In viability test, MA-10 cell surviving rate significantly decreased as the dosage (10 μM to 5 mM) and duration (3–24 h) of cordycepin treatment increased (P < 0.05). Cordycepin at 100 μM and 1 mM for 24 h treatment induced significant DNA fragmentation (P < 0.05). In addition, the percentage of G1 and G2/M phase cell significantly declined by cordycepin (100 μM and 1 mM) for 24 h treatment, while the percentages of subG1 phase cell increased by 100 μM and/or 1 mM cordycepin in 6, 12 and 24 h treatments (P < 0.05), respectively, which highly suggested that cordycepin induced MA-10 cell apoptosis. In mechanism study with the treatments of caspases, c-Jun NH2 terminal kinase (JNK) or reactive oxygen species (ROS) inhibitors plus cordycepin for 24 h, only caspases inhibitor suppressed subG1 phase in MA-10 cells. Moreover, western blotting results showed that cordycepin induced caspase-9, -3 and -7 protein expressions, but not caspase-8, in time- and dose-dependent manners. In conclusion, cordycepin induced apoptosis in MA-10 mouse Leydig tumor cells through a caspase-9 and -3 and -7 dependent pathway.In the present study, the apoptotic effect of cordycepin on MA-10 cells, a mouse Leydig tumor cell line, was investigated. Results demonstrated that the number of rounding-up cell increased by cordycepin (10  Cordyceps sinensis (CS), and was considered as an active component with effects altering cytokines secretion, improving lung function, increasing energy levels and sex drive [Cordycepin (3′deoxyadenosine), the analog of adenosine, is a constituent from the mycelia of ex drive –3. It haex drive , 5. Moreex drive –11. Howe2 terminal kinase (JNK) pathway also participates in apoptosis. JNK, a stress-activated protein kinase, is a subgroup of the MAPK superfamily, which can be activated by cell stress such as ultraviolet, TNF and interleukin-1 [The activation of cystein aspartic-specific proteases (caspases) is commonly thought to be one of the earliest points in the no-return pathway of apoptosis. Caspases are broadly categorized into upstream regulatory caspases and downstream effector caspases . The upsleukin-1 , 18. Furleukin-1 , 20. Excleukin-1 –23.C. sinensis, induce MA-10 cell death. Although some reports have demonstrated that cordycepin possesses anti-tumor action, limited reports about cordycepin-mediated apoptosis in testicular tumor cells have been illustrated. In addition, it has been demonstrated that between 1973 and 1995 the incidence of testicular cancer in the United States increases 51% , and orchidectomy is the common protocol to treat testicular and Leydig cell cancers [We have previously demonstrated that CS could induce MA-10 cell apoptosis . It is p cancers , 26. Thuβ-actin and the monoclonal antibody against caspase-3, -7, and -9 were purchased from Cell Signaling . The polyclonal antibody against caspase-8 was purchased from Santa Cruz .Cordycepin, bovin serum albumin (BSA), Waymouth MB 752/1 medium, 3-ethyl-5-benzyl-2-methyl-4-phenylethylethynyl-6-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (MRS 1191), methylthiazolecterazolium (MTT), SP600125, RNase A, propidium iodide and mercaptoethanol were purchased from Sigma Chemical . Fetal bovine serum (FBS), Dulbecco's phosphate buffered saline, lyophilized trypsin-EDTA and gentamicin sulfate were purchased from Gibco . Sodium hydroxide, hydrochloric acid, sodium dodecyl sulfate (SDS), sucrose, EDTA, isopropyl alcohol, chloroform and Tween 20 were purchased from Merck . Tris base and general caspase inhibitor (zVAD-fmk) were purchased from Calbiochem . Acrylamide was purchased from JT Baker . HEPES was purchased from Mallinckrodt Baker, Inc. . Tissue culture grade sodium bicarbonate, sodium carbonate, sodium chloride, sodium dihydrogen phosphate and potassium chloride were purchased from Riedel-deHaen . Donkey anti-rabbit IgG conjugated with horseradish peroxidase were purchased from Amersham International . Antibody against 2 at 37°C.The MA-10 cell line was a gift from Dr Mario Ascoli , and was maintained with the standard technique . Cells w5) were seeded in 6-cm Petri dish  with 2 mL serum medium. After 70–80% confluence, cells were treated without or with 10 μM, 100 μM, 1 mM, 2 mM and 5 mM cordycepin for 24 h. Cell morphology was then observed and recorded under light microscopy . Apoptosis is characterized by the loss of cellular contact with the matrix and the appearance of plasma membrane blebbing [MA-10 cells (6 × 10blebbing .6) were lysed in a 0.6 mL cell lysis solution containing 20 mM Tris–HCl, 10 mM EDTA, pH 8.0 and 0.3% Triton X-100. DNA was extracted with 0.6 mL phenol/chloroform (1 : 1), and the mixture was centrifuged at 12 500 r.p.m. for 10 min. DNA in the aqueous phase was extracted with phenol/chloroform (1 : 1) again. The aqueous phase with DNA was mixed with isopropanol at −20°C overnight. After centrifugation, DNA pellets were washed with 70% ethanol and air-dried. DNA pellets were dissolved in TE buffer , and RNase A (3 mg/mL) was added to remove RNA at 37°C for 30 min. DNA electrophoresis was carried out in 2% agarose gel. The gel was stained with ethidium bromide. DNA fragments were visualized under UV light and photographed.In order to investigate if cordycepin could induce cell apoptosis, DNA fragmentation was determined first. After treatment, MA-10 cells  was added into each well to dissolve the crystals by shaking the plate weakly for 20 min in dark. The OD values in each treatment were then determined at λ = 590 nm by an ELISA reader .Methylthiazoletetrazolium test was used to determine cell viability with the treatment of cordycepin . MA-10 c5) were seeded in 6-cm dish with 2 mL serum medium. After 70–80% confluence, cells were treated with free medium containing various concentrations of cordycepin for 3, 6, 12, and 24 h, respectively. Cordycepin-treated cells were harvested with trypsin, washed with PBS, and mixed in 75% ethanol for at least 2 h at −20°C. After fixation, cells were washed with cold PBS and then collected by centrifugation, mixed with 100 μg/mL RNase, and stained with PI (propidium iodide) solution containing 40 μg/mL in PBS. The stained cells were analyzed using a fluorescence activated cell sorter  at λ = 488 nm using Cell-Quest software . The DNA content distribution of normal growing cells is characterized by two peaks G1/G0 and G2/M phase. G1/G0 phase possesses normal functioning and resting state of cell cycle with most diploid DNA content, while the DNA content in G2/M phase are more than diploid. Cells in subG1 phase have least DNA content in cell cycle distribution, called hypodiploid. The hypoploid DNA contents represent the DNA fragmentation [To further confirm whether cordycepin could induce cell apoptosis, the redistribution of cell cycle by flow cytometric analysis was used with propidium iodine stain , 30. MA-6) were seeded in 6-cm dish. After treatment, cells were rinsed with cold PBS. Cells were then harvested by 100 μL lysis buffer . Cell lysate was centrifuged at 32 000 r.p.m. for 10 min at 4°C. The pellet was collected by 10 μL lysis buffer and was centrifuged again at 12 000 g. The supernatant, which contains total protein, was collected and stored at −20°C. Protein concentration of the cell lysates were determined by the Lowry method [μg) were separated in 12% SDS–polyacrylamide gel, that performed at 100 V for 2.5 h using standard running buffer , and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane at 400 mA for 4 h in transfer buffer . The membranes were blocked with 5% nonfat milk, washed by TBST , and subsequently incubated with primary caspase-3 or -7 antibodies at 1 : 4000 dilutions and caspase-8 or -9 antibodies at 1 : 2000 dilutions overnight at 4°C. After washing, the membrane was incubated with horseradish peroxidase-conjugated sheep anti-mouse antibody or donkey anti-rabbit antibody, and then visualized by enhanced chemiluminescence (ECL) detection kit . The optical density of each protein band was quantitated by a Quantity One  computer-assisted image analysis system [β-actin in each lane was also detected as a control to correct the expression of caspases proteins.Cells  comparison procedure. Statistical significance was set at μM, 100 μM, 1 mM, 2 mM, and 5 mM) for 24 h, and morphological changes were observed under light microscopy. Cells without cordycepin treatment showed polygonal shape with healthy appearances, blurred outline and firm attachment, which is normal cell growth phenomenon  of cordycepin for different time points . MTT tests showed that cordycepin induced death effect on MA-10 cells in a time- and dose-dependent manner , respectively, in MA-10 cells. After 6 h treatment with 1, 2, and 5 mM cordycepin, cell viability reduced to 78.8 ± 2.6%, 66.6 ± 2.6%, and 53.9 ± 3.4%, respectively (P < .05)   (μM and 1 mM were chosen for the subsequent experiments.P < .05) . Treatme < 0.05) . MoreoveP < .05) . The effμM, 100 μM, 1 mM, 2 mM, or 5 mM) for 3 h . After 12 h treatment, subG1 phase significantly increased from 2.8% in control group to 23.1 and 11.4% in 100 μM and 1 mM cordycepin treatment groups, respectively (P < .05). After 24 h treatment, subG1 phase significantly increased from 4.4% in control group to 38.2 and 63.1% in 100 μM and 1 mM cordycepin treatment groups, respectively (P < .05). In G1 phase, there were no significant differences by different dosages of cordycepin after 3, 6, and 12 h treatments . 100 μM and 1 mM cordycepin treated MA-10 cells for 24 h did significantly decrease G1 phase from 56.3% in control group to 23.1 and 18.1% in 100 μM and 1 mM cordycepin treatment groups, respectively (P < .05). Moreover, only cordycepin at 1 mM treatment for 24 h could significantly decrease G2/M phase from 39.4% in control group to 18.7% in cordycepin treatment group (P < .05). Statistical analysis from three independent experiments of nt group  , 1 mM ROS inhibitor (NAC) or 100 μM general caspase inhibitor (zVAD-fmk) for 24 h. The change of subG1 ratios determined by flow cytometry was then analyzed. μM cordycepin treatment after 24 h (P < .05). JNK and ROS inhibitors could not suppress the subG1 ratio  back to control levels (P > .05). In caspase inhibitor experiment (P < .05). Interestingly, general caspase inhibitor significantly decreased subG1 ratio to 9.26% (P < .05). These results indicated that caspase activation/pathway played roles in cordycepin-induced apoptosis in MA-10 cells. To investigate possible pathway that cordycepin might activate to induce MA-10 cell apoptosis, cells were co-treated with 100 periment , the subμM and 1 mM) for 0, 1, 3, 6, 12, and 24 h, respectively. P > .05). However, 1 mM cordycepin (12 h) significantly induced caspase-9 expression (P < .05) (μM cordycepin (6 h) and 1 mM cordycepin (24 h) induced caspase-3 expression (P < .05) (μM cordycepin (12 h) significantly induced caspase-7 expression (P < .05). These results indicate that cordycepin induced caspase-9, 3-, and -7 expressions, but not caspase-8, to activate MA-10 cell apoptosis. Previous data implied that caspase protein was involved in cordycepin-activated apoptosis in MA-10 cells. We further determined whether cordycepin could induce caspase expression. P < .05) , 100 μM P < .05)  and 100 pression   significantly increased first at 12 h, and then caspase-3 (the effector) increased at 24 h, which illustrated a logical temporal phenomenon to induce MA-10 cell apoptosis. The possible mechanism of how different dosages of cordycepin regulating MA-10 cell apoptosis will be interesting to be further investigated.In the present study, 100 caspases , 41. It c, ROS and JNK pathway and/or caspase-2 to induce cell apoptosis [c, caspase-2 or other alternative pathways to induce caspase-9 expression in MA-10 cells. This indecision will be further examined.In general, the induction of caspase-9 is due to the activation of mitochondrial apoptotic pathway, which can be activated by cytochrome poptosis , 43. In It has been shown that cordycepin is an analog of adenosine. The possibility of adenosine receptor involved in apoptotic effect on some types of cancer cell is also proposed . Thus, iIn conclusion, cordycepin could induce the decrease of G1 and G2/M cell numbers and the increase of subG1 cell number, followed by significant apoptotic cell death in MA-10 mouse Leydig cell line. The expressions of caspases also demonstrate that cordycepin did induce apoptosis on MA-10 mouse Leydig tumor cell line. Many Chinese herbs and its pure compounds have been demonstrated with the anti-cancer effects , 46. ThiNational Science Council (NSC 96-2320-B-006-059-MY3 to B.-M. Huang), Taiwan, Republic of China.


