Alpinia galanga (Khaa), Boesenbergia rotunda (Kra-chaai), Curcuma longa (Khamin-chan), Curcuma zedoaria (Khamin-oi), Zingiber cassumunar (Plai) and Zingiber officinale (Ginger). Concentrations of total arsenic based on dry weight were 92.4 ± 9.2, 103.5 ± 20.8, 61.7 ± 12.5, 89.8 ± 17.5, 106.7 ± 19.5 and 69.3 ± 11.8 ng/g, respectively and inorganic arsenic were 48.8 ± 7.0, 66.3 ± 12.7, 25.5 ± 5.0, 38.7 ± 4.7, 71.2 ± 11.6, and 38.5 ± 5.5 ng/g, respectively. Among these, Plai and Kra-chaai exhibited the highest levels of total arsenic and inorganic arsenic accumulation that remind consumers to be aware of excess consuming of these rhizomes. On the contrary, the lowest value found in Khamin-chan indicating natural dietary supplements and herbal medicines comprising Kamin-chan are safe from arsenic poison. All investigated amounts of total and inorganic arsenic were much lower than limits recommended by Thai Food and Drug Administration.The arsenic accumulation in rhizomes of Zingiberaceous plants was determined by atomic absorption spectrometry interfaced with hydride generation system (HG-AAS). The raw herbal materials, rhizomes, were collected from different regions of Thailand between December 2011 and January 2012. Six well-known Zingiberaceous plants, 16 samples from each and a total of 96 samples, were analyzed  The μg/day . Further per day . Root or rhizome is a part of the plant that has high opportunity to contaminate with arsenic. Current research demonstrated that plants absorbed heavy metals from soil , 13. TheZingiber officinale (Ginger) shows that arsenic level varied from not detected to 0.13 μg/L [Arum ranged from 74.3 to 89.2 mg/kg, in cabbage from 27.12 to 39.39 mg/kg and in pumpkin from 17.28 to 22.05 mg/kg [Phragmites australis (688 mg/kg). Roychowdhury et al. [A. galanga, B. rotunda, C. longa, C. zedoaria, Z. cassumunar, and Z. officinale, were interesting to investigate their total and inorganic arsenic concentrations.A previous study about rhizome of .13 μg/L . Other a.13 μg/L  show tha05 mg/kg . Baroni 05 mg/kg  analyzedy et al. . In geney et al. , 24. Pla3) and hydrochloric acid (HCl) were purchased from Merck Chemicals ; dimethylarsinic acid (DMA), hydrazine sulfate, hydrobromic acid, and other chemicals were obtained from Sigma-Aldrich . All standard solutions, reagents, and samples were prepared using deionized water (18 MΩ cm) throughout the study. To remove possible arsenic residue contamination, all glasswares were washed thoroughly with tap water, air-dried, soaked in 10% (v/v) HNO3 for 20–24 h, and washed three times with deionized water.Standard reference material (SRM) 1568a (rice flour) was purchased from the National Institute of Standards and Technology . Nitric acid  Willd. (Khaa), Boesenbergia rotunda (L.) Mansf. (Kra-chaai), Curcuma longa (L.) (Khamin-chan), Curcuma zedoaria (Berg.) Roscoe (Khamin-oi), Zingiber cassumunar Roxb. (Plai), and Zingiber officinale Roscoe (Ginger). Sixteen samples of each rhizome species were collected during December 2011 and January 2012 from eight provinces  in north, northeast, central and south Thailand. Totally, 96 samples were collected for investigation. These collections were grown in urban and agricultural fields. Growing period was around two years and harvesting time from December to January. The noticed appearances of mature rhizome were strong flavor and pungent odor. The samples were identified by Dr. W. Gritsanapan, and the voucher specimens  were deposited at Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Thailand.Six kinds of plants in the family Zingiberaceae of which rhizomes are widely used for consumption and medication purposes were selected. They were Rhizome samples were washed, cleaned, air-dried, and sliced. Freeze drying is used to dry sliced rhizomes to keep them in a stable condition. The dried samples were grinded into powder with a porcelain mortar and pestle and passed through a fine mesh sieve. Powder of lyophilized samples was kept in air tight containers at 4°C and protected from light until analysis. The moisture contents were calculated using weights of samples before and after lyophilization.3)2·6H2O and 2% (w/v) MgO in water) and 5 mL of 50% (v/v) HNO3. The mixture was evaporated on a hot plate to dryness and mineralized at 450°C in a furnace. The resulting white ash was dissolved in 5 mL of 6 N HCl and 5 mL of a freshly prepared reducing solution (5% (w/v) KI and 5% (w/v) ascorbic acid). The solution was left for 30 min, and then 10 mL of 50% (v/v) HCl was added to the solution. The solution was filtered through a Whatman no. 1 filter paper into a 25 mL volumetric flask and adjusted to volume with 50% (v/v) HCl. The resulting solution was used for the determination of total arsenic. Duplicate analyses were performed for individual samples.The lyophilized sample preparation for determination of total arsenic was performed by acid digestion procedure described by Muñoz et al. . An accug (measured in gravity × force or g-force) for 10 min using an Eppendorf bench top centrifuge 5810 . The chloroform phase was aspirated into another centrifuge tube. The extraction process was repeated twice. To separate some residues from the extraction, the combined chloroform phase was filtered through a syringe filter