Eotetranychus smithiPritchard & Baker (Acari: Tetranychidae), exhibits a facultative diapause that occurs at the egg stage. Diapause was induced by low temperatures alone (≤ 17.5°C) and averted by high temperatures (≥ 20°C). Photoperiod had little effect on diapause induction. This is the first example of temperature-induced diapause in spider mites. The diapause eggs became larger and darker (orange) than non- diapause eggs , suggesting that egg size and egg color are associated with diapause. When mites that were reared from eggs at 25°C and 16:8 L:D were transferred to 15°C and 16:8 L:D just after the start of the teleiochrysalis stage , all females laid non-diapause eggs during the first 30 days and then switched over to laying diapause eggs. The switch to diapause may be caused by the aging of mothers.The spider mite, Aedes vexans(Friedrich 1984), the tobacco hornworm,Manduca sexta, is a multivoltine species that infests various berries, roses, pears, and grapes in the United States, Brazil, and Asia, including China, Korea, and Japan  induce egg diapause independently of photoperiod. The former diapause response may be adaptive forE. smithiinhabiting a strawberry greenhouse, where night temperatures are much lower than day temperatures. The winter temperature in a strawberry greenhouse is around 8°C at night, even with gas heating, and is around 25°C during the day (24-hr average is 15–17°C) under long-day conditions . However, it is unknown whetherE. smithifemales lay non-diapause or diapause eggs under such conditions of fluctuating temperature and long-day photoperiod. If egg diapause ofE. smithiis exclusively induced by low temperature, as in the latter case, allE. smithifemales that underwent juvenile development under low temperatures (15–17°C) in a greenhouse might lay diapause eggs, and also adult females initially laying non-diapause eggs might, in response to low temperatures, switch to laying diapause eggs. Whether adult female mites are sensitive to the diapause-inducing low temperatures and can switch to laying diapause eggs needs to be clarified.These observations indicate that diapause induction is controlled by an appropriate combination of photoperiod and temperature. On the other hand, in theE. smithi. In the first experiment, eggs were kept and juveniles developed to adults under various combinations of photoperiod (16:8 L:D or 10:14 L:D) and temperature  to assess the effect of temperature and photoperiod on egg type laid (diapause or non-diapause eggs). In a second experiment, teleiochrysalis females  that had developed from egg onwards at 25°C and 16:8 L:D photoperiod were transferred to 15°C and a 16:8 L:D photoperiod, and subsequently the type was monitored of all eggs laid to clarify whether adult females can switch from producing non- diapause to diapause eggs, or vice versa.The purpose of the present study was to test the hypothesis that switchover from non- diapause to diapause status occurs in the adult stage ofE. smithiwere collected originally fromRosa multifloraThunb.  on 14 August 2007 at Kuchinotsu, Nagasaki, Japan . Laboratory stocks were reared on leaf discs (ca. 16 cm2) of either mulberry,Morus bombycisKoidz. (Moraceae) (July to September), or strawberry,FragariaxananassaDuchesne (Rosaceae) , placed on water-saturated polyurethane mats in plastic dishes  at 25 ± 1°C, 16:8 L:D photoperiod, and 60–70% relative humidity.Several tens ofE. smithi, groups of 20 inseminated female adults obtained from stock cultures were transferred onto leaf discs (5 cm diameter) of strawberry and kept at 15, 17.5, 20, or 25°C and 10:14 L:D or 16:8 L:D. Females were allowed to lay eggs for 24 hr under each set of these eight regimes. Eggs obtained were reared until all individuals reached the teleiochrysalis stage . Single teleiochrysalis females (n = 32–105) were individually placed on a strawberry leaf disc (1 cm diameter) with two male adults obtained from stock cultures for copulation. All eggs except one were removed per female, and the remaining eggs were observed for 30 days to determine the duration of the egg period under the various conditions. The diameter of a random selection of 20 of these remaining eggs was measured using a binocular stereomicroscope  equipped with an ocular micrometer , and egg color was recorded. Eggs used in this experiment were laid on the third or fourth day after the start of oviposition, that is, on day 17, 15, 6, and 4 of adult life of females kept at 15, 17.5, 20 and 25°C, respectively. On the third or fourth day, oviposition ranged from 1 to 11 eggs/female/day, depending on temperatures.To determine the effects of temperature and photoperiod on induction of the embryonic diapause ofTo assess fecundity and longevity of females reared under the various conditions, females obtained from stock cultures were individually transferred onto strawberry leaf discs (1 cm diameter) and allowed to lay eggs for 24 hr at 15°C/10:14 L:D, 15°C/16:8 L:D, 17.5°C/16:8 L:D, or 25°C/16:8 L:D. On each leaf disc, one egg was left and reared until all individuals reached the teleiochrysalis stage. Two male adults from stock cultures were added to each teleiochrysalis female for copulation. Females were monitored, and eggs were counted daily. If a female had laid one or more eggs since the previous observation, she was transferred onto a new leaf disc (1 cm diameter) and this procedure was repeated until she died. Leaf discs with eggs were moved to the 25°C/16:8 L:D condition and kept there for 10 days to determine whether these eggs would hatch or not. Egg duration of non-diapause eggs ranged from three to five days when females had been reared and oviposited at 25°C/16:8 L:D see.A second batch of 20 teleiochrysalis females that had undergone juvenile development under 25°C/16:8 L:D was transferred to 15°C/16:8 L:D (temperature-switch treatment). Again, two male adults were added for copulation, and eggs were counted daily, as described above. The transfer of adult females to a lower temperature (same photoperiod) may provide more insight in the mite’s developmental phase sensitivity to diapause- inducing stimuli.Data on egg period, egg size, oviposition period, pre- and postoviposition periods, adult longevity, and total fecundity per female were analyzed using oneway analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test for multiple comparisons. Numbers of diapause eggs and non-diapause eggs obtained from the temperature-changed condition were compared using a χ2-test. These statistical analyses were performed using PASW ver. 18 for Windows . To norE. smithiis temperature dependent.At 15 and 17.5°C, all females laid diapause eggs regardless of the photoperiod ; no eggP< 0.05, Tukey’s HSD test;Diapause eggs were larger than non-diapause eggs .Most females that were held at 15 or 17.5°C deposited diapause eggs throughout their lives, but a few females (n = 1–4) began to lay non-diapause eggs near the end of their lives –C. By cP< 0.05, Tukey’s HSD test;P< 0.01, χ2 = 10.632).Under constant temperature and photoperiodic conditions, both the oviposition period and total longevity were shortened with increasing temperature, but they were 1.5 to 3 times longer under the temperature-switch condition  induce diapause in eggs of, Kenya . In temnezarae, entered reproductive diapause under 15°C/16:8 L:D . We feel confident about our findings because we confirmed the diapause status of the eggs at 15 and 17.5°C, both at 10:14 L:D and 16:8 L:D, both in the experiment on egg duration  not react quickly to low temperatures because of extreme fluctuations of autumn climate (E. smithi.The females ofoperiod . The swnunguis, in whinunguis. In the8:6 L:D . By con8:6 L:D . Thus, climate . Female


Objective. To explore the antiprostate cancer effects of Celastrol on prostate cancer cells' proliferation, apoptosis, and cell cycle distribution, as well as the correlation to the regulation of hERG. Methods. DU145 cells were treated with various concentrations of Celastrol (0.25–16.0 μmol/L) for 0–72 hours. MTT assay was used to evaluate the inhibition effect of Celastrol on the growth of DU145 cells. Cell apoptosis was detected through both Annexin-V FITC/PI double-labeled cytometry and Hoechst 33258. Cell cycle regulation was examined by a propidium iodide method. Western blot and RT-PCR technologies were applied to assess the expression level of hERG in DU145 cells. Results. Celastrol presented striking growth inhibition and apoptosis induction potency on DU145 cells in vitro in a time- and dose-dependent manner. The IC50 value of Celastrol for 24 hours was 2.349 ± 0.213 μmol/L. Moreover, Celastrol induced DU145 cell apoptosis in a cell cycle-dependent manner, which means Celastrol could arrest DU145 cells in G0/G1 phase; accordingly, cells in S phase decreased gradually and no obvious changes were found in G2/M phase cells. Through transmission electron microscope, apoptotic bodies containing nuclear fragments were found in Celastrol-treated DU145 cells. Overexpression of hERG channel was found in DU145 cells, while Celastrol could downregulate it at both protein and mRNA level in a dose-dependent manner (P < 0.01). Conclusions. Celastrol exhibits its antiprostate cancer effects partially through the downregulation of the expression level of hERG channel in DU145 cells, suggesting that Celastrol may be a potential agent against prostate cancer with a mechanism of blocking the hERG channel. Tripterygium wilfordii. A general and three-terpene pigment monomer, Celastrol, is found early to have anti-inflammatory and antitumor pharmacological effects on liver cancer, colon cancer, lung cancer, leukemia, cancer of the esophagus, brain cancer, bladder cancer, and so on. Much tumor cell growth and proliferation were inhibited [Ion channels exist widely in all cells in a variety of physiological functions. Cancer is also associated with ion channel dysfunction. Research  recentlynhibited , 7. But Tripterine, molecular formula (C29H38O4), a molecular weight of 450, and purity