Recurrences and deaths are known to occur, even if less frequently, in small, node-negative breast cancer patients, and decision on adjuvant treatments remains controversial. In the present analysis, we evaluate recurrence risk in patients with pT1 a, b, c, node-negative, breast cancer, accordingly with some prognostic biological factors.We retrospectively evaluated 900 node-negative patients  surgery treated between 2000 and 2009 in four Italian oncologic centers. We defined 3 different cohorts: ER positive (ER+); Her-2 positive (Her-2+); and triple negative (TN).p = ns). At multivariate analysis, histologic grade and Ki-67 resulted independent prognostic factors. Overall, 5-year OS was 98 %, without differences among pT1a, b, c, or among the 3 cohorts.pT1a was seen in 7.6% of patients, 37.7 % pT1b, 54.8 % pT1c. Concerning the 3 different cohorts, 58.2 % were ER+; 10.8 % were Her-2+; 8.2 % were TN. Overall, chemotherapy was given to 3.0 %, 27.2 %, 69.8 % of pT1a, b, c, respectively, and to 22.7 %, 58.8 %, 68.9 % of ER+, Her-2+, TN subgroups. At a median follow-up of 67 months, 5-year DFS was 96.3 %, 89.2 %, 89.4 % in pT1a, b, c, respectively  (p < 0.0001); pT1c, but also pT1b, in Her-2+ and TN subgroups, have a worse outcome, and effective chemotherapy treatment should be considered in these unfavorable subgroups.Overall, 5-year DFS was very favorable in this series of small, node-negative breast cancers, but Her-2+ and TN cohorts have a higher recurrence rate than ER+ cohort ( Despite the recent emergence of new prognostic factors and gene expression profiling, tumor size, together with axillary nodal status, is still considered a key factor in breast cancer biology and has important therapeutic implications. Recent knowledge suggest that breast cancer is a heterogeneous disease, with some characteristics, such as Her-2, hormonal receptors, and other variables being a fundamental and critical determinants of clinical outcome, with high prognostic value . Eligibility criteria included complete surgical resection, histologic diagnosis of invasive ductal or lobular carcinoma of 2 cm or less, and no lymph node metastasis; patients with history of previous breast cancer or other malignancies were excluded. Patients who received neoadjuvant chemotherapy or with ductal or lobular carcinoma in situ or microinvasive breast cancer were excluded from the analysis. All patients received locoregional treatment, with breast conservative surgery or mastectomy with axillary lymph node dissection or sentinel node biopsy, and postoperative radiotherapy whenever appropriate; systemic adjuvant treatment was given accordingly to stage and biological characteristics.Pathologic data were obtained by collecting and reviewing pathology reports on tumor size, histologic subtype, modified Bloom-Richardson histologic grade. Immunohistochemical data for hormonal receptors, Her-2, p53, Ki-67 were also collected. Significant expression of hormonal receptors was defined as estrogen and/or progesterone receptor immunostaining of more than 10 % of invasive cells. Her-2 receptor expression level 3+ staining by immunohistochemistry (DAKO Herceptest) was considered positive, and FISH (fluorescence in situ hybridization), or CISH (chromogenic in situ hybridization), or SISH (silver in situ hybridization) evaluation was performed in Her-2 2+ staining. The Ki-67 threshold value was 15 %. Histologic grade and biological features were evaluated on the invasive component of the tumor.Patients were divided into three groups according to their combined hormonal receptor and Her-2 status: ER and/or PgR positive, with Her-2 negative (ER+); Her-2 overexpressed or amplified (Her-2+); triple negative (TN). Survival analysis was performed for patients surgery treated until December 2008 (773 patients).Disease free survival was calculated as the time from surgery to recurrent disease ; for patients who remained alive and disease free, data were censored at the last follow-up. OS was measured from date of surgery to date of death of any cause, or latest follow-up. Contralateral breast cancer was included in recurrent disease.p = 0.10 and p = 0.15, respectively. The assessment of interaction among significant variables was taken into account when developing the multivariate model. The SPSS software  was used for all statistical evaluations.The associations between variables were tested by the Pearson’s chi-square test or Fisher’s exact test, when appropriate. Kaplan–Meier method was used to estimate survival curves and differences among them assessed by the logrank test. The hazard ratio (HR) and the 95 % confidence intervals (95 % CI) were estimated for each variable by the Cox univariate model. A multivariate Cox proportional hazard model was also developed by stepwise regression (forward selection); enter limit and remove limit were Approval for the study was obtained from all institutional review boards.From January 1999 to December 2010, we identified a total of 900 women with surgery-treated pT1N0 invasive breast cancer from 4 Italian oncologic centers. The distribution of patients in the three cohorts was as follows: 524 (58.2 %) ER+; 97 (10.8 %) Her-2+; 74 (8.2 %) TN. The remaining 205 tumors were lacking of complete data for being included in the three cohorts but have been analyzed for other parameters.p < 0.0001). Her-2+ and TN subtypes were more frequent in young (<35 years) patients .Main patient and tumor characteristics on the whole series of 900 patients are reported on Table Breast conservative surgery was performed in 736 patients (81.8 %). Postoperative radiotherapy was given to 79.4 % of the patients. The rate of breast conservative surgery and radiotherapy not significantly differed among ER+, Her-2+, TN subgroups.Adjuvant endocrine treatment (tamoxifen or aromatase inhibitors) was given to 97.1  % of the patients in the ER+ and to 73.2 % of the patients in the Her-2+ cohort.Overall, adjuvant chemotherapy has been delivered to 3.0 %, 27.2 %, 69.8 % of pT1a, b, c, patients, respectively. In ER+ cohort, chemotherapy  has been administered to 119 (22.7 %) patients . In Her-2+ subset, chemotherapy alone has been delivered to 26 (26.8 %) patients , whereas chemotherapy plus trastuzumab was given to 31 (32 %) patients . In the TN cohort, chemotherapy has been administered in 51 (68.9 %) patients  . Grade 3 was found in 52.9 % of TN, 47.7 % of Her-2+, and only in 12.6 % of ER+ tumors (p < 0.0001).Ki-67 was higher than 15 % in 62.3 % of TN and in 59.5 % of Her-2+ tumors, whereas in ER+ subtype, this issue was recorded in 26.8 % of the tumors (p 0.35). In ER+ subtype, 5-year DFS was 100 %, 93.6 %, 89.8 % in pT1a, b, c tumors (p 0.12). In Her-2+ cohort, 5-year DFS was 100 %, 78.7 %, 85.0 % in pT1a, b, c, respectively (p 0.40), whereas in TN subtype, these issues were 100 %, 76.8 %, 85.2 %, in pT1a, b, c tumors (p 0.65).On the whole cohort of patients, with a median follow-up of 67 months , the 5-year DFS was very favorable, being 96.3 %, 89.2 %, 89.4 %, in pT1a, b, c, respectively (p 0.0009), and Ki-67 .Figure p < 0.0001. The subgroups of pT1b in the Her-2+ and TN cohorts showed a slightly higher, even if not significant for the small sample size, recurrence rate than larger tumors (pT1c) ; this may be presumably related to the higher percentage of patients treated with chemotherapy in the pT1c groups. We cannot discern in the Her-2+ cohort how much of the benefit can be ascribed specifically to trastuzumab versus chemotherapy administration, giving the small sample size.Overall, patients with small, node-negative breast cancer have an excellent prognosis, but, as expected, Her-2+ and TN appear to have a higher recurrence rate than ER+ cohort, being 13.4 % and 27 % versus 9.6 %, p 0.009) and Ki-67  were confirmed to be independent prognostic factors .The 5-year OS was 98 %, without differences among tumor size . The 10-year OS was 95.5 %, being 98.2 %, 97.5 %, 93.8 % in pT1a, b, c. The 5- and 10-year OSs were not significantly different among the 3 different cohorts, being 97.3 % in ER+ group, 100 % (5-year) and 95.2 % (10-year) in the TN cohort, 96.6 % in Her-2+ subgroup . A retrospective, single institution, sequential cohort study of node-negative, small (≤2 cm), Her-2 positive patients showed that trastuzumab-treated patients derived significant benefit in RFS and OS in comparison with the no-trastuzumab-treated patients  and biological variables. These risk categories are often useful for assessing prognosis and evaluating risk in groups of patients, even if their role in individual patient is more limited, and recently, various molecular techniques, particular a gene expression profiling, have been increasingly employed to assess prognosis and response to treatment. International guidelines clearly recommend adjuvant systemic treatment for node-negative breast tumors larger than 1 cm, particularly in Her-2 positive or triple negative subgroups, but definite indication for smaller tumors are lacking NCCN 2012). Some retrospective studies of outcome for patients with T1a, b, node-negative breast cancer, treated with local therapy and commonly without adjuvant treatments, reported a very favorable 10-year RFS (>90 %), and on these basis, many clinicians might not recommend systemic treatment in view of the small additional benefit balanced against costs and toxicity (Banerjee and Smith . Some re), as a confirmation of the controversies still existent on adjuvant decision making concerning small tumors.A recent review on trials examining the prognostic value of Her-2 in T1a, b, node-negative breast cancers confirmed the worse outcome for Her-2 positive cohort in some but not all the studies, and in some instances, number needed to treat to save one patient from recurrence was higher than number needed to harm for adverse cardiac event  including tumor size and biological variables. Additional prognostic markers are needed to tailor adjuvant systemic treatment particularly for patients at “low-risk”, such as small, node-negative cancers. Recently, the metastatic potential of pT1 breast cancer was evaluated by the 70-gene MammaPrint signature in 964 early breast cancer patients, resulting the signature an independent prognostic factor (Mook et al. In conclusion, the present study confirms that the prognosis of patients with node-negative small breast cancer depends on variable features. Although the number of recurrence was low in our experience, related to the fact that we are examining a population with low intrinsic metastatic potential, the observation of a worse outcome in a subset of very small breast cancer of specific unfavorable subtypes suggested that an appropriate systemic adjuvant treatment should be considered also for pT1b, Her-2 positive or triple negative breast cancer patients, even if the incremental gain in absolute benefit cannot be actually predicted, and long-term toxicities should be carefully considered on an individual basis during the decision making process.