with a 25 mm PTEF membrane, pore size 0.45 μm , into another tube. Inorganic arsenic in the chloroform phase was back-extracted into an aqueous phase with 10 mL of 1 N HCl and centrifuged at 1,000 ×g for 10 min. The aqueous phase was aspirated into a beaker. The chloroform phase was extracted one more time. The amount of inorganic arsenic in the combined acidic aqueous phase was quantified as described in 3. All samples were analyzed for inorganic arsenic in duplicate.Inorganic arsenic was determined by the nonchromatographic method described by Muñoz et al. . An accu4 in 0.05% (w/v) sodium hydroxide solution) flow rate, 5–7 mL/min, and HCl flow rate, 9–11 mL/min [A PerkinElmer  AAnalyst 300 atomic absorption spectrometer  interfaced with an AS-90 autosampler and a FIAS-400 flow injection system was used to determine total and inorganic arsenic concentrations in the final solutions. The atomic absorption spectrophotometric conditions were as follows: wavelength, 193.7 nm; slit width, 0.70 nm; EDL current, 380 mA; loop sample, 0.5 mL. The hydride generation conditions were as follows: quartz cell, 16 cm path length, 90.7 cm i.d., electrothermal heating, cell temperature, 900°C, carrier gas (argon) flow rate, 50–100 mL/min, reducing agent (0.2% (w/v) NaBH1 mL/min .σ/S, where σ is the standard deviation of y-intercepts and S is the slope of linear regression analysis [The Q2B analytical procedure described by the US FDA was used for determination of the limit of quantification (LOQ). Mixtures of lyophilized samples of all six species with equal weights were used for the determination of the limit of quantification (LOQ) of the method. For the determination of the LOQ for total arsenic, samples were fortified with a standard arsenic mixture (As(III) : dimethylarsinic acid (DMA), 1 : 1 (w/w)) equivalent to total arsenic at concentrations of 250, 500, 1,000, and 2,500 ng/g; blank samples were not fortified with arsenic. All samples were analyzed in duplicates. A total of twelve regression lines  were obtained by the least-square linear regression analyses of the residual peak heights of standard total arsenic versus fortified total arsenic concentrations. The residual peak heights were peak heights of total arsenic-fortified samples minus that of blank sample. The LOQ of the method was calculated using the equation LOQ = 10analysis .σ/S, where σ is the standard deviation of y-intercepts and S is the slope of linear regression analysis [For the determination of the LOQ for inorganic arsenic, samples were fortified with an inorganic arsenic mixture (As(III) : As(V) 1 : 1 (w/w)) at concentrations of 50, 100, 500, and 1,000 ng/g; blank samples were not fortified with inorganic arsenic. Duplicate analyses were performed for individual samples. A total of twelve regression lines  were obtained by the least-square linear regression analyses of the residual peak heights of standard inorganic arsenic versus fortified inorganic arsenic concentrations. The residual peak heights were the peak heights of fortified samples of inorganic arsenic minus the average peak height of blank sample. The LOQ of the method was calculated using the equation LOQ = 10analysis .The accuracy of determination of total arsenic was assessed by analyzing SRM 2568a (rice flour), because no commercial rice standard reference materials for inorganic arsenic are available. The amount of inorganic arsenic in SRM 1568a (rice flour) was determined and compared with the values previously reported.P < 0.05 was accepted for all comparisons. One-way analysis of variance and Tukey's test using the SPSS Statistics version 17.0 software were performed to determine differences in concentrations of total arsenic, inorganic arsenic (both on wet weight and dry weight basis), and percentages of inorganic arsenic with respect to total arsenic of six rhizome types. A significance level of y-intercepts of the linear regression analysis (σ) and the slope (S) by using the equation LOQ = 10σ/S [n = 10, reference value of 290 ± 30 ng/g) and 102 ± 3.7 ng/g (n = 10), respectively [The calculation for LOQs was based on the standard deviation of  = 10σ/S . The LOQectively . The conectively  and 111 ectively , which wn = 3) from China. Average concentration was 77.9 ng/g, whereas the value in this study was 69.3 ng/g. The indicated concentration may imply that the growth environments and soil condition for growing Ginger in Thailand (69.3 ± 11.8 ng/g) were safer than those in China (77.9 ± 8.7 ng/g) [Zhao et al.  determin.7 ng/g) . The stu.7 ng/g) . Our stuC. longa and C. zedoaria were 4.3 ± 1.5 and 6.2 ± 1.5 ng/g (wet wt), respectively. The inorganic arsenic contents in two Curcuma species were not statistically different (P > 0.05) but they were significantly different (P < 0.05) from Zingiber genus. The inorganic arsenic contents in Z. cassumunar and Z. officinale were 11.7 ± 4.5 and 4.4 ± 1.0 ng/g (wet wt), respectively. The well-known species of Alpinia genus, A. galangal, showed the concentration (wet wt) of total arsenic (15.9 ± 5.1 ng/g) and inorganic arsenic (8.4 ± 3.0 ng/g). Finally, Kra-chaai in Boesenbergia genus contained the total arsenic level of 20.8 ± 4.6 ng/g, the inorganic arsenic level of 13.4 ± 3.1 ng/g, and the percentage of inorganic arsenic of 64.4%. In six