more than 95%, was purchased from USA Calbiochem company, dissolved in two dimethyl sulfoxide (DMSO), and kept at −20°C, before using DMEM culture medium diluted to final concentration. DMEM medium and fetal calf serum were from the USA Gibco BRL company. Annexin-V/PI apoptosis kits were purchased from Wuhan Boster Engineering Co., Ltd. Rabbit anti-human hERG1 monoclonal antibody was purchased from Sigma company, HRP standard. Sheep anti free, two anti purchased from USA Santa Cruz products. Bio-Rad protein assay kit, TRIzol kit, and ECL lighting kits originated from Sweden Amersham company. RT-PCR kit was purchased from Fermentas company. Primers were synthesized by Shanghai Sangon company.2, and water saturated humidity condition. Every 1~2 days, for a fluid passage, the logarithm growth period of cell activity of more than 98% cells was used in this study.Prostate cancer cell DU145 was obtained from Tongji Medical College, Department of Immunology. Medium contained 10% fetal bovine serum, penicillin 100 IU/mL, and streptomycin 100 g/mL DMEM, at 37°C, 5% CO5/mL cells were seeded in 96-well plate, adding different concentrations of celastrol (0.25–16.0 μmol/L), with the culture solution containing equal volume DMSO as blank control, each drug concentration group with 3 holes, each hole total volume 200 μL. For 5% CO2, 37°C incubator culture 24–72 h, each hole with 5 mg/mL MTT 20 μL reagent, 37°C incubate for 4 h, carefully to absorb the air in culture supernatant, DMSO solution was added to each well of 150 μL, 10 min oscillation, crystallize fully dissolved in the Bio-Rad M450 enzyme labeled each hole optical density were measured in 492 nm wavelength 1.25 meter value (OD), with each experiment repeated 1.273 times, to calculate the inhibitory rate of cell proliferation. Proliferation inhibition rate (%) =  × 100%.Logarithmic growth phase DU145 cell experiments, cells per hole 2 × 10μmol/L) DU145 cells and blank treated cells in control group, with 4°C ice cold PBS wash 2 times, and then to 1 × 106/mL cell density weight suspended from 100 μL binding buffer, adding 5 μL Annexin-V and 10 μL PI dye solution, mix gently, light reaction temperature 15 min, add 300 μL of the buffer solution, detection of it within 1 h.Operate according to kit, divided into the experimental group and the control group with single standard. A collection of different concentration of tripterine  fixed 15 min, washed two times with PBS; 37°C Hoechst 33258 staining solution (5 mg/L) staining of 15~30 min, PBS wash two times; neutral resin sheet, fluorescent cell morphology was observed under microscope and photographed. Mirror were found 5 does not repeat, apoptotic cell count per 200 cells, the percentage of apoptotic cells is the apoptosis rate.The logarithmic growth phase DU145 cell was 5 × 106, washing with PBS buffer 2 times, with 70% cold ethanol at 4°C fixed overnight, centrifugation, washing 1 time with PBS, adding 20 μL RNase A at 37°C water bath for 30 min and then adding 300~500 μL PI dye solution mixing and placing 4°C under dark condition for 30 min; the cell cycle was detected by flow cytometry, with red fluorescent wavelength at 488 nm.Collection of the treated DU145 cell was 1 × 10TRIzol kit was used to extract total cellular RNA synthesis of cDNA, according to the instructions. In the first chain cDNA cells were used as template, PCR reaction. PCR primer was synthesized by Sangon company in Shanghai, of which hERG gene upstream primer was 5′-CAGCGGCTGTACTCGGGCACAG-3′, downstream primer was 5′-CAGAAGTGGTCGGAGAACTC-3′, amplified fragment is 345 bp; 3-glyceraldehyde phosphate dehydrogenase gene (GAPDH) upstream primer is 5′-GATTTGGTCGTATTGGGGCGC-3′, downstream primer is 5′-CAGAGATGACCCTTTTGGCTCC-3′, amplified fragment is 136 bp. The PCR amplification conditions were 95°C denaturing 5 min, 94°C 1 min, 55°C 50 s, 72°C 1 min, cycle 35, 72°C 10 min end reaction. PCR products were detected by 1.5% agarose gel electrophoresis, UV photography, and scanning analysis, the hERG/GAPDH expression of hERG semiquantitative analysis of the level of.μL pre-cooling (configuration according to molecular cloning method) the ice cracking 30 min, extraction of total cellular protein, quantitative protein by Lowry method. Conventional adhesive preparation, sampling, protein electrophoresis, and then transferring to the membrane were done. Rabbit anti-human hERG1 monoclonal antibodies were added (1 : 1500), 4°C overnight incubation. Rinse after adding HRP-labeled goat anti-rabbit IgG (1 : 2000), at 37°C with shaking and incubation for 1 h. Finally, ECL chemiluminescence reagent, X-ray exposure and development, analysis of computer software. Each concentration was repeated 3 times, taking the mean measurement results.Different concentrations of tripterine treated DU145 cells and control cells, with cell lysate 100  F test, SPSS 11.5 statistical software analysis.Experimental data μmol/L tripterine in DU145 cells after 24~72 h, that cell proliferation activity in different cell groups was lower than that of control cells, but in a concentration less than 1 μmol/L, proliferation effects of tripterine on DU145 cells are small, and when the tripterine concentration reached 1 μmol/L, the proliferation inhibition activity was significantly increased, with significant difference (P < 0.05). And, with the increase of tripterine drug concentration and action time, the inhibitory effects of proliferation were enhanced, and an obvious time dose effect relationship is apparent. The 24 h IC50 value was 2.349 ± 0.213 μmol/L.It can be seen from μmol/L tripterine acid treatment in typical apoptosis morphological changes are as follows: cytoplasmic density, chromosome condensation, marginalization, nuclear condensation, and formation of apoptotic bodies increased %, (40.48 ± 2.34)%, (51.40 ± 1.96)%, and (68.58 ± 2.89)%, while the percentage of cells in S phase was dose-dependently reduced, followed by (49.45 ± 1.67)%, (46.19 ± 1.86)%, (37.67 ± 2.03)%, and (23.29 ± 1.52)%; in contrast, Celastrol effects on G2/M cells were not significant . In order to further clarify the role of Tripterygium wilfordii red on the hERG protein, we examined the changes of hERG protein and mRNA content in the level of gene transcription. Similarly, hERG potassium channel protein level of mRNA was dose-dependently downregulated and obviously higher than the mononuclear cells of normal hERG gene expression level (Compared with normal mononuclear cells, the presence of hERG potassium channel protein expression levels was higher in DU145 cells and the 0.5~2.0 on level . in vitro and in vivo. In the early 1970s, there were reports of tripterine having anti-inflammatory, analgesic, antioxidant, and antiviral effects, and inducing apoptosis of tumor cells is more positive. Further, to determine the effective components of gambogic acid differently from the general characteristics of anticancer drugs, it can selectively kill tumor cells, but normal hematopoietic cells and heart, liver, kidney, and other organs showed no obvious damage, so it is considered to be a safe and effective antitumor drug [People have found that many natural preparations, especially in plants and food components, have significant antitumor activitymor drug , 9 for lμmol/L tripterine treated for 24 h, the apoptosis rate of DU145 cells increased significantly, and the typical morphological changes of cell apoptosis emerged. The Celastrol-induced apoptosis of cycle arrest effect may be related to induction of closely related events. With the increase of tripterine concentration, the percentage of G0/G1 phase cells increased gradually and the percentage of cells in S phase decreased gradually and the tripterine had little effect on the percentage of cells in G2/M phase. That Celastrol operating mainly by blocking DU145 cells at G0/G1 phase to have an apoptosis inducing effect has been reported in the literature [The results show that Celastrol can inhibit the proliferation of DU145 cells and the inhibition is associated with the duration of drug action and drug concentration. At the same time, Celastrol can induce apoptosis of DU145 cells through strong 0.5~2.0 terature . Tripterygium wilfordii on DU145 cells of hERG potassium channel protein regulation. The results show that, compared with the mononuclear cells of normal control, the expression of hERG potassium channel protein levels rises to higher DU145 cell memory, while the normal mononuclear cells hardly show expression. For the effect of tripterine intervention, the protein and gene expression levels were concentration-dependent underground tune. The effect of tripterine on proliferation of DU145 cells and the intracellular expression of hERG potassium channel are closely related. Although confirmed in tumor cells, inhibition of hERG potassium channel protein expression can inhibit the proliferation of tumor cells, inducing tumor cell apoptosis. Tripterine is expected to become the hERG potassium channel protein inhibitor of a new generation.Celastrol has become a hot spot of oncology field expression of ion channel proteins in normal and tumor cells. The ion channel proteins in numerous studies, tumor cell lines by hERG potassium channel protein encoded by the hERG gene as selective surface in different tissues and primary tumor cells, and tumor cell proliferation, differentiation, apoptosis, invasion, and sensitivity to chemotherapy are closely related and are considered to be the molecular target in cancer cells more specifically. In general, the hERG gene was only expressed in the early stages of embryonic development and followed by the inward rectifier potassium channel current it was replaced by . HERG poEffects of hERG potassium channel protein on the biological behavior of the tumor that can inhibit the growth of tumor cells by inhibiting the expression or channel current of IhERG and promote tumor cell differentiation or apoptosis, reduce its invasiveness. There lays a good foundation for tumor targeting therapy and drug screening.