The structure of synthesized complexes was confirmed using elemental analysis, FTIR, UV-visible, 1H NMR, and mass spectrometry techniques. The nanocrystalline nature of complexes was confirmed by powder XRD study. The complexes were evaluated for cytotoxic potential in HeLa , C6 (glioma), and CHO (Chinese hamster ovarian) cell lines. The complex E was found to be more effective cytotoxic agent against HeLa cell line with an IC50 value of 4.06 µM. Furthermore, the effect of synthesized complexes was studied on different stages of the cell cycle in CHO cells. All complexes exhibited the dose dependent increase in cytotoxicity. The results have shown an increase in sub-G0 population with increase in concentration which is an indicative measure of apoptosis.Titanium complexes have been synthesized by the reaction between titanium tetrachloride (TiCl The discovery of cisplatin, a metal (platinum) based anticancer drug by Rosenberg et al. in 1965, has created interest in the development of metal based anticancer drugs . The eff−1on Perkin Elmer 1600. The mass spectrum was recorded by using the electron spray ionization technique on Waters Micromas Q Tof Micro. 1H NMR Spectra were recorded on Brucker Avance 400 MHz spectrometer. Crystalline nature of the complexes has been confirmed by powder XRD technique on Philips 1710 X-ray diffractometer.Ligands and titanium tetrachloride used were obtained from Sigma Aldrich. All the solvents were of AR grade (Merck) and purified by standard procedure before use and stored over 4 Å molecular sieves. Purity of ligands was checked by checking their melting points. Elemental analyses were performed by using Perkin-Elmer, Series 2400. The UV-visible spectra of the complexes were recorded on Perkin Elmer Lambda 750 in the range of 200–800 nm and FTIR Spectra were recorded from 4000–200 cm2(bpome)2] was obtained. Yield: 0.5 g (86.2%). Now, to a solution of TiCl2(bpome)2  in 25 mL of toluene, adamantylamine  in 25 mL of toluene was added dropwise with continuous stirring. The reaction mixture was stirred for 2 h and refluxed for 14 h till the evolution of HCl gas ceased. The evolution of HCl gas was confirmed by passing the gas through an ammonia solution which results in white dense fumes of ammonium chloride. The excess solvent was removed by vacuum distillation and the compound was washed with petroleum ether. The compound was dried under vacuum. A cream colored solid powder was obtained which was recrystallised in methanol. Yield: 0.45 g (64.28%). TiC44H54N6O4: elemental anal. Calcd (%): C 67.67, H 7.17, N 10.76; found (%): C 66.71, H 7.05, N 10.52.FTIR  1H NMR : adamantylamine δ, ppm = 2.1 , 1.86 , 1.66, 1.59, . 4,4′-Dimethoxy-2,2′-bipyridine δ, ppm = 8.66 , 7.96 , 7.37 , 4.09 .To a colorless solution of 4,4′-dimethoxy-2,2′-bipyridyl  in 25 mL of toluene, a pale yellow colored solution of titanium tetrachloride  in 25 mL of toluene was added dropwise with continuous stirring under ice cold conditions. The reaction mixture was stirred for 2 h followed by refluxing for 10 h till the evolution of chlorine gas ceased. The evolution of chlorine gas was checked by passing the gas through a potassium iodide solution which results in reddish brown color of potassium iodide due to liberation of iodine. After removing solvent through vacuum distillationcompound was dried under vacuum. A light yellow colored solid compound [TiCl44H54N6: elemental anal. Calcd (%): C 73.70, H 7.81, N 11.72; found (%): C 73.91, H 7.94, N 11.54. FTIR  1H NMR : adamantylamine δ, ppm = 2.12 , 1.91 , 1.69, 1.64, . 6,6′-Dimethyl-2,2′-bipyridine δ, ppm = 8.5 , 8.23 ,7.68 , 2.89 .The complex was synthesized in accordance to the procedure used for complex A. Yield: 0.4 g (84.38%). TiC32H58N6: elemental anal. Calcd (%): C 66.63, H 10.41, N 14.57; found (%): C 65.77, H 10.23, N 14.72. FTIR  1H NMR : adamantylamine δ, ppm = 2.09 , 1.7, 3J = 4 , 1.63, 1.55, . 1,2-Diaminocyclohexane δ, ppm =3.63 , 3.3, 2.01, 1.76, 1.68, 1.3 .The complex C was synthesized similarly to complex A. Yield = 0.48 g (87.2%). TiC44H46N6: elemental anal. Calcd (%): C 74.55, H 6.77, N 11.85; found (%): C 74.46, H 6.42, N 11.52. FTIR  1H NMR : adamantylamine δ, ppm = 2.04 , 1.75 , 1.61, 1.55, . 1,10-Phenanthroline δ, ppm = 8.56 , 7.7 , 7.4 , 6.8 .The procedure described above for complex A was followed for the synthesis of complex D. Yield: 0.42 g (87.5%). TiC40H48N2O2: elemental anal. Calcd (%): C 71.61, H 7.45, N 4.17; found (%): C 71.70, H 7.27, N 4.10. FTIR  1H NMR : adamantylamine δ, ppm = 2 , 1.71 , 1.58, 1.49, . Benzoylacetone δ, ppm C6H5: δ = 8.08 , 7.96 , 7.6 , 3.92 , 2.5 .The procedure for the synthesis of complex A was followed for the preparation of complex E. However, there was evolution of HCl gas in both the steps. Yield: 0.43 g (86%). TiCμg/mL) at 37°C with 90% humidity and 5% CO2. The complexes were dissolved in dimethyl sulphoxide (DMSO) to prepare the solutions of different concentrations. The selected cell lines were treated with these solutions to calculate the IC50 values by using MTT -2,5-diphenyltetrazolium bromide) assay while control cells received only DMSO.The cytotoxic studies of synthesized complexes were performed on HeLa , C6 (glioma), and CHO (Chinese hamster ovarian) cell line. The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing fetal calf serum (FCS) (10%), penicillin (100 units/mL), and streptomycin  after sterile filtering, was added to each well. Now, culture plates were stirred at 150 rpm for 5 min to thoroughly mix MTT into the media. The plates were further incubated for 4 h at 37°C to allow metabolization of MTT. MTT formazan crystals were resuspended in 100 μL of DMSO and plates were stirred for 20 min in order to dissolve formazan crystals and optical density was measured at 570 nm. The phase contrast imaging was done by using Nikon Eclipse TS100 inverted microscope.The growth inhibitory effect of newly synthesized titanium complexes on HeLa, C6, and CHO cells was determined by MTT assay . For thi6 cells/dish well was plated in 24 well plates which were allowed for adheration for 6 h and then treated with complexes at three different concentrations. After 24 h of treatment, cells were harvested from the plate. The cell suspension having 1 × 106 cells was centrifuged and the resultant cell pellet was resuspended in phosphate buffered saline (1 mL) solution. The cells were fixed in ice cold 70% ethanol and stained with propidium iodide followed by analysis on the FL-2 channel by BD Accuri C6 flow cytometer . DNA content histograms and cell cycle phase distributions were modeled from at least 15,000 single events.1 × 10Synthesis of titanium complexes was carried out in two steps. In the first step, titanium tetrachloride reacted with respective bidentate ligand, that is, 4,4′-dimethoxy-2,2′-bipyridine, 6,6′-dimethyl-2,2′-bipyridine, 1,2-diaminocyclohexane, 1,10-phenanthroline, and benzoylacetone which are coligands in 1 : 2 molar ratio under continuous stirring and refluxing by using toluene as a solvent. There was evolution of chlorine gas during the course of reaction. In the next step, respective titanium complexes reacted with main ligand (adamantylamine) in 1 : 2 molar ratio in the same solvent, which results in evolution of HCl gas  as shown−1 does not change much although the intensity of the band changes and gets weaker upon complexation with titanium metal. The absorption band due to −1 in the bidentate ligand of complexes A, B, and D gets shifted to 1629, 1644, and 1612 cm−1 (−1) having more intensity gets shifted to higher wave number in complexes (around 1000 cm−1). All these changes can be assigned to the coordinated nature of bidentate ligand through nitrogen atoms [−1 due to N–H stretching of adamantylamine ring, while the occurrence of a strong band in the region 1600–1580 cm−1 in complex E may be assigned to stretching modes of −1 assignable to −1 in complexes A, B, C, D, and E shows that ligands are coordinated to the metal atom through nitrogen [−1 due to The bands of FTIR were assigned by comparing the spectra of complexes with those of free ligands and were shown in 612 cm−1 . The shien atoms . The bannitrogen , 38 and ide ions .n → π* transition in complexes A, B, D, and E get shifted to lower wavelength after coordination. However bands due to π → π* around 240–245 nm remain almost at the same position even after coordination. Since in complex C both the ligands are of cyclic nature, so there is no possibility of these transitions.The UV-visible spectra of the complexes  and liga1H NMR spectra of the complexes are consistent with the structures