plants of Zingiberaceae family in this study, the average concentrations of inorganic arsenic in the roots of In this study, the total arsenic concentrations in fresh rhizomes of Kra-chaai, Plai, Khaa, Khamin-oi, Khamin-chan, and Ginger ranged from 11.5 to 27.3, 7.9 to 26.8, 10.0 to 28.3, 6.9 to 21.8, 5.8 to 21.3, and 4.8 to 13.7 ng/g, respectively. These values are in accordance with the fresh vegetable arsenic concentration reported that ranged from 0 to 195 ng/g. Other edible roots, such as carrot and potato were reported high levels of arsenic at 195 and 103 ng/g, respectively . Muñoz eOf the six medicinal rhizomes of Zingiberaceae family analyzed in this study, both of Kra-chaai and Plai exhibited high mean levels (wet weight) of total arsenic (20.8 ± 4.6 and 17.5 ± 6.6 ng/g), inorganic arsenic (13.4 ± 3.1 and 11.7 ± 4.5 ng/g), and percentage of inorganic arsenic (64.4 and 67.4%), respectively. A possible explanation is that these high levels were related to their arsenic accumulative capacity in the rhizosphere soils associated with each species. In contrast, the low levels of inorganic arsenic in Khamin-chan, Khamin-oi, and Ginger (wet weight) ranged from 2.6 to 8.2, 2.5 to 8.4, and 2.6 to 6.0 ng/g, respectively, whereas the average percentages of inorganic arsenic were 41.8, 44.5, and 56.9, respectively. Form the statistical analysis, comparative contents of total arsenic  among six species, Khaa, Kra-chaai, Khamin-oi, and Plai were significantly different from Khamin-chan and Ginger.μg/g) and national limit for arsenic in herbal medicines (4 μg/g) [From our study, the total arsenic level in ninety-six samples complied with Thai regulatory limit (2 (4 μg/g) –32.  TabZ. cassumunar showed a higher level of inorganic arsenic than other species. Arsenic accumulation in rhizomes may be an important risk factor that needed to be taken into account. The results will be valuable for preliminary risk assessment in raw materials of natural products. Therefore, long-term consumption of herbal products that comprised some particular Zingiberaceous rhizomes might cause adverse health effects.All investigated amounts of total arsenic concentrations meet the requirements at a national level. Among six species were grown on urban and agricultural areas. We found that dried root of


Cyanophora paradoxa (Cp) was chemically investigated to identify pigments efficiently inhibiting malignant melanoma, mammary carcinoma and lung adenocarcinoma cells growth. Cp water and ethanol extracts significantly inhibited the growth of the three cancer cell lines in vitro, at 100 µg·mL−1. Flash chromatography of the Cp ethanol extract, devoid of c-phycocyanin and allophycocyanin, enabled the collection of eight fractions, four of which strongly inhibited cancer cells growth at 100 µg·mL−1. Particularly, two fractions inhibited more than 90% of the melanoma cells growth, one inducing apoptosis in the three cancer cells lines. The detailed analysis of Cp pigment composition resulted in the discrimination of 17 molecules, ten of which were unequivocally identified by high resolution mass spectrometry. Pheophorbide a, β-cryptoxanthin and zeaxanthin were the three main pigments or derivatives responsible for the strong cytotoxicity of Cp fractions in cancer cells. These data point to Cyanophora paradoxa as a new microalgal source to purify potent anticancer pigments, and demonstrate for the first time the strong antiproliferative activity of zeaxanthin and β-cryptoxanthin in melanoma cells.The glaucophyte  Cyanophora paradoxa may have interest as anticancer agents, particularly to cure malignant melanomas, which are frequent cancers with bad prognosis.Microalgae have a great potential for the production of bioactive molecules of pharmaceutical interest ,2,3,4,5.Cp extracts 100 µg·mL−1, to discriminate antiproliferative extracts .−1. Interestingly, F3 and F5 inhibited more than 90% of the A-2058 melanoma cells growth at 100 µg·mL−1.Fractions 3–6 were identified as the most active, significantly inhibiting the growth of the three cancer cell lines at 100 µg·mLThe cytotoxicity and pro-apoptotic activity of F3 and F5 were confirmed in the three cancer cell lines by the observation of cell condensation and blebbing , and demCp fractions was based on the cross-check analysis of polarity, pigmentation, absorption spectra, maximal absorption wavelengths, band III/II ratios, coherence of the presence of the pigment in Cp, high resolution mass spectrometric data, and when possible, comparison with pigments standards, as recommended in the Jeffrey monograph +) and a fragment at m/z 371.2270. The identification of 5,6-epoxy-β-cryptoxanthin could not be unequivocally confirmed because of the lack of standard.F5 contained two peaks corresponding to molecules with a median polarity as they eluated in the middle of the flash chromatography gradient. The yellow color and absorption spectra of the two molecules indicated they were carotenoids with very close absorption maxima and polarities, but different band III/II ratios , polarity, and abundance [max = 433.0, 451.2 and 485.3 nm; III/II: 50%). The presence of this carotenoid was not confirmed by HRMS analysis, which only gave ions corresponding to chlorophyll a and pheophytin a.F6 contained one major