Objective. This study investigated whether thyroid hormone (TH) levels are correlated to cell proliferation (Ki67), in euthyroid breast cancer patients. Design and Methods. 86 newly diagnosed breast cancer patients with estrogen receptor (ER) positive tumors, who referred for surgery, were included in the study. Results. FT3, FT4, and TSH were within normal range. No correlation was seen between Ki67 and FT3 , FT4 , or TSH  in all patients studied. However, subgroup analysis showed that, in HER2(+) patients, a negative correlation existed between FT3 levels and Ki67 (r = −0.60 and P = 0.004) but not between Ki67 and FT4 (r = 0.04 and P = 0.85) or TSH (r = −0.23 and P = 0.30). In HER2(−) patients, there was no significant correlation between Ki67 and FT3 , FT4 , or TSH . Phospho-p44/total p44 ERK levels were found to be increased by 2-fold in HER2(+) versus HER2(−) tumors. No difference was detected in phospho-p42/total p42 ERK levels. Conclusions. TH profile is not altered in patients with newly diagnosed breast cancer. However, FT3 levels, even within normal range, are negatively correlated with cell proliferation in HER2(+) breast cancer tumors. This response may be due to the interaction between ERK and TH signaling. Thyroid hormone (TH) may be critical in the pathogenesis and progression of diseases due to its regulatory role on cell maturation . There iA total of 86 patients with newly diagnosed breast cancer with estrogen receptor (ER) positive breast tumors, who referred for surgery, were included in the study.β-blocking agents, aspirin, heparin, phenytoin, steroids, or dopamine in the month before or during the study. In addition, they were not given iodine-containing contrast agents in the six months before and during the study. Thyroid hormone levels and serum measurements were performed prior to surgery in all patients.Patients who had radiation or chemotherapy administration before surgery, hormone replacement therapy, any kind of previously diagnosed thyroid disease, and chronic kidney failure were not included. These patients were not on therapy with Clinical history, age, and menopausal status together with tumor characteristics including tumor histological grade, lymphovascular invasion, lymph node stage, metastases, and tumor size were studied.Consent has been obtained from each patient or subject after full explanation of the purpose and nature of all procedures used. The investigation was approved by the local ethical committee, functioning according to the 3rd edition of the Guidelines on the Practice of Ethical Committees in Medical Research issued by the Royal College of Physicians of London and in accordance with the Declaration of Helsinki (1964).Determination of ER, progesterone receptors (PR), HER2, and Ki67 in breast tumor tissues was performed by immunohistochemical staining. The anti-human antibodies used were ER , PR , HER2 , and KI67 . Their detection was made feasible using Bond Refine Polymer Detection kit and Bond Epitope Retrieval Reagents on a Bond-maX fully automated IHC system (Leica Biosystems GmbH company).For this study, 86 frozen breast tumor tissues were obtained during surgery with informed patient consent. Usage of these tissues complied with the regulations set by our Institutional Review Board (IRB) for research purposes. All the tumors were diagnosed as invasive carcinoma during the diagnostic workup by certified pathologists.μIU/mL with normal range between 0.270 and 4.2 μIU/mL. FT4 is expressed in ng/mL with normal range between 0.8 and 2 ng/mL and FT3 is expressed in pg/mL with normal range between 1.8 and 4.6 pg/mL.Serum TSH, free L-thyroxine (FT4), and free 3,5,3′ tri-iodothyronine (FT3) quantitative measurements were performed using electrochemiluminescence immunoassay (ECLIA) method in COBAS 6000 automated analyzer . TSH is expressed in μg/mL leupeptin. 200 μL of 10% IGEPAL was added and samples were left in ice for 30 min. Homogenization was repeated and the homogenate was centrifuged at 1000 g for 5 min, at 4°C; the pellet was discarded, while the supernatant containing the cytosolic-membrane fraction was stored at −80°C. Protein concentrations were determined by the BCA method.Tumor samples were homogenized in ice-cold buffer (A) containing 10 mM HEPES (pH: 7.8), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, 1 mM DTT, and 10 μg (cytosolic fraction) of total protein was loaded onto 7% or 10% (w/v) acrylamide gels and subjected to SDS-PAGE in a Bio-Rad Mini Protean gel apparatus. For Western blotting, following SDS-PAGE, proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond ECL) at 100 V and 4°C, for 1.5 h using Towbin buffer. After Western blotting, filters were probed with antibodies against total and phosphorylated ERK (p-ERK)  and total and phosphorylated Akt (p-Akt)  overnight at 4°C. Filters were incubated with appropriate anti-rabbit  HRP secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence using Lumiglo reagents (New England Biolabs). Chemiluminescence was detected by the digital image analysis system FluorChem HD2  equipped with a CCD camera and analysis software. Five samples from HER2(+) and ten samples from HER2(−) group were loaded on the same gel. Results were expressed as the ratio of phosphorylated kinase levels to total kinase levels.Samples were prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by boiling for 5 min in Laemmli sample buffer containing 5% 2-mercaptoethanol. 30 χ2 test using Pearson equation. Analysis of quantitative measurements between groups was performed using one-way ANOVA. One-way analysis of variance with Bonferroni or Dunnett correction was used for multiple comparisons. Potential correlations between continuous variables were evaluated by the Pearson product-moment (Pearson r) or by Spearman's rank correlation coefficient (Spearman r), as appropriate. Continuous variables are expressed as mean (SEM), whereas categorical variables are reported as percentages. Significance was set at 0.05. Statistical analysis was performed using the statistical software package SPSS 17.0.Analysis of qualitative measurements between groups was performed with Patient age ranged from 27 to 89 years with a mean age of 60.3 (1.6) years. A percentage of 70.9% of patients (61 out of 86) were menopausal (amenorrhea for at least 1 year). A small percentage of 8.1% (7 out of 86) were found to have distant organ metastases at diagnosis. Concerning the tumor histological grade, 9.3% were identified as grade 1, 77.9% as grade 2, and 12.8% as grade 3.22 patients out of 86 studied were positive for HER2, while 64 were negative for HER2. No significant difference was found in age and clinical stage of the disease between HER2(+) and HER2(−) breast cancer patients . FurtherPhospho-p44/total p44 ERK levels were found to be increased by 2-fold in HER2(+) tumors in comparison to HER2(−) tumors. No difference was detected in phospho-p42/total p42 ERK levels between the two groups. Furthermore, a 3.0-fold reduction in phospho-Akt/total Akt was found in HER2(+) in comparison to HER2(−) tumors .α positive breast cancer patients. No difference in FT3, FT4, and TSH levels was found between premenopausal and postmenopausal patients and between different clinical stages of the disease and histopathological grades , FT4 , or TSH  when all patients enrolled were included in analysis. Furthermore, subgroup analysis showed that, in HER2(+) breast cancer patients, there was no correlation between tumor size and FT3 (r = −0.15 and P = 0.54), FT4 (r = 0.01 and P = 0.99), or TSH (r = −0.21 and P = 0.37). In HER2(−) breast cancer patients, a weak, borderline correlation was found between tumor size and TSH , but not between tumor size and FT3  or FT4 .No correlation was found between tumor size and FT3 , FT4 , or TSH  when all patients enrolled were included in analysis (r = −0.60 and P = 0.004) but not between Ki67 and FT4 (r = 0.04 and P = 0.85) or TSH (r = −0.23 and P = 0.30) , FT4 , or TSH .No correlation was seen between ki67 and FT3  . In HER2This study has provided evidence that changes in thyroid hormone levels within normal range may be associated with proliferative activity of breast tumors in euthyroid patients with breast cancer.A series of 86 consecutive patients who first presented with ER (estrogen receptor) positive breast cancer and without history of thyroid disorder or TH treatment were included in this study. A high percentage of those women were menopausal. Assessment of TH profile was performed by measuring FT3, FT4, and TSH levels in serum. TH levels in serum were within normal range in all patients with newly diagnosed breast cancer. Furthermore, no differences in TH profile were observed across patients with different clinical stage, histopathological grades, menopause status, and presence of HER2 staining.α1, implicating TH signaling in growth development [α1, results in suppressed cell proliferation [In this group of patients, we sought to determine potential correlations of FT3, FT4, and TSH levels with proliferation index Ki67) and tumor size. FT3, FT4, and TSH were not found to be correlated to either proliferation index or tumor size. However, subgroup analysis revealed a negative correlation between proliferation index (Ki67) and FT3 levels only in HER2 positive patients, while no correlation of FT4 or TSH to proliferation index was observed. Furthermore, tumor size was not correlated to FT3, FT4, and TSH levels in serum. HER2 positive breast cancer patients had comparable characteristics  to HER2 negative counterparts but greater tumor size and cell tumor proliferation index. These data clearly show that changes of FT3 even within normal range may be of physiological relevance in HER2 positive breast cancer patients. This differential association of HER2 positive and HER2 negative tumors to thyroid hormone is not fully understood. However, overexpression of HER2 receptor can lead to the activation of growth kinase signaling such as ERK –14. Cons7 and tumferation .In conclusion, the TH profile is not altered in patients with newly diagnosed breast cancer without concomitant thyroid disease. However, FT3 levels even within normal range are negatively correlated with cell proliferation in HER2 positive breast cancer tumors. This may be of important physiological relevance.


Dysfunction of renal tubule epithelial cells is associated with renal tubulointerstitial fibrosis. Exploration of the proteomic profiles of senesced tubule epithelial cells is essential to elucidate the mechanism of tubulointerstitium development.β-galactosidase staining were used to detect cell senescence. p53, p21, and p16 were detected by Western blot analysis. Liquid chromatography mass spectrometry (LC-MS) was used to examine differentially expressed proteins (DEPs) between P6 and P3 cells. The expression of DEPs was examined by Western blot analysis. Bioinformatics analysis was performed by protein-protein interaction and gene ontology analyses.Primary human proximal tubule epithelial cells from passage 3 (P3) and passage 6 (P6) were selected for evaluation. EdU and SA-β-galactosidase, whereas passage 3 (P3) cells were negative. Senescence biomarkers, including p53, p21, and p16, were upregulated in P6 cells relative to P3 cells. EdU staining results showed a lower rate of EdU positive cells in P6 cells than in P3 cells. LC-MS was used to examine DEPs between P6 and P3 cells. These DEPs are involved in glycolysis, response to stress, cytoskeleton regulation, oxidative reduction, ATP binding, and oxidative stress. Using Western blot analysis, we validated the down-regulation of AKR1B1, EEF2, EEF1A1, and HSP90 and the up-regulation of VIM in P6 cells seen in the LC-MS data. More importantly, we built the molecular network based on biological functions and protein-protein interactions and found that the DEPs are involved in translation elongation, stress, and glycolysis, and that they are all associated with cytoskeleton regulation, which regulates senescent cell activities such as apoptosis and EMT in tubule epithelial cells.The majority of tubule cells from passage 6 (P6) stained positive for SA-We explored proteomic profile changes in cell culture-induced senescent cells and built senescence-associated molecular networks, which will help to elucidate the mechanisms of senescence in human proximal tubule epithelial cells. Aging and aging-related diseases are associated with various health problems worldwide. Renal aging begins around approximately 40 years old and is accompanied by decreased renal blood flow (~10 % per year). The decline in renal function and susceptibility to age-related renal insufficiency may contribute to chronic progressive kidney failure. Senescence of tubule epithelial cells involves multiple complex activities including apoptosis, cell shape enlargement, decreased motility, aberrant energy metabolism, and epithelial-to-mesenchymal transition (EMT), and it is the major cause of renal tubulointerstitial fibrosis [The known senescence-associated cell activities are mainly based on changes in protein components. It has been reported that the upregulation of nuclear factor kappa B (NF-κB), tumor growth factor beta (TGF-β) and hypoxia-inducible factor (HIF) affect tubule epithelial cell proliferation, cellular apoptosis and EMT, which is associated with senescence in tubule epithelial cells , 3. HoweTM Invitrogen, Barcelona, Spain) and Ham’s F-12 media  supplemented with 10 % heat-inactivated fetal bovine serum (FBS) , 10 ng/ml EGF , 5 mg/ml human transferrin, 5 mg/ml bovine insulin , 100 U/ml penicillin, and 100 μg/ml streptomycin . Passage 3 was defined as the young control, and passage 6 was defined as cellular senescence.Segments of macroscopically and histologically normal renal cortex were obtained under aseptic conditions from patients undergoing nephrectomy for small (<6 cm) tumors in the Department of Urology, Chinese PLA General Hospital. Patients were accepted into the study if they had no history of renal or systemic disease associated with tubulointerstitial pathology. Tubular fragments were derived from the segments of renal cortex by collagenase digestion and were isolated by centrifugation in 45 % Percoll . The PTECs were re-suspended in a 1:1 (vol/vol) mixture of Dulbecco’s modified Eagle’s  and DAPI  for nuclear staining. Cells were examined using a Nikon fluorescence microscope (Japan).2) overnight at 37 °C without CO2. The cells were then examined under a microscope.Cells were fixed with 2 % formaldehyde and 0.2 % glutaraldehyde for 15 min and stained with freshly prepared senescence-associated β-galactosidase  , 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgClProliferative activity was detected using an EdU labeling kit  following the manufacturer’s recommendations. Fluorescent images were obtained by florescence microscopy.Antibodies against p53 , p53, AKR1B1, EEF1A1, EEF2, HSP90 (Proteintech Group Inc.), p21, and p16  were used for Western blot analysis. β-Actin  served as a control. Approximately 30 μg of protein were subjected to 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After incubation in primary and secondary antibodies, images were acquired using an Opti-Chemi 600 .Protein 50 μg) was separated by 12 % SDS-PAGE. Gels were stained with R250 Coomassie Brilliant Blue. Each lane of the gel was cut into four fragments, and each fragment was trypsin-digested as described previously . Peptide0 μg was http://bioinfo.capitalbio.com/mas3/), BiNGO and 2.44 STRING 9.0 (http://string.embl-heidelberg.de) software. The ClueGo and BiNGO 2.44 software and plug-ins for Cytoscape 2.7 were used to analyze the biological functions. String and the MAS 3.0 system were used for protein-protein interaction (PPI) analysis (score >0.6) and PPI network building. Cytoscape 2.7 was used to modify the network.Data analysis was performed using MAS 3.0 . Immunofluorescence of CK18 in the cytoplasm confirmed the purity of tubule cells Fig. . Next, SLC-MS was used to examine the DEPs between P3 and P6 renal tubule epithelial cells. Thirty-four proteins were downregulated and 36 proteins were upregulated in P6 cells compared to P3 cells Tables  and 2. WTo better explore the mechanisms involved in PTEC senescence, we built molecular networks based on PPI Fig. . In the In this study, the specific protein changes involved in human PTEC senescence were explored. The altered proteins were found to be involved in regulating senescence-associated biological functions including cytoskeleton regulation, glycolysis, stress and metabolism. More importantly, these biological functions can affect each other via PPI, which provide new insights on the mechanism of senescence in PTECs.Aberrant energy metabolism, such as glucose hysteresis, is an important cause of aging. In this study, we determined that key enzymes such as PKLR, ALDOA, GAPDH, and LDHA were disrupted and contributed to a disturbance in glycolysis. ALDOA is a key enzyme that catalyzes the reversible conversion of fructose-1,6-bisphosphate to glyceraldehydes-3-phosphate (GAPDH) and dihydroxyacetone phosphate in glycolysis . LDHA caStress plays a crucial role in senescence. In our study, we revealed that most stress-associated proteins including HYOU1, HSP90, NQO1, and XRCC were downregulated in aging renal tubule epithelial cells. Specifically, HYOU1 is associated with endoplasmic reticulum stress , HSP90 iEukaryotic translation elongation factors are also closely related to senescence. It was reported that EEF1A1 and EEF1B2 could serve as senescence-associated biomarkers, which are downregulated during cellular senescence . In thisMore importantly, we explored molecular networks to define the role of biological functions in PTECs. In the network, translation elongation, stress, and glycolysis were associated with cytoskeleton regulation by PPI. We found that DEPs mediating cytoskeleton regulation were closely associated with regulating cell activities such as EMT and apoptosis in aging PTECs. For example, upregulation of DEPs VIM, IQGAP1, and moesin is closely related to EMT and renal fibrosis, , 16 and We identified specific proteomic profiles involved in cell culture-induced senescence of renal tubule epithelial cells and built a senescence-associated biological function network involved in regulation of PTEC senescence activities. These results will aid in understanding the mechanisms involved in renal tubule epithelial cell senescence.