proposed in the reaction scheme. We find that bidentate ligands of synthesized complexes show a considerable downfield shift of protons after complexation with titanium. This shift may be due to transfer of electron density from ligand protons to the metal atom [The tal atom , 40. Howm/z = 152 due to C10H17N fragment ion and 4,4′-dimethoxy-2,2′-bipyridine ligand in the complex showed peak at m/z = 217 with relative intensity of 25%. We find that this complex also shows a peak at m/z = 478 due to TiC24H24N4O4 fragment ion. In case of complex B, one peak at m/z = 185 due to C12H12N2 fragment ion and another peak at m/z = 152 due to C10H17N fragment ion were found with a relative intensity of 18%. In complex C, peaks were formed at m/z = 98, 115, 230 due to C6H12N, C6H14N2, and TiC10H19N2 fragment ions. The complex D shows peaks at m/z = 304 and 335 due to TiC16H22N3 and TiC16H22N3 fragment ions. In addition to these peaks, complex D shows a molecular ion peak at m/z = 708 with very low intensity. In complexes C, D, and E, formation of the base peak takes place due to C10H17N fragment ion at m/z = 152. The complex E, in addition to base peak, also shows fragment ion peaks, in which one peak is formed at m/z = 401 due to TiC22H32N2O2 fragment ion. The complexes A, B, C, and E show their molecular ion peaks at m/z = 780, 716, 577, and 670 indicating the formation of complexes. The existence of these different fragment ion peaks, base peaks, and molecular ion peaks supports the stoichiometric formulation of synthesized complexes [The structure of complexes was further confirmed by recording electron spray mass spectrum. The complex A showed base peak at λ × 0.9)/(β × Cosθ) [λ as the wavelength of incident radiation , and β as full width half maximum (FWHM), was used to calculate the crystallite size of complexes. The calculated crystallite size for complexes A, B, C, D, and E was 69, 26.5, 19.1, 115, and 76.6 nm, respectively, which falls in nanorange. Unit cell parameter of the complexes has been calculated by using Powder X software [1H NMR, and mass spectrometry, an octahedral geometry may be proposed for the synthesized titanium complexes [The powder X-ray diffraction study was performed to understand the lattice structure of the complexes. × Cosθ) , 41, witsoftware  and the omplexes .50 values were calculated by using best fit regression model and results have been tabulated in 50 values, it has been observed that complex E with benzoylacetone ligand shows (4.06 μM) better activity than other complexes against the HeLa cell line, which is even better than known anticancer drug camptothecin as seen in 50 values of main ligand, that is, adamantylamine along with its complexes, has been shown in The IC50 values which caused the decrease in the number of cells with an increase in dose due to induction of apoptosis. It has been observed that all complexes increases cells in hypo-diploid cells of cell cycle and also increased the cell death with increase in concentration. Among all the complexes, complex E having benzoylacetone ligand showed 44.3% cell death at 80 μM, which is the maximum for all the complexes. However, known anticancer drug Camptothecin showed 45.5% cell death at 6 μM. Abundant evidences suggest that mitochondria plays a key role in the initiation of apoptosis by releasing Cytochrome C [1 population) which indicates apoptosis. Previous studies showed that titanium affects polymerase proteins and transcription factors which inhibits protein synthesis and causes cytotoxicity [For cell cycle analysis, CHO cells were treated with the complexes at three concentrations almost near to their ICchrome C . It has toxicity .1H NMR, and mass spectrometry techniques. Cytotoxic studies were done on different cell lines and it has been found that complex E with benzoylacetone ligand was a more potent cytotoxic agent. The morphological analysis on CHO cells indicates characteristics features of apoptosis and cell cycle analysis indicate increase in hypo-diploid cells. The mechanism of action has been certainly established in vitro; however, the efficacy of these complexes with their action mechanisms should also be demonstrated in vivo.We have reported the synthesis of mixed ligand titanium complexes having nitrogen containing ligands. The structure of the complexes has been confirmed by elemental analysis, FTIR, UV-visible, Supplementary material: The FTIR spectra of ligands and titanium complexes are included.Click here for additional data file.


The combination of chitosan as a renewable heterogeneous catalyst and ionic liquid as a “green” solvent was employed for the Knoevenagel reaction. The chitosan catalyst was characterized by various techniques, including X-ray powder diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), and elemental analysis. Excellent conversions were achieved under mild conditions without the need for an inert atmosphere. There was no contribution from leached active species, and conversion was only being possible in the presence of the solid catalyst. The chitosan catalyst as well as the ionic liquid solvent could be recovered in essentially pure form after being used in the reaction, and each of them could be reused several times without a significant degradation in efficiency. Room temperature ionic liquids have been considered as potential green alternatives to conventional volatile organic solvents during the last decade –5. They The Knoevenagel condensation between aldehydes or ketones with activated methylene compounds is one of important carbon-carbon forming reactions in organic synthesis , 40. Conα radiation source on a D8 Advance Bruker powder diffractometer. Scanning electron microscopy studies were conducted on a JSM 740 Scanning Electron Microscope (SEM). Transmission electron microscopy studies were performed using a JEOL JEM 1400 Transmission Electron Microscope (TEM) at 100 kV. The chitosan samples were dispersed on holey carbon grids for TEM observation. Fourier transform infrared (FT-IR) spectra were obtained on a Bruker TENSOR37 instrument, with samples being dispersed on potassium bromide pallets. 1H and 13C NMR spectra were recorded using a Bruker AV 500 spectrometer. MS spectra were performed on a Thermo Finigan TSQ7000 triple quadrupole.All reagents and starting materials were obtained commercially from Sigma-Aldrich and Merck, and they were used as received without any further purification unless, otherwise, noted. Chitosan was kindly donated by MKVN Chemicals Company . Nitrogen physisorption measurements were conducted using a Quantachrome 2200e system. Samples were pretreated by heating under vacuum at 150°C for 3 h. A Netzsch Thermoanalyzer STA 409 was used for thermogravimetric analysis (TGA) with a heating rate of 10°C/min from 30 to 900°C in air. X-ray powder diffraction (XRD) patterns were recorded using a Cu Kμm). The temperature program for GC analysis heated samples from 60 to 200°C at 20°C/min and held them at 150°C for 1 min; then heated them from 150 to 160°C at 1°C/min and held them at 200°C for 2 min; then heated them from 200 to 300°C at 50°C/min and held them at 300°C for 4 min. Inlet and detector temperatures were set constant at 300°C. p-Xylene was used as an internal standard to calculate reaction conversions. GC-MS analyses were performed using a Hewlett Packard GC-MS 5972 with an RTX-5MS column . The temperature program for GC-MS analysis heated samples from 60 to 280°C at 10°C/min and held them at 280°C for 2 min. Inlet temperature was set constant at 280°C. MS spectra were compared with the spectra gathered in the NIST library.Gas chromatographic (GC) analyses were performed using a Shimadzu GC 17-A equipped with a flame ionization detector (FID) and an DB-5 column  60%  and water (50 mL) were then added dropwise to prevent the temperature from rising significantly. After stirring for 12 h at room temperature, the upper acidic aqueous layer was separated by decantation and the lower ionic liquid phase was washed with cold water (10 × 50 mL) until the washings were no longer acidic [6]) (83% yield).1-Butyl-3-methylimidazolium bromide ([BMIM][Br]) was prepared from the reaction of rocedure , 61. A p1H NMR : δ = 0.91 , 1.26 , 1.77 , 3.85 , 4.16 , 7.67 , 7.73 , 9.07 . 13C NMR : δ = 13.14 (C–CH3), 18.71 (CH2), 31.27 (CH2), 35.65 (N–CH3), 48.51 (N–CH2), 122.19 (C=C–N), 123.54 (C=C–N), 136.44 (N–C=N). MS (ESI): m/z 139 [BMIM]+, 423 [(BMIM)2PF6]+.6]), and 1-octyl-3-methylimidazolium hexafluorophosphate ([OMIM][PF6]) ionic liquids were synthesized in a yield of 83% and 85%, respectively.Using a similar procedure, 1-hexyl-3-methylimidazolium hexafluorophosphate  for [HMIM][PF6]: δ = 0.87 , 1.27 , 1.78 , 3.85 , 4.15 , 7.77 , 7.73 , 9.07 . 13C NMR : δ = 13.71 (C–CH3), 21.80 (CH2), 25.09 (CH2), 29.27 (CH2), 30.49 (CH2), 35.65 (N–CH3), 48.79 (N–CH2), 122.19 (C=C–N), 123.54 (C=C–N), 136.43 (N–C=N). MS (ESI): m/z (%) 167 [HMIM]+, 479 [(HMIM)2PF6]+.1H NMR  for [OMIM][PF6]: δ = 0.86 , 1.27 , 1.78 , 3.85 , 4.15 , 7.67 , 7.74 , 9.08 . 