peak and two minor peaks. The major peak corresponded to a major bundance . The HRMbundance ,20,21, iCp pigment and five minor peaks, but had low cytotoxic activity in cancer cells. The major F7 Cp pigment presented absorption maxima at 430.6 and 663.0 nm, was slightly more apolar than chlorophyll a, and was identified as chlorophyll a epimer. HRMS analysis confirmed the presence of chlorophyll a related ions in F7 , according to its polarity, absorption spectrum, and band III/II ratio [Cp is coherent because it is a β,β-carotene derivative. The third minor peak absorbed at λmax = 408.9 and 669.1 nm, and was identified as monovinyl pheophytin a according to its absorption maxima and polarity [max = 401.5 and 669.1 nm) [F7 was green, contained one major ns in F7 . The firphytin a ,22. ThisII ratio . Althougpolarity ,22. No M69.1 nm) . No stan69.1 nm) ).Cp pigment, unequivocally identified as β,β-carotene, according to its color, absorption maxima, hydrophobicity, band III/II ratio, abundance in Cp [F8 only contained a major ce in Cp , and HRMPigments standards were obtained from DHI Lab Denmark and Sigma-Aldrich France. Ultra-pure water was obtained using a Milli-Q system . All reagents were of HPLC grade. Flash chromatography columns were obtained from Interchim, Montluçon, France and RP-HPLC columns from Phenomenex, Le Pecq, France.Cyanophora paradoxa (Cp) is a freshwater glaucophyte containing two chloroplasts (cyanelles), relics of cyanobacterial endosymbionts, as demonstrated by their thylakoid organization and the presence of peptidoglycan layers separating the cyanelle from the cytoplasmic content of the protist host cell. It has been widely studied as a model species to understand how oxygenic photosynthesis was established in eukaryotes and how the transfer of endosymbiont genes to the protist host cell allowed plastid establishment and conservation. The pigments described in Cp are chlorophyll a, β-carotene, allo-phycocyanin, c-phycocyanin, β-cryptoxanthin, and zeaxanthin [axanthin ,24,25.Cyanophora paradoxa SAG 29.80  was cultivated at IFREMER PBA, Nantes, in 10L flasks under continuous illumination at an average light intensity of 180 µmol·m−2·s−1 . Cells were frozen at −20 °C, sent to laboratory LIENSs, La Rochelle, and freeze-dried at −55 °C and P < 1 hPa, on a freeze-dryer equipped with a HetoLyoPro 3000 condenser and Heto cooling trap .The xenic strain ·m−2·s−1 . Growth Cp phycocyanin is currently under study in our lab and will be discussed later. Flash liquid chromatography fractionation was performed from the ethanol extract 2, using an Interchim Puriflash PF430 system. One mL of Cp ethanol extract 2 (0.25 g·L−1) was added to the top of a PF-C18 column . The column was then washed with a mobile phase consisting of a ternary gradient of solvent A (Methanol/water (80/20)); solvent B (Acetonitrile/water (90/10)) and solvent C (ethyl acetate). The gradient flow program was set as follows: 0 min—100% A, 3 min—100% B, 35 min—30% B and 70% C, 38 min—100% C, 41 min—100% C, 43 min—100% B, 45 min—100% A. The flow rate was 5 mL·min−1 and elution was monitored from 400 to 800 nm, with an automatic collector (20 mL per tube).The pigments extraction sequence is described in −1 and elution was monitored at 435 nm. Purification of single peaks for mass spectrometry was also performed in semi-preparative conditions on a Phenomenex Luna C18 (2) column  with a flow rate fixed at 5 mL·min−1 and with the same gradient and detection conditions.The number of molecules contained in flash chromatography fractions and their absorption spectra were determined using an analytical RP-HPLC system composed of a binary pump , an autosampler , a thermostated (20 °C) column compartment, and a photodiode-array detector . A 100 µL sample was injected into a Phenomenex Luna C18 (2) analytical column . The mobile phase and gradient flow program were simplified compared to flash chromatography to obtain a quick separation. The mobile phase consisted of a binary gradient of solvent A (methanol/water (80/20)) and solvent B (ethyl acetate) and the gradient flow was set as follows: 0 min—100% A, 8 min—95% A, 5% B, 9 min—100% B, 13 min—100% A, and 15 min—100% A. The flow rate was 1 mL·min−1 as follows: 0–0.2 min, 2% B; 0.2–5.2 min, 2%–100% B; 5.2–5.70 min, 100% B; 5.70–10.70 min, 100% B–100% C; 10.70–11.20 min, 100% C; 11.20–11.25 min, 100% C–100% B; 11.25–11.50 min, 100% B; 11.50–11.55 min, 100%–2% B; 11.55–15 min, 2% B. ESI conditions were: source temperature 120 °C, desolvation temperature 500 °C, cone gas flow 50 L·h−1, desolvation gas flow 1000 L·h−1, capillary voltage 0.5 kV, sampling cone 35 and Source offset 80. The instrument was set to acquire over the m/z range 50–950 with scan time 0.15 s. Data were collected in the positive (ESI+) electrospray ionization mode, in MSE continuum mode. Leu-enkephalin (MW = 555.62 Da) (1 ng·µL−1) was used as lock mass. The mass spectrometer was calibrated before analyses using a 0.5 mM sodium formate solution.Mass spectrometry analyses were performed using an Acquity UPLC  coupled to a xevo G2 S Q-TOF mass spectrometer . Ten microliters of sample were injected into a Waters Acquity UPLC BEH C18 column , at 25 °C. Separation consisted of a gradient of solution A (0.01% formic acid (FA), 0.05% ammonia