Saccharum officinarum L.) is an important sugar crop which belongs to the grass family and can be used for fuel ethanol production. The growing demands for sugar and biofuel is asking for breeding a sugarcane variety that can shed their leaves during the maturity time due to the increasing cost on sugarcane harvest.Sugarcane  and leaf packaging sugarcane varieties (Q2 and B). Initially, 275,018 transcripts were assembled with N50 of 1,177 bp. Next, the transcriptome was annotated by mapping them to NR, UniProtKB/Swiss-Prot, Gene Ontology and KEGG pathway databases. Further, we used TransDecoder and Trinotate to obtain the likely proteins and annotate them in terms of known proteins, protein domains, signal peptides, transmembrane regions and rRNA transcripts. Different expression analysis showed 1,202 transcripts were up regulated in leaf abscission sugarcane varieties, relatively to the leaf packaging sugarcane varieties. Functional analysis told us 62, 38 and 10 upregulated transcripts were involved in plant-pathogen interaction, response to stress and abscisic acid associated pathways, respectively. The upregulation of transcripts encoding 4 disease resistance proteins , 6 ABC transporter G family members and 16 transcription factors including WRK33 and heat stress transcription factors indicate they may be used as candidate genes for sugarcane breeding. The expression levels of transcripts were validated by qRT-PCR. In addition, we characterized 3,722 SNPs between leaf abscission and leaf packaging sugarcane plants.Our results showed leaf abscission associated genes in sugarcane during the maturity period. The output of this study provides a valuable resource for future genetic and genomic studies in sugarcane.The online version of this article (doi:10.1186/s12864-016-2552-2) contains supplementary material, which is available to authorized users. Saccharum officinarum L.) is an important sugar crop, which is widely grown in the tropical and subtropical areas [Sugarcane  gene SEEDSTICK (STK) and the bHLH transcription factor gene HECATE3 (HEC3) [Arabidopsis floral organ AZs is regulated by BLADE-ON-PETIOLE1 (BOP1) and BOP2 which encode BTB/POZ domain and Ankyrin repeat containing NPR1-like proteins [qSH1  [SH4  [HATTERING ABORTION1  [SHATTERING1 homologue  [CTD phosphatase-like protein1 (OsCPL1) [Although some genes have been found to regulate the development of AZs tissues (step 1), they are different across species and organs. In 3 (HEC3) , 14, butproteins . In tomaproteins , 15. In obox TF) , SH4 (a protein) , HATTERImily TF) , the ricmily TF) , and CTD(OsCPL1) . OverallRNA-Seq, a next generation sequencing technology, is used for profiling gene expression and plant breeding programs in many plants including rice , maize  and millS. officinarum accessions ranged from 7.50 to 8.55 Gb with an average size of 7.88 Gb [In order to understand the mechanism of leaf abscission in sugarcane, six transcriptome libraries for two parent sugarcane varieties Q1 and Q2), two F1 generation sugarcane varieties (T1 and T2), which can shed their leaves during the maturity time, and another two F1 generation sugarcanes (B1 and B2), which are packed by the leaves during the maturity, were constructed and sequenced. As shown in Table  and Q2,  7.88 Gb , our dat 7.88 Gb , resulti−5. As expected, in the NR mapping results we found 64,902 (58.98 %), 25,410 (23.09 %), 9,433 (8.57 %) and 2,606 (2.37 %) assembled transcripts were aligned to Sorghum bicolor, Zea mays, Oryza sativa Japonica Group and Oryza sativa Indica Group, respectively , UniProtKB/Swiss-Prot, GO and KEGG pathway. The numbers of transcripts aligned to each database can be seen in Fig. notation , 32. GO notation , 35.Fig.Then, we extracted likely coding sequences from the assembled transcripts using TransDecoder. In total, 100,813 likely protein sequences, 44,041 5’-prime-UTRs and 57,521 3’-prime-UTRs Fig.  were obtWe next aligned the clean reads of all six samples to the assembled transcripts using Bowtie2  and estip-values (<0.05). By using this critical, differentially expressed transcripts were shown in red in the volcano plots of Fig. We next used edgeR  to identp-value: 2.20E-16), “limonene and pinene degradation” , “plant hormone signal transduction”  and “phenylpropanoid biosynthesis” . However, transcripts upregulated in T (compared to B) were enriched in the pathways of “plant-pathogen interaction” , “one carbon pool by folate”  and “homologous recombination” . As we know, internal and environmental signals can influence and proceed the leaf senescence and death, including abiotic factors like drought, nutrient limitation, extreme temperature, and oxidative stress by UV-B (ultraviolet B) irradiation and ozone [We were surprised that Q1-up-regulated transcripts and T-up-regulated transcripts were enriched in different KEGG pathways , transcripts, 7 RPP13 (recognition of Peronospora parasitica 13) transcripts and 6 RPP13-like transcripts. Both RPM1 and RPP13 can trigger the plant defense process [Arabidopsis, RPM1 acts through its interaction with RIN4 (RPM1-interacting protein 4), an essential regulator of plant defense, and triggers plant disease resistance when RIN4 is phosphorylated by AvrRpm1 [Arabidopsis, WRK33 is involved in regulation of the antagonistic relationship between defense pathways mediating responses to the bacterial pathogen P. syringae and the necrotrophic pathogen B. cinerea promoters [First, we analyzed the transcripts of “plant-pathogen interaction” because it was the most significant KEGG pathway of commonly upregulated transcripts. Unlike in animals, pathogen interactions often trigger programmed cell death in plants –52. Amon process , 54. In  AvrRpm1 , 56. Aft AvrRpm1 . In addi AvrRpm1  and func AvrRpm1 . In Arabromoters . The up Several stresses can lead cell death and leaf abscission in plants, like drought , 62 and Arabidopsis, ABA transport can be mediated by PDR-type ABC transporter, ABG25 and AB40G [In the abscission process, ABA plays a critical role and involves in different pathways . In citrnd AB40G , 69. ZEPnd AB40G .Further, 2,031 of the assembled transcripts were annotated to encode TFs in this study. There were 603 and 58 transcripts were upregulated in Q1 and T relatively to Q2 and B, respectively. Of the upregulated transcripts encoding TFs, 16 were in common, including two transcripts encoding MADS-box transcription factors, six transcripts encoding heat stress transcription factors, two transcripts encoding transcription factor DIVARICATA, one WRKY33 transcript, two transcripts encoding NAC transcription factor 29, and three transcripts encoding ethylene-responsive transcription factor. Although the function of these TFs in sugarcane is not clear, they have been reported to regulate the leaf senescence and abscission in Arabidopsis –73.Overall, pathways of plant-pathogen interaction, response to stresses and ABA signaling are important pathways in plants and involve in several bio-activities including cell development and death , 74. Alt2 First Strand Kit (QIAGEN) and PCR mix (QIAGEN) to perform the cDNA synthesis and real-time PCR experiment. Forward and reverse primers for these 20 candidate transcripts were designed by Primer Express Software  and actin gene was used as the reference gene. The information of primers used in this study can be found in Additional file Differentially expressed transcripts between LASP and LPSP identified by RNA-Seq were confirmed by quantitative real-time PCR (qRT-PCR) experiment. A total of 20 transcripts were randomly selected as candidates. We used RTEucalyptus grandis [Brassica napus [Medicago sativa [Specificity and high abundance of genes support we can call single nucleotide polymorphisms (SNPs) using RNA-Seq . RNA-Seq grandis , Brassicca napus , and Medo sativa . In thiso sativa  and bcftIn current study we employed next generation sequencing method RNA-Seq to analyze the transcriptome of sugarcane leaves and characterize candidate genes related to the leaf abscission in sugarcane during the maturity time. A total of 215,019,578 transcripts were initially assembled with N50 of 1,177 bp in length. We annotated them by mapping them to several known databases such as NR, UniProtKB/Swiss-Prot, GO and KEGG pathway databases. Further annotations including signal protein, rRNAs, protein domains and membrane proteins were performed by Transdecoder and Trinotate pipelines. Based on the assembled transcriptome, we identified several transcripts differentially expressed between LASP and LPSP, which were annotated to involve in the plant-pathogen interaction, stress response and ABA-associated pathways. qRT-PCR was used to validate the expression levels of 20 randomly selected transcripts. The results showed high consistency between qRT-PCR and RNA-Seq methods. Furthermore, a total of 1,544,787 SNPs were identified successfully, of them 1,134 were nonsynonymous SNPs and 2,588 were synonymous SNPs. The transcriptome produced by this study will provide a valuable resource of molecular information for future investigations and process understanding the roles of genes in the leaf abscission of sugarcane. Our study may also help develop sugarcane varieties which may shed their leaves during the maturity time and benefit the sugarcane farmers.th December of 2014 we performed the sugar content test for all six sugarcane plants and found the sugar content of each plant reached (or was close to) the peak. New born leaf tissues approximately 5 cm above the growing point were taken on 5th December of 2014. The leaf tissues were stored in the liquid nitrogen before RNA extraction. The F1 generation sugarcane varieties were proved to have stable agronomic characteristic on leaf shedding by 5 years of filed observation.The sugarcane plants were grown and maintained in the experimental filed of Sugarcane Research Institute in Nanning, Guangxi Province of China. ROC-26 (from Taiwan) is a precocious and productive sugarcane variety, but the sugar cane is wrapped very tight at the physiological maturity . GT96-16Total RNA was isolated from sugarcane leaves by using TRIzol® reagent (Invitrogen) according to the manufacture’s protocol. Briefly, 1 ml of TRIzol® reagent was added into 100 mg of leaf sample, the sample was homogenized using power homogenizer and centrifuged at 12,000 × g for 10 min at 4 °C. After the fatty layer was removed and discarded, the cleared supernatant was transferred into a new tube and mixed with 0.2 ml of chloroform. The tube was shaken vigorously for 15 s, followed by an incubation for 3 min at room temperature. Next, the sample was centrifuged at 12,000 × g for 15 min at 4 °C and the aqueous phase was moved into a new tube for RNA precipitation. For precipitating RNA from each sample, 10 μg of RNase-free glycogen