13C NMR : δ = 13.87 (C–CH3), 22.39 (CH2), 26.09 (CH2), 28.77 (CH2), 28.84 (CH2), 30.14 (CH2), 31.50 (CH2), 36.64 (N–CH3), 50.00 (N–CH2), 121.86 (C=C–N), 123.64 (C=C–N), 137.08 (N–C=N). MS (ESI): m/z 195 [OMIM]+, 535 [(OMIM)2PF6].p-xylene (0.2 mL) as an internal standard was placed into a 25 mL flask containing 3 mL [BMIM][PF6]. The catalyst concentration was calculated with respect to the amino/benzaldehyde molar ratio. The reaction vessel was stirred for 30 min to disperse the chitosan catalyst in the liquid phase. Malononitrile  was then added, and the resulting mixture was stirred at room temperature for 6 h. Reaction conversion was monitored by withdrawing aliquots from the reaction mixture at different time intervals, quenching with acetone, filtering through a short silica gel pad, analyzing by GC with reference to p-xylene, and further confirming product identity by GC-MS. The reaction mixture was washed with diethyl ether (5 × 15 mL). The chitosan catalyst was then separated by simple centrifugation, washed with copious amounts of anhydrous ethanol, dried at 60°C overnight, and reused if necessary. For the leaching test, a catalytic reaction was stopped after 0.5 h, analyzed by GC, and centrifuged to remove the solid catalyst. The reaction solution was then stirred for further 5.5 h at room temperature. Reaction progress, if any, was monitored by GC as previously described.The Knoevenagel reaction between benzaldehyde and malononitrile using the chitosan catalyst was carried out in a magnetically stirred round bottom flask. Unless, otherwise, stated, a mixture of chitosan , benzaldehyde , and θ = 10.5° and 2θ = 20.5° were observed on the XRD diffractogram of the catalyst (The chitosan catalyst was characterized using a variety of different techniques, including SEM, TEM, XRD, FT-IR, TGA, nitrogen physisorption measurements, and elemental analysis. The morphology of the chitosan was observed from the SEM micrograph, exhibiting an uneven surface with straps and shrinkage . As expecatalyst . These tcatalyst . The the−1 were attributed to the O–H stretching vibration of the hydroxyl groups, as well as the inter- and extramolecular hydrogen bonding of chitosan molecules  ionic liquid as solvent at room temperature. The ionic liquid solvent was synthesized and characterized according to a literature procedure , [HMIM][PF6], and [OMIM][PF6], respectively. It was found that the reaction rate decreased with the solvent order: [BMIM][PF6] > [HMIM][PF6] > [OMIM][PF6]. The reaction carried out in [HMIM][PF6] afforded more than 99% conversion after 5 h, while 99% conversion was observed for the reaction in [OMIM][PF6] (The effect of different solvents on organic transformations using heterogeneous catalysts could normally be crucial, depending on the nature of the catalyst material , 76. MacIM][PF6] .For a liquid-phase organic transformation using a solid catalyst, an important problem that should be taken into account is the possibility that some of active sites could dissolve into the solution during the course of the reaction. In fact, these leached species might contribute significantly to the catalytic reaction, and the reaction might not be truly heterogeneous with respect to the substrates and products. . It shou6] ionic liquid at room temperature. Aliquots were withdrawn from the reaction mixture at different time intervals and analyzed by GC to observe the kinetic data during the course of the reaction. After the reaction, product and unreacted starting materials were separated from the reaction mixture by extraction with diethyl ether. The mixture of chitosan catalyst and ionic liquid solvent was then washed several times with diethyl ether and reused in further reaction under identical conditions to those of the first run. Interestingly, it was found that the catalyst and solvent system could be reused without significant degradation in catalytic activity. Quantitative reaction conversion was still achieved at the fifth run using the recovered catalyst and solvent system (When using solid catalysts for organic transformations, issues that should be considered are the ease of separation as well as the deactivation and reusability of the catalyst. In fact, active lifetimes should be an important characteristic for both heterogeneous and homogeneous catalysts. Ionic liquids have been considered as green solvents not only due to their nonvolatile nature, minimizing emission of toxic organic compounds, but also because of their recyclability. The chitosan catalyst and the ionic liquid solvent were, therefore, investigated for recoverability and reusability in the Knoevenagel reaction over five successive runs. The Knoevenagel reaction was carried out using 20 mol% chitosan catalyst in the [BMIM][PFt system .As mentioned earlier, research works have been mostly focused on homogeneous catalysis in ionic liquids, and the number of reports covering the heterogeneous catalysis in ionic liquids are limited in the literature. Santamarta and coworkers previously carried out the Knoevenagel reaction using 20 mol% glycine as a homogeneous catalyst in ionic liquid . The mixIn conclusion, we have demonstrated the application of chitosan as a renewable solid catalyst for the Knoevenagel condensation between benzaldehyde with malononitrile to form benzylidene malononitrile as the principal product. The chitosan catalyst was characterized using a variety of different techniques, including FT-IR, TEM, SEM, XRD, TGA, and elemental analysis. The reaction was carried out in ionic liquid as a “green” solvent. Excellent conversions were achieved under mild conditions without the need for an inert atmosphere. The reaction could only proceed in the presence of the solid chitosan catalyst, and there was no contribution from leached active species in the reaction solution. To the best of our knowledge, the combination of chitosan as a renewable heterogeneous catalyst and ionic liquid as a “green” solvent for the Knoevenagel reaction was not previously mentioned in the literature. The chitosan catalyst as well as the ionic liquid solvent could be recovered in essentially pure form after being used in the reaction, and they could be reused several times without a significant degradation in efficiency. Current research in our laboratory has focused on the application of several heterogeneous catalysts for a wide range of organic transformations in ionic liquids.


IIa) and 4-(4-methoxyphenyl)-6-phenyl-2,3,3a,4-tetrahydro-1H-pyridocarbazole (IIb) were elucidated by single crystal X-ray diffraction. Compound (IIa), C28H25N, crystallizes in the triclinic system, space group P-1, with a = 8.936(2) Å, b = 10.490(1) Å, c = 11.801(1) Å, α = 102.69(5)°,  β = 103.27(3)°, γ = 93.80(1)°, and Z = 2. The compound (IIb), C28H25NO, crystallizes in the monoclinic system, space group P21/a, with a = 11.376(5) Å, b = 14.139(3) Å, c = 13.237(4) Å, β = 97.41(3)°, and Z = 4. In both the structures, the pyrido ring adopts a twist boat conformation and the carbazole molecule has the twisted envelope structure with C3 and C13 at the flap. No classical hydrogen bonds are observed in the crystal structures. Details of the preparation, structures, and spectroscopic properties of the new compounds are discussed.The crystal structures of 4-(4-methylphenyl)-6-phenyl-2,3,3a,4-tetrahydro-1H-pyridocarbazole ( Bacillus anthracis and Mycobacterium tuberculosis, indicating that indolo  carbazoles could serve as promising leads in the development of new drugs to combat anthrax and tuberculosis infections [C. albicans and the emerging pathogen Candida glabrata [Many natural products with carbazole moiety are found to have antitumor properties . The Schindolo 2,-a carbazfections . A serieglabrata . Althougglabrata . This paIIa), a mixture of 2-(3-oxo-1-(4-methylphenyl)-3-phenylpropyl)cyclohexanone (3 mmol) and phenylhydrazine hydrochloride (6 mmol) in ethanol (20 mL) was stirred for 2 hrs. After the completion of the reaction, the mixture was poured into excess water and the precipitate was filtered off and recrystallized from ethanol . For the preparation of compound (IIb), a mixture of 2-(1-(4-methoxyphenyl)-3-oxo-3-phenylpropyl) cyclohexanone (3 mmol) and phenylhydrazine hydrochloride (6 mmol) in ethanol (20 mL) was stirred for 2 hrs. After the completion of the reaction, the mixture was poured into excess water and the precipitate was filtered off and recrystallized from ethanol [For the preparation of the compound (IIa) and (IIb) were collected using a Nonius CAD-4 MACH 3 diffractometer with MoKα (0.71073 Å) radiation at room temperature (293 K). The data reduction was performed with XCAD4 [ψ-scan method [F2 taking all the unique reflections using SHELXL97 [2) Å or 0.96 Å (–CH3) Å with Uiso(H) = 1.2Ueq (parent C atom). The crystal data, experimental conditions, and structure refinement parameters for the compounds (IIa) and (IIb) are presented in Tables IIa) and (IIb) showing the atom numbering scheme using ORTEP 3 [Single crystal X-ray intensity data for compound -3-phenylpropyl) cyclohexanones following the procedure of Moskovkina and Thilichenko [The title compounds, 4-(4-methylphenyl/4-methoxyphenyl)-6-phenyl-2,3,3a,4-tetrahydro-lichenko . The schIIa), bands appear at 3051 cm−1 (aromatic C–H stretching), 1567 cm−1 (olefinic C=C stretching), 2844 cm−1, and 2923 cm−1 . The identity of the above compound has been ascertained by NMR spectral data. A detailed analysis of the two-dimensional NMR spectra has helped to assign the hydrogens and carbons of interest in these compounds. The numbering for carbons and hydrogens given here for NMR data and crystal structure is for convenience and not exactly the correct way of numbering according to IUPAC rules. Compound (IIa) has a broad quartet at 1.34 ppm , a multiplet between 1.75–1.78 ppm , a multiplet between 1.80–1.87 ppm , a multiplet at 2.07–2.12 , a strong triplet with further fine coupling at 2.73 ppm , and a doublet of doublet at 2.82 ppm . The H-3a proton appears as a broadened triplet at 3.06 ppm with coupling constant J = 11.7 Hz, while H-4 proton appears as a doublet of doublet at 3.31 ppm (J = 12.2 & 2.4 Hz). A doublet appears at 5.39 ppm, (J = 2.4 Hz), which is assigned to H-5 proton. The H-8 proton appears as a doublet at 6.33 ppm (J = 8.1 Hz) followed by two triplets centering at 6.85 ppm and 7.02 ppm (J = 7.8 Hz). A multiplet appears between 7.14 to 7.24 ppm accounting for five hydrogens. There are seven CH carbons at 112.6, 116.8, 118.2, 119.6, 121.1, 128.5, and 129.1 ppm. Two CH carbon signals appear at 128.1 ppm (merging