in water) in solution B (0.01% FA in acetonitrile) and C (0.01% FA in ethyl acetate) at a flow rate of 200 µL·min2–95% air humidified atmosphere, in DMEM  supplemented with 10% heat-inactivated  FCS  to which were added penicillin 100 U·mL−1 and streptomycin 100 µg·mL−1. The algal extracts and related fractions were evaporated to dryness using a Büchi rotavapor and solubilized in DMSO before being diluted in the cell culture medium to a final concentration of 100 µg·mL−1. The final DMSO concentration was lower than 1% and tested as a negative control. Cell viability at 72 h was studied using the MTT assay in 96-well microplates as previously described [MCF-7 human mammary carcinoma cells, A549 human lung adenocarcinoma cells, and A2058 human melanoma cells  were grown as monolayers, at 37 °C in a 5% COescribed ,27.−1 pheophorbide a (positive apoptosis control), or microalgae fractions diluted to 100 µg·mL−1 in cell culture medium. Phosphatidylserines translocation onto the outer side of the plasma membrane of early apoptotic cells was detected using an Annexin V-Alexa-568 fluorochrome , and at the same time DNA was labeled using the cell-permeant Syto® green fluorescent nucleic acid stain kit . Cells were incubated for 4 h min at 37 °C with the labeling mix solution, and observed using a Leica epifluorescence microscope, equipped with an I3 epifluorescence filter block (blue excitation 450–490 nm) and a numeric camera. Control cells were labeled in green with a round shaped nucleus while apoptotic cells, having exposed phosphatidylserines, appeared as red spots. Some early apoptotic cells also exhibited a mixed green and red fluorescence, the red fluorescence being located in the cytoplasmic membrane.Five thousand cells were grown for 24 h on epifluorescence live cell array slides  and treated for 72 h with control culture medium (negative control), culture medium containing 100 µg·mLCp extracts cytotoxicy was determined using the unpaired Student t test .The significativity of Cyanophora paradoxa in human melanoma, breast and lung cancer cells, and presents an updated analysis of Cp pigment composition, suggesting the presence of mutatochrome, 5,6-epoxy-β-cryptoxanthin and an unidentified carotenoid in this species. The major result of this study is the demonstration and first report that zeaxanthin and β-cryptoxanthin exert a strong antiproliferative activity in A-2058 melanoma cells. As zeaxanthin and β-cryptoxanthin antiproliferative activity has already been documented in other cancer cells [This study establishes the high antiproliferative activity of pigments from er cells ,29,30, o


Long-term exposure to high levels of fatty acids impairs insulin secretion and exaggerates glucagon secretion. The aim of this study was to explore if the antihyperglycemic agent, Isosteviol (ISV), is able to counteract palmitate-induced α-cell dysfunction and to influence α-cell gene expression.p<0.01) increase in glucagon secretion. Concomitantly, the TG content of α-cells increased by 78% (p<0.01) and cell proliferation decreased by 19% (p<0.05). At 18 mM glucose, ISV (10−8 and 10−6 M) reduced palmitate-stimulated glucagon release by 27% (p<0.05) and 27% (p<0.05), respectively. ISV (10−6 M) also counteracted the palmitate-induced hypersecretion of glucagon in mouse islets. ISV (10−6 M) reduced α-TC1–6 cell proliferation rate by 25% (p<0.05), but ISV (10−8 and 10−6 M) had no effect on TG content in the presence of palmitate. Palmitate (0.5 mM) increased Pcsk2 (p<0.001), Irs2 (p<0.001), Fasn (p<0.001), Srebf2 (p<0.001), Acaca (p<0.01), Pax6 (p<0.05) and Gcg mRNA expression (p<0.05). ISV significantly (p<0.05) up-regulated Insr, Irs1, Irs2, Pik3r1 and Akt1 gene expression in the presence of palmitate.Long-term incubation studies with clonal α-TC1–6 cells were performed in the presence of 0.5 mM palmitate with or without ISV. We investigated effects on glucagon secretion, glucagon content, cellular triglyceride (TG) content, cell proliferation, and expression of genes involved in controlling glucagon synthesis, fatty acid metabolism, and insulin signal transduction. Furthermore, we studied effects of ISV on palmitate-induced glucagon secretion from isolated mouse islets. Culturing α-cells for 72-h with 0.5 mM palmitate in the presence of 18 mM glucose resulted in a 56% (ISV counteracts α-cell hypersecretion and apparently contributes to changes in expression of key genes resulting from long-term exposure to palmitate. ISV apparently acts as a glucagonostatic drug with potential as a new anti-diabetic drug for the treatment of type 2 diabetes. Nearly four decades ago, the bihormonal-abnormality hypothesis Elevated levels of free fatty acid (FFA) are apparent in patients with diabetes and prediabetes, suggesting that FFAs may be involved in the pathophysiology of type 2 diabetes e.g. using GLP-1) or inhibiting glucagon action (e.g. with glucagon receptor antagonists) have received much attention as therapeutic strategies for the treatment of excess glucose production in type 2 diabetic patients However, the cellular and molecular mechanisms whereby FFAs modulate the coupling of α-cell stimulus secretion remain an enigma Stevioside, a diterpene glycoside isolated from leaves of the plant Stevia Rebaudiana Bertoni, represents