells (non-metastatic). The tyrosine phosphorylation of SHP2 was examined by immunoprecipitation. The activation of ERK1/2 and p38 induced by P2Y receptor agonist ATP was analyzed by Western blot with phospho-specific antibodies against the dually phosphorylated, active forms of ERK1/2 and p38. The in-vitro invasive ability through Matrigel was measured by boyden-chamber assay. RESULTS: ATP induced significant SHP2 phosphorylation, which was stronger and lasted longer in 1E8 than in 2B4. SHP2-wt enhanced the ERK1/2 activation induced by ATP in 2B4 cells, while SHP2-cs delayed and decreased this effect in 1E8 cells. Both SHP2-wt and SHP2-cs had no obvious influence on p38 activation. ATP stimulated cell invasion of both 1E8 and 2B4, while transfection of SHP2-wt into 2B4 cells further increased the invasive-stimulating ability of ATP (18.7% increase compared with ATP treatment alone). Transfection of SHP2-cs into 1E8 cells, however, antagonized the invasive-stimulating ability of ATP (40.9% decrease compared with ATP treated group). Up-regulation of MKP5-wt inhibited phosphorylation of p38 by ATP and reduced cell invasion stimulated by ATP (22.4% and 28.7% decrease compared with ATP treated group of 1E8 and 2B4, respectively). CONCLUSIONS: Both SHP2 and MKP5 play some roles in P2Y receptor-mediated activation of MEK/ERK, p38 signaling pathways and prostate cancer invasion. SHP2 positively regulates ERK activation and prostate cancer invasion, whereas MKP5 inhibits the invasion by suppressing p38 activation.

[ERK1/2 and p38 kinases are important regulators in P2Y receptor-mediated prostate cancer invasion].

OBJECTIVE: To explore whether ERK1/2 and p38 pathways mediate P2Y receptor-induced prostate cancer invasion. METHODS: The two subclones from the PC-3 human prostate carcinoma cell line: 1E8 (highly metastatic) and 2B4 (non-metastatic), were cultured and transfected with the plasmid pcDNA3-KA-MEK1 containing the dominant negative mutant of MEK1 (KA-MEK1) and wild type MKP-5 (a dual-specificity phosphatase of p38). P2Y receptor-activated ERK1/2 and p38 kinases were detected using phospho-specific antibodies directed against the dually phosphorylated active forms of these kinases by Western blotting. P2Y receptor agonists ATP and P2Y receptor antagonist suramin were used respectively to observe their effects on the activity of ERK1/2. The roles ERK1/2 and p38 pathways play in P2Y receptor-induced in vitro invasion were detected by in vitro invasion assay. The cells were pre-treated with ATP, SB203580, a p38 inhibitor, and PD98059, a blocker of ERK1/2 pathway, respectively. RESULTS: ATP activated ERK1/2 and p38 kinase time-dependently. Suramin significantly inhibited the activation of ERK1/2 and p38 kinase by ATP. ATP stimulated prostate cancer cell invasion. The stimulated cancer cell invasion was significantly inhibited by pretreatment of the cells with PD98059 or SB203580. Transfected of 1E8 cells with KA-MEK1 or up-regulation of MKP-5 both, while inhibiting phosphorylation of ERK1/2 or p38, significantly reduced the invasion of prostate cancer cells in vitro. CONCLUSION: P2Y receptor-induced prostate cancer cell invasion is mainly regulated through ERK1/2 and p38 pathways.

In vitro study of HIF-1 activation and VEGF release by bFGF in the T47D breast cancer cell line under normoxic conditions: involvement of PI-3K/Akt and MEK1/ERK pathways.

Hypoxia-inducible factor (HIF) is critical in the modulation of tumour angiogenesis in response to hypoxia. In the present study, the mechanisms underlying basic fibroblast growth factor (bFGF)-induced activation of HIF-1 and the subsequent release of vascular endothelial growth factor (VEGF) in a human breast cancer cell line (T47D) under normoxic conditions were explored. The data show that HIF-1alpha expression is induced by bFGF in a dose- and time-dependent fashion, while increased HIF-1alpha protein expression and transactivity of HIF-1 are due to the phosphorylation of Akt by bFGF, as indicated by application of the phosphatidylinositol 3-kinase (PI-3K) inhibitor LY294002. The data also show that the MEK1 (mitogen-activated protein kinase kinase-1)/ERK (extracellular signal-regulated kinase) pathway is only involved in bFGF-induced transactivity of HIF-1, but not HIF-1alpha expression, indicating roles for both the PI-3K/Akt and the MEK1/ERK pathways in bFGF activity. In addition, the translation inhibitor cycloheximide confirmed that bFGF-induced HIF-1alpha protein expression was due to de novo protein synthesis. In contrast, p38 was not required for the expression of HIF-1alpha or HIF-1 transactivity, although significant phosphorylation of p38 was observed after bFGF treatment. Treatment of the cells with bFGF increased the amount of VEGF release, and this could be suppressed by either PD98059 or LY294002, suggesting the presence of a HIF-1alpha-dependent pathway for bFGF-induced VEGF production. In conclusion, the PI-3K/Akt and MEK1/ERK pathways, in a potentially independent and co-operative fashion, can modulate HIF-1 activation by bFGF. Further studies will pinpoint whether HIF-1 is the transcriptional factor responsible for the increased VEGF production following bFGF treatment of breast tumour cells.