was added into the aqueous phase as a carrier, followed by 0.5 ml of 100 % isopropanol, then, samples were incubated at room temperature for 10 min and centrifuged at 12,000 × g for 10 min at 4 °C. To wash the RNA pellet, we added 1 ml of 75 % ethanol into the tube, vortexed the tube gently, centrifuged the tube 7,500 × g for 5 min at 4 °C and discarded the wash. The RNA pellet was air-dried, suspended in RNase-free water, water bathed at 60 °C for 10 min, quality controlled by Agilent 2100 Bioanalyzer and used for cDNA library construction and deep sequencing.http://trace.ncbi.nlm.nih.gov/Traces/sra/) under the accession number of SRA291189.Equal amount of total RNA (20 μg) was used for cDNA library construction using TruSeq™ RNA Sample Preparation Kit v2 (Illumina) and for transcriptome sequencing on the Illumina HiSeq 2000 platform, according to protocols. Briefly, total RNA was used to isolate poly(A) mRNAs using Dynal Oligo(dT) beads (Invitrogen). Then, mRNAs were chemically fragmented to ~200 nt fragments by using divalent cations  under elevated temperature. Cleaved RNA fragments were then copied into first strand cDNA by using reverse transcriptase and random primers, followed by the second strand cDNA synthesis using DNA Polymerase I (Invitrogen) and RNase H (Invitrogen) treatment. The cDNA fragments were next end repaired by using End Repair Mix (Illumina) reagent, purified and enriched with PCR to create the final cDNA libraries. Six cDNA libraries were sequenced by using pair-end (2 × 90 bp) sequencing technology with an Illumina HiSeq™ 2000 sequencer. The sequencing raw data (FASTQ formatted files) for six samples can be accessed in the NCBI Sequence Read Archive (SRA) database (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) before assembly. De novo assembly of the transcriptome was performed by Trinity software (release 2014-07-17) [Raw reads of six libraries were cleaned by removing adapter sequences and low-quality sequences (reads with ambiguous bases ‘N’ and reads with more than 10 % Q < 20 bases). The resulting high quality reads of each library were quality controlled by using the program FastQC (4-07-17) , accordi4-07-17) . InitialWe used TransDecoder, which is included in the Trinity software distribution, to extract potential coding sequences from assembled transcripts. Briefly, the longest open reading frame (ORF) was first identified within the assembled transcripts. Using a Markov model based on hexamers, a subset corresponding to the very longest transcripts were identified from all the longest ORFs. Then, all the longest ORFs identified were scored according to the Markov Model  in each of the six possible reading frames. For a particular transcript, the ORF was reported when its proper coding frame score calculated by GeneID software  was posihttp://trinotate.github.io/). Briefly, deduced proteins were used to search against UniProtKB/Swiss-Prot database (ftp://ftp.broadinstitute.org/pub/Trinity/Trinotate_v2.0_RESOURCES/uniprot_sprot.trinotate_v2.0.pep.gz) to identify known protein sequences. Functional domains were identified by mapping them to the PFAM domain database (ftp://ftp.broadinstitute.org/pub/Trinity/Trinotate_v2.0_RESOURCES/Pfam-A.hmm.gz) [http://eggnogdb.embl.de) [After the ORFs were extracted from the assembled transcripts, the deduced proteins were annotated using Trinotate   using HM.hmm.gz) . Potenti.hmm.gz) , TMHMM S.hmm.gz)  and RNAM.hmm.gz) , respectembl.de)  which en−5). The resulting transcripts were then processed to retrieve associated Gene Ontology (GO) items describing biological processes (BPs), cellular components (CCs) and molecular functions (MFs) by BLAST2GO software [To identify the assembled transcripts related with Gene Ontology (GO) and biology pathways, they were annotated by comparing to previously annotated genes in three public databases, NCBI non-redundant (NR) database, Swiss-Prot database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We used BLAST software  to map asoftware . By usinp-value (<0.05).The abundance of each transcript was evaluated by Bowtie2 and RSEM (RNA-Seq by Expectation-Maximization) tools in every sample. First, high quality reads of each library were mapped to Trinity assembled transcripts by Bowtie2 . Then, ahttps://github.com/samtools/samtools/releases/download/1.2/samtools-1.2.tar.bz2) [https://github.com/samtools/bcftools/releases/download/1.2/bcftools-1.2.tar.bz2) to find possible single nucleic polymorphisms (SNPs). In brief, clean reads of each sample were aligned against the assembled transcriptome reference by Bowtie2, generating BAM formatted files. The BAM files were then indexed and processed by the mplieup function of samtools to produce a BCF file that contains all the locations in the genome. The BCF file was then used to call genotypes and reduce the list of sites to those found to be variant by passing this file into bcftools call. Finally, after filtering low quality SNPs, reliable SNPs were left and stated by a self- developed Perl script.As the phenotypes of sugarcanes used in this study were different, we used samtools   and bcftp-value (Fisher’s exact test) and q- value [p-value < 0.05 and q-value < 0.9. Detected pathways only related to animal or human GO items and KEGG pathways were filtered.Functional analysis was performed using the Gene Ontology and KEGG pathway annotations for the transcripts. To find enriched GO items and KEGG pathways, we used q- value  to show 2 First Strand Kit (QIAGEN) and qRT-PCR was performed by using PCR mix (QIAGEN), according to the manufactures’ protocols. Briefly, genomic DNA elimination mix (10 μl) was first made by using total RNA (4 μg), Buffer GE and RNase-free water. After incubated at 42 °C for 5 min and on ice for 5 min, the genomic DNA elimination mix was mixed with 5x Buffer BC3 (4 μl), control P2 (1 μl), RE3 Reverse Transcriptase Mix (2 μl) and RNase-free water (3 μl). The final reverse transcription mix (20 μl) was incubated at 42 °C for exactly 15 min and at 95 °C for 5 min to finish cDNA synthesis. Primers for qRT-PCR were designed by Primer Express Software  and synthesized by BGI  numbers of each transcript in every samples were evaluated, mean CT values were calculated for subsequent analysis. Base on the mean CT value, ΔCT was used to present and normalize the expression of a candidate transcript. Relatively normalized expression  was used to show the expression change of a transcript in two samples. CT values greater than 35 were set to 35.Quantitative real-time PCR (qRT-PCR) experiment was used to validate the expression patterns of RNA transcripts in different sugarcane varieties. Total RNA (4 μg) which was used for RNA-Seq previously described was used for cDNA synthesis by using RT


Antidiabetic effects of Ber-NS relative to efficacy of bulk Ber were evaluated in streptozotocin (STZ) induced diabetic C57BL/6 mice. The particle size and zeta potential of Ber-NS were 73.1 ± 3.7 nm and 6.99 ± 0.17 mV, respectively. Ber-NS (50 mg/kg) treatment via oral gavage for 8 weeks resulted in a superior hypoglycemic and total cholesterol (TC) and body weight reduction effects compared to an equivalent dose of bulk Ber and metformin . These data indicate that a low dosage Ber-NS decreases blood glucose and improves lipid metabolism in type 2 diabetic C57BL/6 mice. These results suggest that the delivery of Ber as a nanosuspension is a promising approach for treating type 2 diabetes.Berberine (Ber), an isoquinoline derivative alkaloid and active ingredient of Coptis, has been demonstrated to possess antidiabetic activities. However its low oral bioavailability restricts its clinical application. In this report, Ber nanosuspension (Ber-NS) composed of Ber and D- Type 2 diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia resulting from relative insulin deficiency and insulin resistance in various tissues. The worldwide prevalence of diabetes is estimated to rise to more than 200 million people and may subsequently reach 300 million by 2025 –3. SeverBerberine (Ber), an isoquinoline derivative alkaloid isolated from several Chinese medicines, such as Berberidis Radix (Chinese name: Sankezhen), Phellodendri chinensis cortex (Chinese name: Huangbo), Coptidis Rhizoma (Chinese name: Huanglian), and Mahoniae Caulis (Chinese name: Gonglaomu), is commonly used as a quality control marker –9. In reCmax as high as 3.01 mg/ml and the bioavailability of 82% in beagle dogs, while the commercially available danazol suspension with the average particle size of 10 mm could only obtain the Cmax of 0.20 mg/ml and the bioavailability of 5%. It could be found that NS significantly enhanced the oral absorption of danazol, a poorly water-soluble drug. A few techniques have been used to prepare drug loaded NS, including nanoprecipitation, pearl-milling, high speed homogenization, sonication, and high-pressure homogenization (HPH) [Nanocrystal suspension, nanosuspension (NS) for short, is a carrier-free nanoparticles system containing only pure drug crystal and minimum surfactant and/or polymer for stabilization . Reduction (HPH) . Among tα-tocopheryl polyethylene glycol 1000 succinate  of 1.0% and hypromellose  of 0.5% were dissolved in distilled water. Ber  powder of 0.5% was dispersed in the aqueous surfactant solution using high speed homogenization 5000 rpm for 10 min . Then the premix was passed through a Lab HPH ; 5 cycles were performed at 500 bar and 15 cycles at 1500 bar.HPH technique was applied to prepare Ber-NS. Briefly, D-2 flow (100 ml/min).The particle size, polydispersity index, and Zeta potential measurements were performed on a Nano-ZS90  thermostated at 25°C. The sample was diluted 50 times with bidistilled water before the measurements. Each value reported is the average of three measurements. DSC was recorded on a DSC 204 F1 . 