a new class of insulin secretagogues chemically unrelated to previous oral antidiabetic drugs, such as sulphonylureas. Interestingly, we have shown that stevioside is able to counteract α-cells hypersecretion caused by palmitate and to enhance the expression of genes involved in fatty acid metabolism The present study was, thus, designed to elucidate the potential role of ISV on α-cell function and its effects on expression of specific genes following long-term exposure to palmitate. Our findings provide evidence that ISV decreases palmitate-induced hyperglucagonemia in both the mouse pancreatic α-cell line, αTC1–6 cells, and in isolated mice islets. ISV concomitantly restores the palmitate-induced impairment of insulin secretion from islets, affects intracellular insulin signal and α-cell proliferation.−6 M). To mimic hyperglycemia characteristic of the diabetic state, the islets were incubated in fresh KRB medium containing 18 mM glucose for 2 h after incubating for 72 h in conditions as described above. It was found that pre-treatment with palmitate substantially increased glucose induced-glucagon secretion by 73%  significantly suppressed palmitate-induced hyper-secretion of glucagon by 32%  at high glucose (18 mM glucose), 0.5 mM palmitate increased glucagon secretion by 52%  had no effect on glucagon secretion by palmitate-treated cells , without or with 0.5 mM palmitate, on glucagon secretion in the presence of 2 mM glucose (p<0.05) and 56% (p<0.01), respectively. ISV alone (10−8 and 10−6 M) had no effect on glucagon secretion. However, in the presence of 0.5 mM palmitate, the palmitate-induced glucagon release was inhibited by 18% (p<0.05) in the presence of 10−8 M ISV at 2 mM glucose. Furthermore, at 18 mM glucose, glucagon secretion was decreased by 27% (p<0.05) and 27% (p<0.05), respectively, by 10−8 M and 10−6 M ISV. glucose  or 18 mM glucose , respectAs illustrated at p<0.01), however, the addition of ISV  in the presence of palmitate had no impact on the TG content.The effect of palmitate and ISV on TG content in α-TC1–6 cells cultured for 72 h is shown in 3H-thymidine incorporation was used to assess cell proliferation. Within 72 h at 18 mM glucose, 0.5 mM palmitate reduced the 3H-thymidine incorporation by 19% compared with glucose alone (p<0.05). At 0.5 mM palmitate, 10−6 M ISV caused a 25% , insulin receptor substrate 2 , fatty acid synthase , sterol regulatory element binding factor 2 , acetyl-Coenzyme A carboxylase alpha , paired box gene 6  and glucagon . ISV alone significantly increased expression of all the selected genes, in the absence of palmitate . Compared with cells cultured with palmitate alone, cells cultured with ISV (10−8 M) had a significantly higher expression of insulin receptor (Insr), insulin receptor substrate 1 (Irs1), and phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1  (Pik3r1) genes in the presence of palmitate, while cells cultured with a higher concentration of ISV (10−6 M) displayed significantly higher expression levels of Irs1, Irs2, Pik3r1 and thymoma viral proto-oncogene 1 (Akt1) gene in the presence of palmitate. Glucagon receptor (Gcgr) gene expression was undetected in α-TC1–6 cells (unpublished data).To gain further insight into the counteractive effect of ISV on palmitate-induced hypersecretion of glucagon, the expression of a few selected genes was examined in α-TC1–6 cells . Palmitap<0.001, p<0.01, p<0.001, To investigate the interaction between α-cells and β-cells, α-TC1–6 cells and MIN6 cells were co-cultured. The effects of co-culture on the expression of the insulin1 (Ins1) and insulin2 (Ins2) genes (specific to β-cells) and on the expression of Gcg gene (specific to α-cells) were examined. The co-culturing was found to result in significantly decreased expression levels of Ins1 p<0.001, , Ins2 (p−8 and 10−6 M) counteracted the palmitate-induced glucagon secretion. Short-term incubation of α-TC1–6 cells with 0.5 mM palmitate resulted in enhanced glucagon secretion under conditions of high glucose, while ISV (10−8 M and 10−6 M) had no effect on glucagon secretion during short-term incubation. As expected, exposure of murine islets to 0.5 mM palmitate for 72 h resulted in decreased glucose-stimulated insulin secretion (GSIS) and glucagon hypersecretion. Our results show that ISV (10−6 M) simultaneously counteracts the decrease in GSIS and abolishes glucagon hypersecretion from murine islets. ISV (10−6 M) concomitantly reduces α-cells proliferation. ISV did not change the TG or glucagon content of α-cells in the presence of 0.5 mM palmitate. The expression of Pax6, Pcsk2, Gcg, Irs2, Acaca, Fasn and Srebf2 were up-regulated during lipotoxicity, while acetyl-Coenzyme A carboxylase beta (Acacb/ACC2), Insr, Irs1, Pik3r1 and Akt1 remained unchanged. Interestingly, ISV  significantly increased the expression of Insr, Irs1, Irs2, Pik3r1 and Akt1 during lipotoxicity. However, expression of Pax6, Pcsk2, Gcg, Acaca, Acacb, Fasn and Srebf2 genes were not affected by these conditions. Gcgr expression was undetected in α-TC1–6 cells but was detected in liver tissue from mice, using the same Gcgr assay (unpublished data). Whether or not it is expressed in normal α-cells has yet to be determined.Here we have demonstrated that incubation of α-TC1–6 cells