together). The quaternary carbons appear at 110.7, 128.9, 134.9, 136.1, 136.3, 136.5, 138.2, and 140.1 ppm. IIa) based on the connectivity studies is shown in In the IR spectrum of  and the benzylic hydrogen (3.31 ppm) have poor coupling between them indicating that they may probably be nearly orthogonal to each other. This is confirmed in the crystal structure. The considerable coupling of 12.0 Hz between the benzylic hydrogen and the ring junction hydrogen (3.06 ppm) amounts to that they are antiparallel to each other. This is again found to be corroborated in the solid state, with the torsion angle between these hydrogens being 177.6°. This ring junction hydrogen has a broad triplet with coupling nearly 11.7 Hz suggesting that it assumes an axial-like arrangement with another axial coupling with the ring vicinal hydrogen. The inspection of the crystal structure suggests this could be the axial hydrogen vicinal to the ring junction hydrogen which also experiences the anisotropic shielding of the p-methylphenyl ring.It is clear that the phenyl substituent at the olefinic carbon goes out of plane to the rest of the ring. This ultimately leads to the shielding of theIIb) are very similar to that of compound (IIa), and IIb), and IIb). The significant IR bands observed for (IIb) are 1428, 1450, 1504, 1567, 1619, 2922, 2835, 3016, and 3043 cm−1.The NMR data for compound (IIa) and (IIb) has the twisted envelope structure with atoms C3 and C13 at the flap, respectively, and they have the distance of 0.777(3) Å and 0.651(2) Å from the mean plane of the carbazole. The most favoured orientation of the phenyl and methylphenyl/methoxyphenyl rings in the structure is that their least-square mean planes are inclined toward the opposite directions with respect to least-square mean plane of the carbazole rings and they are essentially planar and perpendicular to each other by the planar angles, 80.72(3)° and 82.33(3)°, in compounds (IIa) and (IIb), respectively. In the crystal structure of (IIa), H3A and H4 have the transconformation with the torsion angle of 176.60(2)°. In compound (IIb) too, similar conformation is found. The structures are stabilized by weak intermolecular interactions. Further, no classical hydrogen bonds are observed in the crystal structure. From the crystal structures, it is seen that there is no change in the ideal nature of the moiety due to the substitution of methylphenyl or methoxyphenyl groups. The pyrido ring of both the compounds adopts a twist boat conformation. The carbazole molecule of compounds (


Penicillium occitanis mannanases could be almost doubled as compared to a batch cultivation on acacia seeds (76 versus 41 U/mL). Also, a 10-fold increase of enzyme activities was observed from shake flask fermentation to the fed-batch fermentation. These production levels were 3-fold higher than those obtained on coconut meal. The high mannanase production using acacia seeds powder as inducer substrate showed the suitability of this culture process for industrial-scale development.By applying a fed-batch strategy, production of  The Pol6 strain is a hypercellulolytic mutant selected by Jain et al. [The n et al.  after eiLocust bean gum (LBG) was procured from Sigma . Commercial coconut meal was procured from local market in Tunisia. Samples of acacia were collected during April-May, in 2010, from Sfax (Tunisia). The dried seeds were transferred to a mortar, crushed with a pestle, and sieved with a standard 0.5 and 1 mm sieves to obtain two different particle sizes (P1 (<0.5 mm ) and P2 ( 0.5-1 mm)). The chemical composition of coconut and acacia seeds is presented in β-mannan-degrading activity of P. occitanis Pol6. Its composition was (g/L) KH2PO4, 2; NaNO3, 5; MgSO4·7H2O, 0.3; CaCl2, 0.3; yeast extract, 1 g; tween 80, 1 mL/L; trace elements solution, 1 mL/L. The trace elements solution contained (in g/L) CoCl2, 2; MnSO4·H2O, 1.6; ZnSO4· H2O, 1.4; FeSO4· 7H2O, 5.0. The initial pH value was adjusted to 5.5 with orthophosphoric acid (2 M) before sterilisation at 121°C for 20 min. The medium (100/500 mL Erlenmeyer flasks), added with glucose at 2 g/L as a carbon substrate, was inoculated with a mycelial culture grown on potato dextrose agar plates for 1 week. The liquid culture was then incubated for 3 days at 30°C on a rotary shaker at 180 rpm. Mandels and Weber's medium modified by Ellouz Chaabouni et al.  was usedCultures with carbon mannan sources  were carried out in Erlenmeyer flasks (50/250 mL). 1 mL sample of the above inoculum was added to each flask. Samples were removed after 6 days and analysed for mannanase activity.2 of 20% of air saturation. A 30-L fermenter  was used for all experiments. The fermenter was equipped with automatic control of agitation, temperatures, pH, dissolved oxygen concentration (pO2), and foaming. Batch cultures were carried out by adding a 7% inoculum to 7 L sterile medium in a presterilized fermenter . The pH was maintained at 5.5 by adding sodium hydroxide (2 M) or orthophosphoric acid (2 M). The temperature was kept constant at 30°C. Antifoam (1%) was added when required. Aeration automatically varied from 0.1 to 1.0 volume of air per fluid volume per min (v/m) to maintain a POThe fed-batch culture was started with a batch growth phase, followed by a feeding phase. Acacia seeds powder were added by intermittent feeding at a final concentration of 20 g/L. The feeding procedure of the fermenter with coconut meal or acacia seeds powder (0.75 g/h) began after 3 days. Broth samples were taken regularly during the course of fermentation. After centrifugation , supernatants were used to determine mannanase activities. β-mannanase was determined as described by Blibech et al. [h et al. . The relh et al. .Cell growth was followed indirectly by determination of the protein content of the cultivated material by the Kjeldahl method . Commercial mannans such as locust bean gum, guar gum, and konjac flour substrates were largely used for fungal mannanase production , 12, 13.Acacia seeds contained considerable amounts of carbohydrate 53.4%) and organic nitrogen sources (23.37%)  essentia.4% and oP. occitanis growth and enzyme production. Maximum mannanase yields were noticed at, 6th day of incubation (about 8 U/mL), and further increase in fermentation time resulted in the reduction of mannanase activities.The mannanase production data presented in The enzyme production pattern, however, varied with acacia particle size. Enhanced mannanase-production (7.9 ± 0.15 U/mL) was recorded with substrate particles size ranging from 0.5 to 1 mm. It was three times higher than the production yield obtained with substrate particles of an average size <0.5 mm. This may be due to an increase in interparticle porosity which provided better aeration and efficient nutrient availability for mycelium growth.The mannanase yield obtained on acacia seeds in shake flask culture was higher than those obtained by other mannanase-producer fungi cultivated in various agricultural substrates/byproducts-based media .Batch fermentation experiment was carried out in 30 L fermentor scale. The mannanase activity and productivity reached the maximum of 41.1 U/mL (at 72 h) and 571.1 U/l/h, respectively, when using acacia seeds as substrate. These values were threefold higher than those obtained using coconut meal . The sigIn order to improve the production of mannanase on acacia seeds, fed-batch fermentation was performed at a feed rate of 0.75 g/l/h. The profile of mannanase activity, cell growth, and pH was shown in βwindhager et al. [Sclerotium rolfsii.The highest mannanase activity and productivity obtained by fed-batch fermentation using acacia seeds as substrate were 76 ± 3.1 U/mL and 649.5 ± 53.6 U/l/h respectively. These values were about 10-fold higher than those obtained in the Erlenmeyer flask cultures. These results were in agreement with others; for example, Gror et al.  reportedP. occitanis in fed-batch using acacia seed as substrate. The highest mannanase activity reached 76 ± 3.11 U/mL which was about 10-fold in the shake flask culture and about three-fold in coconut meal. So, acacia seeds could be used as a less expensive substrate for an efficient mannanase production at industrial scale.The results obtained in the present study indicate high mannanase production and productivity by culturing