5–10 mg of samples were sealed in aluminum pans and heated at 10°C/min from 30°C to 300°C and under Nn = 10), and metformin  via oral gavage daily for 8 weeks or treated with vehicle control , respectively. Normal mice as control  were administered citrate buffer alone and treated with vehicle control for 8 weeks. Ber and Met were dissolved in saline. All the mice were allowed to continue to feed on their respective diets until the end of the study. Then, biochemical parameters were performed as described below.Female C57BL/6 mice 7-8 weeks old, body weights 15–20 g, were purchased from the medical experimental animal center of Guangdong Province, China. The National Institute of Health guidelines for the care and use of laboratory animals were followed in all animal experimental procedures. The animals, housed individually in plastic cages, were allowed free access to food and water at all time and were maintained on a 12 h light/dark cycle in a controlled temperature (20 to 25°C) and humidity (50 ± 5%) environment for 1 wk before use. The normal chow diet consisted  of 12% fat, 60% carbohydrate, and 28% protein and a high-fat diet (HFD) consisted of 41% fat, 41% carbohydrate, and 18% protein. C57BL/6 mice with normal diets and with HFD are generally used as nondiabetic controls and a diabetic model, respectively . After 4Fasting blood samples collected from the orbital cavity were added into precooled tubes containing EDTA  and centrifuged at 2,500 ×g for 20 min at 4°C. Plasma glucose, cholesterol, and triglyceride levels were measured with commercial kits .t-test was used to compare the mean differences between samples using the statistical software SPSS version 16.0 . In all cases P < 0.05 was considered statistically significant.Results were expressed as mean ± standard deviation (SD). Student's The mean particle size and polydispersity index (PDI) were measured immediately after the preparation of the NS. The mean particle size with PDI 0.302 was 72.4 nm. At eight weeks later, the particle size with a PDI of 0.462 was 294.6 nm . The PDIDSC spectrograms are given in To evaluate the effect of Ber-NS on glucose and lipid metabolism, control and diabetic mice were treated with metformin (Met), Ber, Ber-NS, and plasma glucose, total cholesterol, and triglycerides analyzed. As shown in Ber is the major active ingredient of rhizome coptidis, a popular traditional Chinese herb used for the treatment of infection and inflammation. Many animal studies and clinical trials have proved that Ber has significant hypoglycemic effect, even comparable to metformin . AlthougMore than 95% of the Ber-NS particles showed a small particle size (107.4 nm) . NS wereIn the present study, the low solubility and poor membrane permeability of Ber  were enh


Lactobacillus sakei (L. sakei) strain FBL1, isolated from mukeunji purchased at the Gwangju World Kimchi Culture Festival in 2012, South Korea. The total draft genome size was 2,032,158 bp with a G+C content of 41.2%.This report describes the 2,032,158-bp draft genome sequence of  Lactobacillus sakei (L. sakei) is a facultative heterofermentative and Gram-positive anaerobic lactic acid bacterium (LAB). This bacterium, originally described in rice alcohol or sake, is commonly found in fresh meat and fish and is used as a starter culture (L. sakei 23K (GenBank accession no. CR936503.1) was reported in 2005  16S rRNA gene. Comparative genomic analysis revealed 84% similarity with L. sakei 23K.The genome sequence of d kimchi , was anaL. sakei 23K as the reference genome. Assembly of the reads resulted in 44 contigs . The total draft genome size was 2,005,160 bp with a G+C content of 41.0%. A total of 1,972 coding sequences, 76 RNA genes, and 296 subsystems were revealed by the RAST server  and quality was evaluated by the Agilent 2100 bioanalyzer with the high-sensitivity DNA kit. Draft genome sequencing was performed using an Ion Torrent Personal Genome Machine (PGM) system  and a 318 semiconductor chip with 400-bp sequencing reads. A total of 4,048,495 reads, with an average read length of 320 bp, were generated. Reference-based assembly was carried out using SPAdes version 3.1.0, with LSFF00000000. The version described in this paper is the first version, LSFF01000000.This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number


In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme “cocktail”, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60 to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy. Trichoderma reesei , endoglucanase (EC 3.2.1.4), and β-glucosidase (EC 3.2.1.21) for cellulose hydrolysis; xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) to hydrolyze xylan; and α-L-arabinofuranosidase (EC 3.2.1.55) and α-glucuronidase (EC 3.2.1.39) to hydrolyze side chains in hemicellulose  was used to identify β-xylosidase . In total, four genes from three fosmid clones were selected for further study: Xyl522, coding for β-xylosidase; Xyn526, coding for xylanase; Bgl8520, coding for β-glucosidase; and Cel1753, coding for cellulase. Phylogenetic analysis was performed by MEGA5.1 with the best hits in BLASTP and characterized protein sequences from the same protein family, using the neighbor-joining algorithm.The genomic DNA of the thermophilic methanogenic digester community, as described previously  . Recombinant cells were cultured in Lysogeny Broth medium with 50 μg/mL kanamycin and induced by 1 mM IPTG at 37°C for 4 h. Cells were harvested and subjected to freeze-thaw lysis. Next, the cell lysate was centrifuged at 4°C, 12,000 × g for 30 min, and the supernatant was collected for protein purification though Ni-NTA resin  according to the manufacturer's instructions. The protein concentration was determined using the BCA Protein Assay Kit .The four target genes were PCR amplified from the corresponding fosmids with the primers listed in Supplementary Table pNPX  and pNPG  were mixed with 1 μg Xyl522 and 1 μg Bgl8520, respectively, each to a final volume of 40 μL (pH 5.0). After incubation at 50°C for 5 min, 50 μL 1 M Na2CO3 was added to terminate the reaction. The concentrations of p-nitrophenol product were determined by detecting the absorbance at 410 nm using spectrophotometer UV1700 . The specific activities (U) of β-xylosidase and β-glucosidase were shown in micromole p-nitrophenol produced from pNPX and pNPG per minute per milligram of protein. Similarly, beech wood xylan  and CMC  were mixed with 5 μg Xyn526 and 5 μg Cel1753, respectively, to a final volume of 200 μL (pH 5.0). After incubation at 50°C for 15 min, the reaction mixtures were added to 500 μL dinitrosalicylic acid and boiled for 5 min to terminate the reactions , Tris-HCl (pH 8.0–10.0) and Na2HPO4-NaOH (pH 10.0–12.0) were used. For determination of optimal temperatures, pH was maintained at 5.0, for consistency with conditions in the methanogenic digester community from which the enzymes originate. To test for pH stability, the four enzymes were incubated in the buffer solutions mentioned above with corresponding pH values at 4°C for 24 h, after which their residual enzyme activities were measured as described above. For example, Xyn526 was incubated at 4°C in buffer solutions ranging from pH 3.5 to 12.0 for 24 h, after which mixtures containing 5 μg xylanase protein were used for activity determination as described above. To test the thermostability of the four enzymes, Xyl522 and Bgl8520 were incubated at 50, 55, 60, and 75°C; Xyn526 incubated at 50, 65, 70, and 75°C; and Cel1753 incubated at 50, 60, 65, and 70°C for 2 h in buffer solutions of pH 4.5, 6.0, 5.0, and 5.0, respectively. The residual activities of all enzymes were tested at 50°C and pH 5.0. Briefly, mixtures containing 1 μg Xyl522 and Bgl8520 each, or 5 μg Xyn526 and Cel1753 each were sampled every 20 min until 120 min, and enzyme activity was determined for samples taken at each time point. In addition, CMC, microcrystalline cellulose (MIC), beech wood xylan, pNPG, pNPC (pNP-β-D-cellobioside), pNPX, and pNPA (pNP-α-L-arabinofuranoside) were used to investigate the substrate specificity of the enzymes. mK and maxV were calculated by Lineweaver–Burk plot based on enzyme activities at their optimal conditions with different substrate concentrations .The pH and temperature optima of the four enzymes were determined in the range of pH 3.0–12.0  and 20–90°C . To maintain suitable pH values, citric acid-Naw/v) and Xyn526 and Xyl522 cocktail against xylan  were assayed by detecting the products using thin-layer chromatography (TLC). After the mixtures were incubated at 50°C, pH 5.0 for 4 h, respectively, 5 μL of each culture was spotted on TLC Silica gel 60   as the mobile phase, and dyed with chromogenic agent containing 4 g diphenylamine, 4 mL phenylamine, 20 mL 85% phosphoric acid, and 200 mL acetone for 15 s followed by heating at 80°C for 10 min.The activities of Cel1753 and Bgl8520 cocktail against CMC  were incubated with Cel1753 , Xyn526 (200 U/g), a cocktail of Cel1753 (40 FPA/g), and Bgl8520 (50 U/g), a cocktail of Xyn526 (200 U/g) and Xyl522 (50 U/g), and a cocktail of all four enzymes at 50°C, pH 5.0 for 72 h. Enzyme dosages used above were the minimum ones to achieve the saturated hydrolysis of corncobs by itself. The concentrations of glucose, cellobiose, xylose, and xylobiose were measured using a Prominence Liquid Chromatograph-20A (Shimadzu) according to the manufacturer's instructions. Briefly, a fluorometric detector and a shim-pack ISA-07 column were used. The system was operated at 65°C and a flow rate of 0.6 mL/min using H3BO3-KOH as the mobile phase with 1% w/v L-arginine for product derivatization. Calculation of saccharification efficiency was performed as described previously , 1,4-β-xylanase from Caldicoprobacter oshimai , β-glucosidase from bacterium UASB270 , and cellulase from an uncultured bacterium . Xyl522 and Bgl8520 did not have signal peptides, while Xyn526 and Cel1753 had, suggesting that Xyl522 and Bgl8520 were intracellular, while Xyn526 and Cel1753 were functional only when secreted. It is notable that the alanine, proline, arginine, and glutamic acid residue contents in our proteins were generally around 1–4% higher than those of the mesothermal enzymes, whereas the glycine, serine, lysine and aspartic acid content was around 1–4% lower  of cellulase, xylanase and β-glucosidase were 1/8.1, 1/10.8, and 1/4.4, respectively, which were higher than those metagenomes from a 1.5 year enriched biogas digester (Yan et al., 8 TIM barrel fold structure. Xyl522, a member of the GH 3 family, also contained an immunoglobulin-like region, which has been proven to contribute to both substrate binding and dimerization in this family (Mcandrew et al., The predicted models of enzyme structure (Supplementary Figure pNPX, pNPG, and CMC (Table pNPX more effectively than pNPA or pNPG. Bgl8520 could hydrolyze pNPG 50 times more efficiently than pNPC (Table mK for pNPG indicated a higher affinity and efficiency for glycosidic bonds in cellobiose. In addition, Bgl8520 has a higher affinity toward pNPC than other β-glucosidases whose Km are normally between 4.8 and 17.6 mM (Del Pozo et al., pNPG, xylan from beech wood, pNPX, and CMC were the optimal substrates for Xyl522, Xyn526, Bgl8520, and Cel1753, respectively.Xyl522, Xyn526, Bgl8520, and Cel1753, without their signal peptides, were heterologously expressed and purified. Plate-based function screening confirmed each protein was responsible for the assumed activities, further supporting the results of sequence alignment. Xyn526 and Cel1753 showed 79.6 U/mg and 174.0 U/mg specific activities for the hydrolysis of beech wood xylan and CMC, respectively. However, Xyn526 had relatively weak hydrolyzing activity against MC Table . Xyl522 The pH value influenced