for 72 h with 0.5 mM palmitate significantly increased their glucagon secretion. Concomitantly, at high glucose concentrations the TG content was increased, while α-TC1–6 cell proliferation was decreased. In addition, their overall glucagon content was unchanged. Interestingly, ISV , zinc ions and glutamate, the central and autonomic nervous systems and several hormones −8 M and 10−6 M) had no effect on glucagon secretion, long-term incubation with ISV (10−8 M and 10−6 M) resulted in a significant reduction in glucagon hypersecretion elicited by palmitate. It is noteworthy that ISV (10−8 M and 10−6 M), in the presence of palmitate, also enhanced Insr, Irs1, Irs2, Pik3r1 and Akt1 expression. The highest ISV dose (10−6 M) elicited more pronounced changes in gene expression than the lower dose in the absence of palmitate  is the enzyme responsible for processing proglucagon to the active glucagon in pancreatic α-cells et al. reported that elevated plasma glucose levels in diabetic mice is accompanied by increased pancreatic α-cell numbers et al. reported that ISV inhibited human cancer cell growth Besides the regulation of glucagon secretion, α-cell growth and survival are of increasing interest in diabetes research. Liu i.e. Fasn, Acaca and Srebf2. ISV was found not to reduce TG content and/or alter the expression of fatty acid metabolism-related genes in the presence of palmitate. Surprisingly, we found no change in the glucagon content of α-TC1–6 cells in presence of palmitate. This is, however, in line with Hong et al. i.e. the glycosylated form of ISV.In the present study we demonstrated that long-term exposure to palmitate resulted in TG accumulation in α-cells and increased expression of genes that are involved in fatty acid synthesis and oxidation In summary, our study demonstrates that chronic exposure to palmitate inhibits insulin secretion, increases glucagon secretion from murine islets and from α-TC1–6 cells, while increasing Pax6, Pcsk2 and Gcg gene expression and TG accumulation. The inhibitory effect of ISV on excess glucagon secretion from murine islets may be due to augmentation of GSIS, which suppresses glucagon secretion. Evidence suggests that the effect of ISV on α-TC1–6 cells may be related to enhanced expression of key regulatory genes involved in insulin signal transduction pathways, thereby increasing insulin sensitivity in α-cells, and concomitantly counteracting hyperglucagonemia. As ISV possesses insulinotropic and glucagonostatic effects, it is likely that ISV may serve as a valuable new therapeutic agent in type 2 diabetes. To further confirm ISV antihyperglycemic effects, clinical trials including consenting people with type 2 diabetes are now warranted.All chemicals were purchased from Sigma-Aldrich  if not stated otherwise.−3 M) prepared in 99% ethanol.Isosteviol (ISV)  was added to the medium from a stock solution .Modified Krebs-Ringer Buffer (M-KRB): 125 mM NaCl, 5.9 mM KCl, 1.2 mM MgClSYTO 24 solution: 5 mM SYTO 24 green fluorescent nucleic acid stain  was dissolved in dimethyl sulfoxside ≥99.9% to a final concentration of 0.01 mM.Adult female Naval Medical Research Institute (NMRI) mice  were maintained on a 12-h light/dark cycle and had free access to water and an ordinary laboratory chow diet. At the time of experiments, they had similar body weights (31–38 g). This study was approved by the Danish Council for Animal Experiments (2010/561-1805).2 in 10 ml RPMI 1640 containing 11.1 mmol/l glucose supplemented with 10% fetal bovine serum (FBS), 2.06 mM L-glutamine, 100 IU/ml penicillin G and 100 µg/ml streptomycin . In each experiment, islets were obtained from 8–10 mice to compensate for inter-individual differences.Islets were isolated by the collagenase digestion technique −6 M). Palmitate solution (0.5 mM) was freshly prepared before each experiment.This part of the studies aimed to determine whether ISV could prevent lipotoxicity caused by palmitate in mice islets. After overnight culture, the islets were incubated in 4-well plates (NUNC) with fresh medium. Thus, ten islets were incubated in 1 ml RPMI 1640 containing 11.1 mM glucose in the absence or presence of 0.5 mM palmitate, and with 0.5 mM palmitate plus ISV . Consequently, batches of five islets were handpicked and incubated in a 37°C water bath with 250 µl of M-KRB containing 0.5% BSA with 18 mM glucose. After 2 h, 150 µl of incubation medium from five islets was collected on ice and frozen for the subsequent analysis of glucagon and insulin secretion. The results of glucagon and insulin were expressed as pg/ml/5 islets, and ng/ml/5 islets, respectively. Glucagon and insulin were analysed by radioimmunoassay (detailed below).2, 95% air) atmosphere. Cells were passaged once a week and replaced with fresh medium twice weekly. These were seeded at 0.25×106 cells/well (24-well plate) and 0.75×106 cells/well (6-well plate) 24 h before an assay.The α-TC1 cell line was derived from an adenoma developed in transgenic mice expressing the SV40 large T-antigen oncogene −8 M and 10−6 M ISV, 0.5% BSA, 0.5 mM palmitate with or without 10−8 M and 10−6 M ISV at 18 mM glucose. Subsequently, supernatants (300 µl each) were collected, centrifuged, and 200 µl supernatants were kept frozen at −20°C for glucagon analysis.