To obtain initial evidence that MHY908 acts as a PPAR α/γ dual agonist, ChIP and reporter gene assays were conducted in AC2F rat liver cells, and to investigate the anti-diabetic effects and molecular mechanisms, eight-week-old, male db/db mice were allowed to eat ad libitum, placed on calorie restriction, or administered MHY908 (1 mg or 3 mg/kg/day) mixed in food for 4 weeks. Age-matched male db/m lean mice served as non-diabetic controls. It was found that MHY908 enhanced the binding and transcriptional activity of PPAR α and γ in AC2F cells, and it reduced serum glucose, triglyceride, and insulin levels, however increased adiponectin levels without body weight gain. In addition, MHY908 significantly improved hepatic steatosis by enhancing CPT-1 levels. Remarkably, MHY908 reduced endoplasmic reticulum (ER) stress and c-Jun N-terminal kinase (JNK) activation in the livers of db/db mice, and subsequently reduced insulin resistance. The study shows MHY908 has beneficial effects on type 2 diabetes by simultaneously activating PPAR α/γ and improving ER stress, and suggests that MHY908 could have a potent anti-diabetic effect as a PPAR α/γ dual agonist, and potential for the treatment of type 2 diabetes.Peroxisome proliferator-activated receptor (PPAR) α/γ dual agonists have been developed to alleviate metabolic disorders and have the potential to be used as therapeutic agents for the treatment of type 2 diabetes. In this study, we investigated the effects of a newly synthesized PPAR α/γ dual agonist, 2-[4-(5-chlorobenzo [ The worldwide prevalence of obesity is steadily growing, and various systems that modulate the balance between energy intake and energy expenditure have been suggested to stave off obesity PPARs are a group of three homologous transcription factors that belong to the nuclear receptor superfamily, members of which are activated by fatty acids and fatty acid metabolites. The different PPAR subtypes, such as, PPAR α, PPAR γ, and PPAR β/δ, have important physiological functions that are in part determined by their tissue distributions Elevated triglyceride and low high-density lipoprotein cholesterol (HDL-C) levels can be addressed by administering PPAR α agonists, and the insulin-sensitizing effects of these agonists have been demonstrated in a variety of experimental models. Accordingly, PPAR γ agonists are now being used to treat type 2 diabetes A number of PPAR α/γ dual agonists have been recently developed and others are being evaluated for their effects on insulin resistance in animal models and in type 2 diabetes patients 4, filtered, and evaporated under reduced pressure. The resultant residue was purified by silica gel column chromatography using hexane and ethyl acetate (7:1) as eluent to give compound 1 : 1H NMR  δ 9.70 , 7.62 , 6.73 , 4.05 , 1.50 , 1.03 ; 13C NMR  δ 190.7, 173.4, 161.0, 131.6, 130.4, 117.8, 79.6, 61.7, 25.4, 14.0; LRMS (ESI) m/z 237.2 (M+H)+;1 N sodium ethoxide  was added to a stirred solution of 4-hydroxybenzaldehyde  and ethyl–bromoisobutyrate  in ethyl alcohol (50 mL) and refluxed for 14 h. After evaporating volatiles, the residue was partitioned between ethyl acetate and water and the organic layer was dried over MgSO4, filtered, and evaporated under reduced pressure. The resulting solid was filtered and washed with methanol and water to give the benzothiazole 2 : 1H NMR  δ 7.95 , 7.91 , 7.70 , 7.26 , 6.88 , 4.22 , 1.64 , 1.21 ; 13C NMR  δ 174.0, 169.6, 158.6, 155.3, 133.4, 132.4, 129.0, 127.0, 125.5, 122.9, 122.4, 118.7, 79.6, 61.9, 25.6, 14.3; LRMS (ESI) m/z 376.1 (M+H)+.A solution of 2-amino-4-chlorobenzenethiol  and compound 1  in dimethylformamide (10 mL) was then heated at 80°C in the presence of Sodium metabisulfite  for 11 h. After evaporating volatiles, the residue was partitioned between ethyl acetate and water and the organic layer was dried over anhydrous MgSO2  in 1,4-dioxane (4 mL) and stirred at room temperature for 17 h. The reaction mixture was then partitioned between methylene chloride and water and the aqueous layer was acidified with 6 N HCl to pH 2. The precipitate generated was then filtered and washed with water to give MHY908  as a white solid: melting point, 190.8–192.0°C; 1H NMR  δ 8.03 , 7.98 , 7.92 , 7.37 , 6.92 , 1.55 ; 13C NMR  δ 175.2, 169.8, 159.0, 155.2, 133.7, 131.9, 129.4, 126.3, 125.8, 124.2, 122.5, 118.9, 79.5, 25.8; LRMS (ESI) m/z 348.1 (M+H)+; HRMS(ESI) m/z 3-(5-{[(thiophen-2-ylmethyl)amino]methyl}furan-2-yl)benzoic acid chloride (M+H)+ calcd 348.0461, observed 348.0458.1 N NaOH  was added to a stirred solution of compound 1  in 1,4-dioxane (50 mL) stirred at room temperature for 4 h. After evaporating volatiles, the residue was partitioned between methylene chloride and water and the aqueous layer so obtained was acidified with 12 N HCl to pH 2. The aqueous layer was then extracted with methylene chloride and the organic layer was dried over MgSO4, filtered, and evaporated under reduce pressure to give compound 3 : 1H NMR  δ 10.54 , 9.83 , 7.79 , 6.95 , 1.69 ; 13C NMR  δ 191.7, 178.5, 161.0, 132.0, 130.5, 118.5, 79.5, 25.5; LRMS (ESI) m/z 209.1 (M+H)+. A solution of 2-amino-4-chlorobenzenethiol  and compound 3  in acetic acid (0.4 mL) was refluxed in the presence of sodium acetate  for 1 h. After cooling, the reaction mixture was partitioned between ethyl acetate and water and the organic layer was evaporated under reduced pressure. The precipitates generated were filtered and washed with methylene chloride to give MHY908 .Alternative synthetic method for MHY908: 1 N NaOH  was added to a stirred solution of compound 5′-ACAGGGTTAGCGGTTGTCAC-3′; reverse 5′-AGAGCAAAGTCTGGGGTGTC-3′), and PPAR γ  promoters.Chip assays were performed using an EZ ChIP Chromatin immunoprecipitation Kit , according to the manufacturer's instructions. Immunoprecipitation was performed using an antibody directed against PPAR α , or PPAR γ , or using rabbit IgG (provided in the Millipore kit) as a negative control. After immunoprecipitation, associated DNA was amplified using a primer pair containing PPARs binding site, PPAR response elements (PPREs), PAPR α , and measured using a TECAN GENios luminescence plate reader .For luciferase assays, 0.1 μg of plasmid was transfected into 5×10in silico protein-ligand docking, AutoDock4.2 is most commonly used because of its automated docking feature http://www.cambridgesoft.com).The crystal structures of PPAR α and γ were extracted from the PDB archives  A pharmacophore is an ensemble of ligand features required for interaction with a specific receptor. A pharmacophore model was generated using the LigandScout 3.0 program Male, 8-week-old, C57BLKS/J-lean and C57BLKS/J-db/db mice were purchased from Japan SLC. Mice were maintained under a 12 h light/dark cycle at 23±1°C and 50±5% RH under specific pathogen-free conditions. db/db mice were allocated to four groups of 8 mice. Animals in the db/db control group were maintained on a normal diet . Feeding levels in the calorie restriction (CR) group were calculated by feeding animals with 60% of the individual mean food intake in (db/db control group). Animals in the two MHY908 groups were administered MHY908 mixed in food . Animals were euthanized after four weeks and livers were collected for Western blot analysis. The animal protocol used in this study was reviewed and approved beforehand by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC) with respect to the ethical of procedures and scientific care .Blood samples were collected after sacrifice of animals from each group. Kits were used to measure the concentrations of metabolites serum metabolites/cytokine such as insulin , glucose and triglyceride , adiponectin , and leptin .TM . Pre-stained protein markers were used for molecular weight determinations.Homogenized samples of liver were boiled for 5 min in gel-loading buffer  at a volume ratio of 1∶1. Total protein equivalents in samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 10% acrylamide gels, and then transferred to PVDF membranes at 80 V and 1.5 h, using a semi-dry transfer system. Membranes were immediately placed in blocking buffer (10 mmol Tris (pH 7.5), 100 mmol NaCl, 0.1% Tween 20, and 5% non-fat milk). Blots were allowed to block at room temperature for 30 min, and then membranes were incubated with specific primary antibody  at 4°C overnight, followed by horse radish peroxidase-conjugated anti-rabbit antibody  at room temperature for 1.5 h. Antibody labeling was detected using a WestsaveP values <0.05.All results are expressed as means ± SEMs. Treatments were compared by one-way ANOVA followed by Dunnett's test. Statistical significance was accepted for A chromatin immunoprecipitation (ChIP) assay was used to determine whether PPAR α and γ activation by MHY908  results The docking simulation exercise produced significant scores. The binding energies of MHY908 and PPAR α or PPAR γ were −9.10 and −8.88 kcal/mol, respectively, and the binding energies of fenofibrate and rosiglitazone for PPAR α and PPAR γ were −8.80 and −8.03 kcal/mol, respectively. The docking score between a ligand and receptor is arrived at by summing energy terms, such as, electrostatic energy, van der Waals energy, and solvation energy, and in the present study docking simulation showed that the binding affinities of MHY908 were greater than those of fenofibrate and rosiglitazone . In addiWe also investigated the pharmacological effects of MHY908 in db/db mice. Serum glucose, TG, and insulin profiles were analyzed to determine whether MHY908 attenuates metabolic abnormalities in obese and diabetic subjects. In db/db mice, MHY908 significantly reduced serum glucose, TG, and insulin levels . FurtherFatty liver is a complication of type 2 diabetes. Since MHY908 was developed and identified as a PPAR α/γ dual agonist, we examined whether MHY908 attenuates fatty liver in db/db mice. Histological analysis showed that lipid droplets accumulated in the livers of db/db mice, but that 3 mg/kg of MHY908 in food daily for 4 weeks markedly reduced this accumulation . To confIt has been recently suggested that ER stress plays a central role in the development of insulin resistance and diabetes via the JNK-mediated inhibition of insulin activity An elevated circulating leptin level is a marker of leptin resistance and is independently associated with insulin resistance, and is commonly observed in the obese. Therefore, we examined whether MHY908 down-regulates serum leptin levels in db/db mice . Becausein vitro and in vivo.Type 2 diabetes and its related complications are serious worldwide health problems, and thus, an aggressive approach that modulates diabetes using anti-diabetic agents that ameliorate insulin resistance and hyperlipidemia is needed. The therapeutic advantages of PPAR α and/or PPAR γ agonists for the treatment of metabolic abnormalities prompted the development of PPAR α/γ dual agonists, such as, razaglitazar, tesaglitazar, and muraglitazar, MHY908 showed agonistic activities for PPAR α and γ. Interestingly, MHY908 more potently activated PPAR α and PPAR γ than fenofibrate and rosiglitazone, respectively . The higMany authors have concluded that PPAR α/γ dual agonists have been identified and tested obese and insulin resistant individuals Fatty liver is a common complication in obese As ER is a central organelle involved in lipid synthesis, protein folding, and protein maturation, ER stress is receiving greater research attention as a cause of insulin resistance via JNK activation, and thus, the inhibition of insulin signaling Leptin levels reflect the amount of stored fat and degree of energy imbalance. A prolonged over-nutritional state greatly increases fat stores In summary, MHY908 exhibited greater PPAR α and γ agonistic activities than fenofibrate and rosiglitazone, which are known PPAR α and γ agonists, respectively. In addition, MHY908 regulated glucose and lipid metabolisms, and ameliorated ER stress and insulin signaling in diabetic animal model. Furthermore, MHY908 also modulated serum leptin and adiponectin levels. Therefore, we concluded that MHY908 has the potential to prevent the up-regulation of ER stress and insulin resistance resulting from overnutrition-induced lipid accumulation by activating PPAR α and γ . In addi