hydrolysis activity and enzyme stability differently among the four enzymes. The optimal pH values were 4.5, 5.0, 6.0, and 5.0 for Xyl522, Xyn526, Bgl8520, and Cel1753, respectively Figure . Xyl522 Xyn526 had the highest optimal temperature of 75°C Figure ; the corCel1753 could hydrolyze CMC to produce mainly cello-oligosaccharides along with small amounts of glucose and cellobiose, whereas the Bgl8520 could hardly hydrolyze CMC Figure . When a Trichoderma reesei cellulase resulted in a relative increase of 20% glucose yield from corn stover at 50°C (Del Pozo et al., Synergistic action of these enzymes was also detected when steam-exploded corncob was used as substrate. Compared with Cel1753 alone, addition of Bgl8520 significantly increased the yield of glucose from steam-exploded corncob from 11.8 to 52.3%, while the yield of cellobiose in this cocktail decreased dramatically Figure . MoreoveClostridium absonum (Rani and Nand, Thermoascus aurantiacus (Hong et al., In the biofuel industry, increasing the thermostability of enzymes is important for reducing operational cost. Many attempts have been made to isolate thermostable enzymes, from organisms such as The four enzymes we isolated from a long-term enriched thermophilic microbial community had higher thermostability Figure  than thoin situ. The presence of a signal peptide in Xyn526 indicates that it is secreted out of cells to hydrolyze xylan. The resulting oligosaccharides, such as xylo-oligosaccharides and arabino-oligosaccharides, could be then transported into the cytoplasm by carbohydrate ABC transporters, which were found to be enriched in F52. Glucoside hydrolases including Xyl522 could then degrade these oligosaccharides into monosaccharides like xylose and arabinose for further metabolism (Supplementary Figure Using a cocktail composed of the four lignocellulose hydrolases, we observed enhanced hydrolysis of steam-exploded corncob after incubation at 50°C for 72 h, suggesting the compatible thermostabilities and synergistic cooperation of these enzymes. It is reasonable that Xyl522 and Bgl8520 could prevent product inhibition of Xyn526 and Cel1753, respectively. As cellulose is often embedded in hemicellulose and lignin (Jeoh et al., In summary, four novel thermostable glycoside hydrolases were isolated and identified from a metagenomic library constructed from a long-term dry thermophilic methanogenic digester community. The isolated enzymes and their mixed cocktail were highly effective in the hydrolysis of lignocellulose such as steam-exploded corncob. Since enzymes from the same metagenome may have high compatibilities and act synergistically, these experiments offer a way of screening compatible enzymes to form highly efficient enzyme cocktails.MW, YN, ZS, and XW designed and coordinated the study and write the manuscript. MW, GS, GL, and BZ carried out the experiments and analyzed the results. LL and XW contributed preparing the manuscript. All authors read and approved the final manuscript. ZS, YN, and XW contributed equally to this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The emerging 3q13.31 microdeletion syndrome appears to encompass diverse neurodevelopmental conditions. However, the 3q13.31 deletion is rare and few adult cases have yet been reported. We examined a cohort with schizophrenia (n = 459) and adult control subjects  using high-resolution microarray technology for deletions and duplications at the 3q13.31 locus.GAP43, LSAMP, DRD3, and ZBTB20. No overlapping 3q13.31 deletions or duplications were identified in control subjects.We report on the extended adult phenotype associated with a 3q13.31 microdeletion in a 41-year-old male proband with schizophrenia and a nonverbal learning disability. He was noted to have a speech impairment, delayed motor skills, and other features consistent with the 3q13.31 microdeletion syndrome. The 2.06 Mb deletion overlapped two microRNAs and seven RefSeq genes, including Later-onset conditions like schizophrenia are increasingly associated with rare copy number variations and associated genomic disorders like the 3q13.31 microdeletion syndrome. Detailed phenotype information across the lifespan facilitates genotype-phenotype correlations, accurate genetic counselling, and anticipatory care. DRD3, ZNF80, TIGIT, MIR568, and ZBTB20[DRD3 (OMIM #126451), which codes for the D3 subunit of the dopamine receptor, and ZBTB20 (OMIM #606025), a DNA transcription repressor expressed in hippocampal neurons, may contribute to the neurodevelopmental features of the 3q13.31 microdeletion syndrome [LSAMP, GAP43) was identified in a 7-year-old female with ADHD, hypotonia, and postnatal growth above the mean [LSAMP (OMIM #603241) encodes a limbic system-associated membrane protein (LAMP) and has been shown to regulate anxiety-like phenotypes in mice [GAP43 (OMIM #162060) is almost exclusively expressed in neuronal tissue and is a candidate gene for ASD [Preliminary evidence suggests that individuals with microdeletions at chromosome 3q13.31 OMIM #615433) may be predisposed to a broad spectrum of neurodevelopmental/neuropsychiatric conditions including global developmental delay/intellectual disability (DD/ID), speech delay, autism spectrum disorder (ASD), and attention deficit hyperactivity disorder (ADHD) [433 may bDRD3, ZBTB20, LSAMP, and GAP43.Later-onset conditions, including neurodevelopmental diseases like schizophrenia, may also be associated with rare copy number variations (CNVs) . To dateth-90th percentile). There was no evidence of hypotonia. The patient was noted to have delayed motor developmental milestones and “clumsiness” prior to age 3 years, when he was hospitalized for suspected viral meningoencephalitis with decreased level of consciousness and dystonic movements. A speech impairment and enuresis was noted thereafter and persisted into adulthood. At age 9 years, fine motor and perceptual motor skills were noted to be under-developed. A head CT (computed tomography) scan at age 15 years showed focal hypoplasia of the superior cerebellar vermis; a repeat CT scan at age 33 years was read as normal.The male proband Figure  was concHe was enrolled in a special education program beginning at age 7 years, with particular difficulties noted in mathematics and writing. At age 33 years, clinical neuropsychological testing revealed a full-scale IQ in the borderline range. Research-based testing at age 39 years using the WASI  demonstrated a full-scale IQ of 75, with a marked difference between verbal (90) and performance (62) scores consistent with a nonverbal learning disability.The patient developed schizophrenia and was first treated with standard antipsychotic medications at age 24 years. Overall, he has had an unremarkable course of illness (details available upon request); of note, aggressive behaviour at age 28 years prompted a change in his medication regimen. His past medical history also includes type 2 diabetes mellitus treated with insulin and hypercholesterolemia diagnosed in his 20′s, mild hypocalcemia first identified at age 31 years, bilateral L5-S1 disc protrusion requiring surgery at 34 years, and hypertension diagnosed at age 34 years. At time of last contact, he was 41 years old and living in a supported situation in the community.GAP43 and LSAMP) and Molin et al. (DRD3 and ZBTB20) [http://decipher.sanger.ac.uk) database and 10 patients were found to have deletions overlapping the one presented in this case report  ,3. The 3Including the present case, four adults ≥18 years) with 3q13.31 microdeletions have been reported in the literature to date  have been previously associated with the 3q13.31 microdeletion; however, this is the first report of schizophrenia in an individual with a 3q13.31 microdeletion. Moreover, the patient described in this report had a >25 point discrepancy between his performance and verbal IQ scores, consistent with a nonverbal learning disability . InteresGAP43, DRD3, LSAMP, and ZBTB20) that have been posited as contributing to the brain phenotype of the 3q13.31 microdeletion syndrome are all overlapped by this patient's deletion  searched for 3q13.31 deletions and duplications.Click here for file


CYP19A1 gene, daily levels of oestrogens, mammographic density phenotypes and body mass index (BMI) in premenopausal women.High mammographic density is an established breast cancer risk factor, and circulating oestrogen influences oestrogen-regulating gene expression in breast cancer development. However, less is known about the interrelationships of common variants in the CYP19A1, 17β-estradiol and mammographic density in 202 premenopausal women. DNA was genotyped using the Illumina Golden Gate platform. Daily salivary 17β-estradiol concentrations were measured throughout an entire menstrual cycle. Mammographic density phenotypes were assessed using a computer-assisted method (Madena). We determined associations using multivariable linear and logistic regression models.Based on plausible biological mechanisms related to the oestrogen pathway, we investigated the association of single nucleotide polymorphisms (SNPs) in rs749292 were positively (P = 0.026), and the minor alleles of rs7172156 were inversely (P = 0.002) associated with daily 17β-estradiol. We observed an 87% lower level of daily 17β-estradiol throughout a menstrual cycle in heavier women (BMI >23.6 kg/m2) of rs7172156 with minor genotype aa compared with major genotype AA. Furthermore, the rs749292 minor alleles were inversely associated with absolute mammographic density (P = 0.032). Lean women with rs749292 minor alleles had 70 to 80% lower risk for high absolute mammographic density (>32.4 cm2); Aa: odds ratio (OR) = 0.23 (95% CI 0.07 to 0.75). Lean women with rs7172156 minor homozygous genotype had OR 5.45 for high absolute mammographic density (aa: OR = 5.45 (95% CI 1.13 to 26.3)).The minor alleles of CYP19A1, rs749292 and rs7172156, are associated with both daily oestrogen levels and mammographic density phenotypes. BMI may modify these associations, but larger studies are needed.Our findings suggest that two SNPs in The online version of this article (doi:10.1186/s13058-014-0499-2) contains supplementary material, which is available to authorized users. CYP19A1 is a member of the cytochrome P450 family and is involved in the bioconversion of androstenedione to oestrone and of testosterone to estradiol , and, and3],  15q21.2 . In huma 15q21.2 , owing t 15q21.2 ,9]. Imp. ImpCYP1strogens .rs10995190) in the ZNF365 gene, which promotes genome stability during DNA damage, was associated with both breast cancer risk and mammographic density , an, an12],  density . However density .Mammographic density is also influenced by several well-known major breast cancer risk factors such as age, body mass index (BMI), parity, and hormone therapy . PreviouCYP19A1 have been associated with higher estradiol levels. Therefore, the main aim of the present study was to elaborate whether hypothesis-driven selected common variants in the CYP19A1 gene are associated with daily 17β-estradiol levels and mammographic density phenotypes among healthy premenopausal women and whether BMI modifies these associations.Few studies have been focused on genetic susceptibility, daily levels of oestrogen and premenopausal mammographic density, but plausible biological mechanisms may exist because functional genetic polymorphisms in the aromatase gene A total of 204 women ranging in age from 25 to 35 years participated in the Norwegian Energy Balance and Breast cancer Aspects I study (EBBA-I) from 2000 to 2002 at the Department of Clinical Research, University Hospital of North Norway, Tromsø (UNN) . Women mParticipants’ characteristics, including reproductive and lifestyle factors, were collected by one trained nurse using questionnaires and interviews at the time of recruitment. Recall and memory-probing aids, including a lifetime calendar, were used to date specific life events. Questionnaires  were used to collect information about birth weight, age at menarche, marital status, education, ethnicity, reproductive history, lifetime total physical activity, previous use of hormonal contraceptives and family history of cancer, smoking and alcohol. Dietary data were collected on 7 different days during the menstrual cycle (days 3 to 6 and 21 to 23) using a previously validated, precoded food diary . Daily aParticipants attended three study visits during one menstrual cycle: first visit, days 1 to 5 of the menstrual cycle, early follicular phase; second visit, days 7 to 12, late follicular phase; and third visit, days 21 to 25, late luteal phase. Measurements included height to the nearest 0.5 cm and weight (in light clothing) to the nearest 0.1 kg on a regularly calibrated electronic scale. BMI in kilograms per square metre was calculated for all participants. Fasting blood samples were drawn at all three scheduled visits during the menstrual cycle.Serum concentrations of 17β-estradiol were measured in fresh sera for all three collection points using a direct immunometric assay  at the Department of Clinical Chemistry, UNN . The sen125I-based radioimmunoassay kit . All cycles were aligned to the day of ovulation, based on the identification of the 17β-estradiol drop, which provides a reasonable estimate of the day of ovulation . Th. Th125I-Bilateral two-view mammograms were obtained from women during the second scheduled visit (between cycle days 7 and 12) at the Centre of Breast Imaging, UNN, using a standard protocol . The lefCYP19A1 genetic polymorphisms that encode the aromatase enzyme. Blood samples from 204 women in the EBBA-I study were frozen at −70°C. DNA was extracted from whole blood using a MagAttract DNA Blood Mini M48 kit  by the Department of Medical Genetics, UNN. DNA was genotyped on the Golden Gate Platform  at the Fred Hutchinson Cancer Research Center (Makar Lab), using the manufacturer’s protocol. In brief, 250 ng of genomic DNA was divided into aliquots in 96-well plates, processed accordingly and scanned on the Illumina iScan reader using BeadStudio software.We analysed r2 threshold of 0.8 and a minor allele frequency >5%, representing variability in the white European population. Tag SNP coverage extended 2 kilobases (kb) upstream and 1 kb downstream of the gene, and 29 SNPs were covered. We further reduced the number of SNPs using power calculations and ended up with a final selection of eight common SNPs with minor allele frequency >0.2: rs10046, rs17703883, rs2414097, rs2445761, rs4646, rs7172156, rs727479 and rs749292  was quantified by direct assay using enzymes modified by polyethylene glycol and dextran sulphate.CYP19A1 gene, hormone levels  and mammographic density phenotypes  using multivariable linear regression models. Associations were assessed for the selected SNPs, and the selected SNPs were coded as AA = 0 (major homozygous), Aa = 1 (heterozygous) and aa = 2 (minor homozygous) and were included as ordinal variables in the models. We compared the linear response between the categories of genotypes by including indicator variables for Aa and aa, using AA as the reference.On the basis of the plausible biological mechanisms related to the oestrogen pathway, we investigated the associations between eight SNPs in the rs7172156 and rs749292) and stratified the women by major, heterozygous and minor genotypes. We then compared the genotype groups using different characteristics of the study population , and we used one-way analysis of variance for continuous variables and the χ2 test for categorical variables.Age, parity and BMI are known to be associated with mammographic density phenotypes, are possibly associated with hormone levels and/or CYP19A1 variants, and were therefore considered as potential confounders and included as covariates in all models . Further2) and median percent mammographic density (28.5%) as cutoff values. Mammographic density was used as a dependent variable, and rs7172156 and rs749292 were used as independent variables, adjusted for age, parity and BMI. In addition, we analysed in detail whether BMI variations influenced our results , but only dichotomised BMI by median BMI values gave additional information and thus were included in the final analysis.The multivariable logistic regression models were run using median absolute mammographic density .Our candidate polymorphisms were based on plausible biological hypotheses, and all All participants underwent informed consent procedures and signed a consent form. The study was approved by the Norwegian Data Inspectorate and the Regional Committee for Medical Research Ethics.2 ) for age of 30.7 (3.07) years and BMI of 24.4 (3.77) kg/m2 Table . When wers749292, rs7172156) and both salivary estradiol and absolute mammographic density. Moreover, a positive association was observed between rs749292 and midmenstrual salivary 17β-estradiol (P = 0.026), and an inverse association between rs7172156 and midmenstrual salivary 17β-estradiol (P = 0.002), after adjustment for age, BMI and parity (Table rs749292 and absolute mammographic density (P = 0.032) after adjusting for age, BMI, parity, salivary midmenstrual 17β-estradiol and serum HDL-C.We observed an association between two SNPs  and salivary 17β-estradiol . For rs7172156, we observed an inverse association between the minor homozygous genotype and salivary 17β-estradiol  (Table 2). For rs7172156, the minor homozygous genotype (aa) was inversely associated with 17β-estradiol levels  in women with a high BMI (>23.6 kg/m2) . Among women with genotype AA and genotype Aa, compared with women with genotype aa, 57% and 56% higher mean 17β-estradiol levels were observed, respectively . We observed an 87% lower level of mean 17β-estradiol throughout a menstrual cycle in heavier women (BMI >23.6 kg/m2) with minor genotype aa of rs7172156 compared with those with major genotype AA  with rs749292 major genotype AA with heavier women (BMI >23.6 kg/m2) with rs749292 minor genotype aa, a 52% higher mean 17β-estradiol level was observed  of the menstrual cycle. In the mixed linear regression models, we found that women with different genotypes of rs749292, we observed an inverse association between minor alleles  and absolute mammographic density  in lean women (≤23.6 kg/m2), but not in women with a BMI >23.6 kg/m2. Among lean women (≤23.6 kg/m2) with rs7172156 genotype aa, we observed a positive association with absolute mammographic density   who had rs749292 minor alleles  had an 80% lower risk for high percent mammographic density (above median: >28.5%) ; aa: OR = 0.17 (95% CI 0.03 to 0.82)). The results were similar but attenuated for absolute mammographic density ). Similar associations were observed for rs7172156 and percent mammographic density  of eight studied in the CYP19A1 gene were related to both daily salivary 17β-estradiol and mammographic density phenotypes. The association with mammographic density was revealed when we used salivary 17β-estradiol as a covariate, and similar results were observed for absolute and percent mammographic density. Furthermore, our results suggest that body weight may modify these associations. We observed an 87% lower level of daily 17β-estradiol throughout a menstrual cycle in heavier women (BMI >23.6 kg/m2) with minor genotype aa (17β-estradiol 12.3 pmol/L) of rs7172156 compared with major genotype AA (17β-estradiol 22.4 pmol/L). Furthermore, lean women with rs7172156 minor homozygous genotype aa had a fivefold higher OR for high absolute mammographic density compared with major homozygous genotype AA. Lean women who had rs749292 minor alleles had 70% to 80% lower risk for high absolute and high percent mammographic density compared with major homozygous genotype AA.In the present study of premenopausal women, two SNPs  have recently been associated with premenopausal percent mammographic density , and, andCYP1isk loci . This doormation . Our resormation  that rs725 kg/m2 . Intereshibitors . These f density , and res density . Few kno density . Despitet cancer -49], bu, buCYP19s1008805  and, recal women .CYP17 polymorphisms were associated with 17β-estradiol levels, especially in women with unfavourable metabolic profiles . Pr. PrCYP19observed . Thus, uobserved , but theobserved . Howeverobserved . A limitCYP19A1 SNPs (rs7172156 and rs749292) and both daily 17β-estradiol throughout an entire menstrual cycle and both absolute and percent mammographic density in premenopausal women, and the results differed between lean and heavier women. This observation suggests that there may be genetic influences on these breast cancer biomarkers and also that the effect of body size may play a major role. Future research on genetic control of mammographic density phenotypes and sex hormones should include exploratory salivary hormone measurements and take body size and adiposity into account.In the present study, we found associations between two Four supplementary tables. Table S1. Allele frequencies and distributions of selected single-nucleotide polymorphisms (SNPs) in CYP19A1: The Norwegian EBBA-I study. Table S2. Population frequencies of single-nucleotide polymorphisms (SNPs) in selected single-nucleotide polymorphisms in CYP19A1.Table S3. Associations between each of eight selected single-nucleotide polymorphisms (SNPs) in the CYP19A1 region and mammographic density . Table S4. Associations between each of eight selected single-nucleotide polymorphisms (SNPs) in the CYP19A1 region and estradiol. (DOC 124 KB)Additional file 1: Authors’ original file for figure 1Below are the links to the authors’ original submitted files for images.