The enaminone methyl 4-(4′-bromophenyl)aminocyclohex-3-en-6-methyl-2-oxo-1-oate (E139) has anticonvulsant activities. It has been reported to have a better safety profile than some anticonvulsant drugs. Since some anticonvulsant drugs are used in the management of neuropathic pain, we evaluated the effects of E139 in rodent models of acute pain and paclitaxel-induced neuropathic pain. The reaction latency to thermal stimuli (hot-plate test) of BALB/c mice was recorded before and after intraperitoneal treatment with paclitaxel , and after treatment with E139 (0.1–40 mg/kg), amitriptyline (10 mg/kg), and gabapentin (10 and 30 mg/kg). Mechanical allodynia in paclitaxel-treated Sprague Dawley (SD) rats was measured using a dynamic plantar aesthesiometer before and after treatment with E139 (10 and 20 mg/kg) or its vehicle for four consecutive days from day 7 after first administration of paclitaxel . Administration of E139 (10–40 mg/kg) produced antinociceptive activity against thermal nociception in naïve mice. Treatment with E139, amitriptyline, or gabapentin reduced paclitaxel-induced thermal hyperalgesia. E139 reduced paclitaxel-induced mechanical allodynia, with the effects lasting longer (24 h) after repetitive dosing. Our results indicate that E139 has antinociceptive activity and attenuates paclitaxel-induced neuropathic pain in rodents. Paclitaxel (taxol), a taxane with mitotic spindle inhibitory activity, is a key component of regimens used in the treatment of various solid tumors including ovarian, breast, and lung cancer. Its use, however, is negatively affected by the development of dose-limiting painful peripheral neuropathy . Neurotoβ-dicarbonyl compounds, have been reported to have anticonvulsant effects in in vitro and in vivo models with minimal side effects  × 100.Mechanical allodynia in rats was measured using the dynamic plantar aesthesiometer , as previously described , 17 and t-test, one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test, Grubbs' test, Kruskal-Wallis test, or two-way repeated measures ANOVA followed by Bonferroni posttests. The differences were considered significant at P < 0.05.Statistical analyses were performed using Student's P > 0.05), whereas higher doses  produced significant percentage increase in reaction latency compared to vehicle-treated mice , 20 mg/kg , and 40 mg/kg . The effect of E139 was time dependent; from the time of administration it rose to a peak between 1.5 and 3 h and was still significantly high at 4 h.The intraperitoneal administration of vehicle or lower doses of E139 (0.1 and 1 mg/kg) did not change the reaction latency to thermal stimulation  on day 7 after first drug administration compared to the baseline latency  and vehicle-only-treated animals in the hot-plate test  and 20 mg/kg  but not 40 mg/kg . The effect of E139 was time dependent; from the time of administration it produced significant effects from 2 h onwards.The intraperitoneal administration of E139  produced significant percentage increase in reaction latency in mice with paclitaxel-induced thermal hyperalgesia compared to vehicle-treated animals . The effect of amitriptyline was also time dependent, producing significant effects earlier than E139 at 1.5 h after administration but was also short lived, having no significant effects 2 h after administration. The peak effect of amitriptyline 10 mg/kg at 1.5 h was 37.7 ± 8.9% increase in reaction latency, which was earlier than but similar in magnitude (P > 0.05) to that of E139 10 mg/kg (at 3 h and 35.0 ± 6.1% increase in reaction latency). A higher dose of amitriptyline (30 mg/kg) caused sedation and thus was not evaluated in the hot-plate test.The intraperitoneal administration of amitriptyline at a dose of 10 mg/kg, which has been shown to reduce thermal hyperalgesia in another model of neuropathic pain , also prP < 0.05, F5,110 = 3.72, P = 0.0038) and 30 mg/kg . The effect of gabapentin was also time dependent, producing significant effects earlier than E139 at 0.5 h after administration but was also short lived, having no significant effects 1.5 h after administration. The peak effects of gabapentin 10 and 30 mg/kg at 1 h were 37.7 ± 9.3% and 41.3 ± 7.9% increase in reaction latency, respectively, which were earlier than but similar in magnitude (P > 0.05) to that of E139 10 mg/kg (at 3 h and 35.0 ± 6.1% increase in reaction latency).The intraperitoneal administration of gabapentin, which has been shown to reduce paclitaxel-induced thermal hyperalgesia , 10 and P < 0.05; F7,128 = 11.08, P < 0.0001). The nadir of the reduction of the withdrawal threshold was on day 9, where paclitaxel treated rats had a withdrawal threshold of 11.6 ± 1.4 g compared to 35.0 ± 2.4 g before paclitaxel treatment, almost 70% reduction in withdrawal threshold.Paclitaxel produced a significant reduction in response withdrawal threshold to mechanical stimuli , as previously described , from 7 P < 0.05, Grubbs' test performed using http://graphpad.com/quickcalcs/Grubbs1.cfm). The values obtained from this animal were presented together with those obtained from the rest of the other animals in this group as median values with interquartile range and the data in this set was analyzed using a nonparametric statistical test, Kruskal-Wallis test.Rats with paclitaxel induced mechanical allodynia were treated with E139 (10 and 20 mg/kg) or its vehicle daily for four consecutive days from day 7 after first administration of paclitaxel. One of the rats with paclitaxel-induced mechanical allodynia treated with vehicle produced values which were significant outliers (P < 0.05) but not at 24 h after drug administration  after drug administration  starting from the third administration of the drug.The intraperitoneal administration of E139 10 mg/kg significantly increased the withdrawal threshold in rats with paclitaxel-induced mechanical allodynia compared to vehicle-treated animals only for the first two doses and at 2 h  of 270 mg/kg in rats [E139 has been shown to have anticonvulsant activities  in rats , which w in rats . In rats in rats . On the c dose TD0 of 270  in rats  had seda in rats ; thus we in rats , 24 and  in rats . Somnole in rats –27 an an in rats , 28.E139 produced antinociceptive effects against thermal nociception in naïve mice similar to what has been observed with amitriptyline or gabapentin –32. We oMechanical allodynia is a well-known feature in rats treated with paclitaxel , 35 and α2-adrenoceptors in in vitro models of epilepsy [α2-adrenoceptors [α-adrenoceptors are possibly responsible for its effects against neuropathic pain, including that induced by paclitaxel [α2δ-1 subunit containing voltage-gated calcium channels also has antinociceptive/antiallodynic effects in rodents with paclitaxel-induced neuropathic pain [E139 has been reported to block TTX-sensitive sodium currents and enhance extracellular levels of GABA possibly via activation of epilepsy , 38, 39.epilepsy –42 and tepilepsy . The antoceptors  and thisclitaxel , 44, 45.clitaxel . Gabapenhic pain , 36, 37.In conclusion the data obtained in this study show that the anticonvulsant enaminone E139 has antinociceptive activity, and attenuates paclitaxel-induced neuropathic pain. The antiallodynic activities of E139 are longer lasting after repetitive treatment; thus, repetitive dosing of E139 has potential therapeutic benefits in the management of paclitaxel-induced neuropathic pain.


Exiguobacterium pavilionensis strain RW-2, isolated from a cold thrombolytic microbialite. The isolate grows at temperatures from 4 to 50°C, at pH levels from 5 to 11, and in media without added NaCl or KCl or with 7% added NaCl.We report the draft genome sequence of  Exiguobacterium with the characterization of E. aurantiacum strain DSM6208T from an alkaline potato processing plant  Pavilion Lake (50.86677°N 121.74191°W) near Lillooet, BC, Canada. The microbialite was also the source of Agrococcus pavilionensis strain RW-1 (3 liter–1), and is permanently cold (4°C to 8°C) in the hypolimnion . The error-corrected and phiX-removed reads were assembled using Ray assembler (version 2.2.0), yielding 23 contigs summing to 3,019,504 bp, with an average contig length of 131,282 bp and a largest contig of 947,149 bp   . The err 52.05%) .E. pavilionensis. Genes putatively encoding a Pho regulon for high-affinity phosphate uptake, carbon starvation, oxidative stress, and cold-shock proteins are consistent with growth in cold oligotrophic waters.Annotation was conducted on the RAST annotation server using the Glimmer-3 option FIGfam database release-59 . The genExiguobacterium (This is the sixth genome to be sequenced from the genus acterium , 8, 16, ATCL00000000. The version described in this paper is version ATCL01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number