α-TC1–6 cells were pre-incubated, for 15 min, in Krebs-Ringer buffer (KRB) and 0.5% BSA (pH 7.4) and were subsequently incubated for 2 h with KRB containing 18 mM glucose in the presence or absence of 10After the acute secretion study, the medium was removed and cells were lysed in 0.1 M NaOH (Sigma). Intracellular protein was measured by Bio-Rad detergent compatible protein assay kit . The glucagon levels in short-term α-TC1–6 cells incubation were adjusted to protein concentration.−8 to 10−6 M ISV. Palmitate solution (0.5 mM) was freshly prepared before each experiment.α-TC1–6 cells were incubated for 72 h at 37°C in DMEM with 18 mM glucose in the presence or absence of 0.5 mM palmitate , 0.5% BSA  with or without 102O and the number of cells was estimated using nuclear staining with 0.01 mM SYTO 24 reagent  and measured by FLUOstar Galaxy . Glucagon levels were normalized to cell number.After 72 h, the cells were pre-incubated for 15 min in KRB and 0.5% BSA (pH 7.4) and were then incubated for 2 h with KRB containing 2 or 18 mM glucose and 0.5% BSA. Subsequently, as before, supernatants (300 µl) were collected, centrifuged and 200 µl were stored at −20°C for glucagon analysis. After the secretion study, the cells were washed once with 1 ml dH−8 or 10−6 M) in six-well plates as 3 ml/well. These culture conditions were used in all subsequent studies with the α-TC1–6 cell line, except where otherwise stated. The cells were washed once with 2 ml of cold PBS (GIBCO), replaced by 1 ml of glycine-BSA  and were then transferred to 1.5 ml tubes. α-cells were disrupted by sonication  on ice for 15 s (twice). After centrifugation at 16,000 rpm for 30 min, the supernatants were collected and stored at −20°C for glucagon and protein assay. Glucagon content levels were adjusted to protein concentration. Intracellular protein was measured by Bio-Rad detergent compatible protein assay kit .Cells were exposed, for 72 h, to DMEM containing 18 mM glucose with or without 0.5 mM palmitate as well as ISV  and mono-125I-(Tyr A14)-labeled human insulin (Novo Nordisk) as tracer and rat insulin as standard (Novo Nordisk). Bound and free radioactivity was separated by ethanol. The inter- and intra-assay variation coefficients were both less than 5%. ISV, like Stevioside and steviol at the concentrations studied, did not interfere with the insulin assay Glucagon was analyzed by radioimmunoassay kit  according to manufacturer's instructions. The glucagon antibody is specific for pancreatic glucagon and has no cross-reaction with other islet polypeptides. The limit of sensitivity for the glucagon assay is 20 pg/mL.To determine TG content, cells were seeded in 24-well plates. After 72 h, the medium was removed, the cells were washed once with 1.0 ml of 0.9% NaCl, and the number of cells was estimated as above. The cells were frozen and stored for 1 h at −80°C. Subsequently, cells were incubated for 20 min with TG reagents . TG content was determined by a TG GPO-PAP kit (Roche) and was normalized to cell number. The recovery of TG content was ∼90%.3H-thymidine incorporation was used to monitor α-TC1–6 cells proliferation and DNA synthesis 4 cells/well) were seeded in 96-well plates  and were cultured in DMEM containing 18 mM glucose and 10% FBS. The next day, the medium was replaced with DMEM containing 5.5 mM glucose, 0.1% BSA and without FBS for 24 h (i.e. serum deprivation). The cells were then cultured for a further 72 h in DMEM containing 0.5% BSA, 18 mM glucose and 0.5 mM palmitate and 1 µCi [methyl-3H] thymidine . After 72 h, the cells were washed twice in cold PBS and then 200 µL liquid scintillator was added. The incorporated [methyl-3H] thymidine was counted by a 1450 MicroBeta TRILUX .The α-TC1–6 cells were cultured in 6-well plate for 72 h (as described in “Glucagon content”) prior to RNA extraction.The MIN-6 cells  produce insulin and have morphological characteristics of pancreatic β-cells Cells were then washed once with cold PBS and RNA was extracted from three different passages of cells using the AllPrep® RNA/Protein kit  according to the manufacturer's instructions. RNA was quantified by measuring absorbance at 260 and 280 nm . The integrity of the RNA was checked by visual inspection of the two ribosomal RNAs, 18S and 28S bands on a 1% non-denaturing agarose gel, stained with SYBR green.Reverse transcription (RT): cDNA was synthesized, from 1 µg of total RNA for each 20 µl RT reaction, using the iScript™ cDNA synthesis Kit  according to the manufacturer's instructions. cDNA was assayed undiluted for Acacb and diluted (1∶8) for Pax6, Pcsk2, Gcg, Insr, Irs1, Irs2, Acaca, Fasn, Srebf2, Pik3r1, Akt1 and Hypoxanthine guanine phosphoribosyl transferase (Hprt) in α-TC1–6 cells alone, (1∶100) for Ins1, Ins2, Gcg and Hprt in 25% α-TC1–6 and 75% MIN6 cells, both or just one or the other.Real-time PCR (qPCR) was subsequently performed using TaqMan® assays using an ABI 7500 FAST machine . Predesigned TaqMan® probes and primers were obtained from Applied Biosystems  for Pax6 (assay Mm00443072_m1), Pcsk2(assay Mm00500981_m1), Gcg(assay Mm00801712_m1), Insr(assay Mm00439693_m1), Irs1(assay Mm01278327_m1), Irs2(assay Mm03038438_m1), Acaca(assay Mm01304277_m1), Acacb(assay Mm01204678_m1), Fasn(assay Mm00662319_m1), Srebf2(assay Mm01306289_m1), Pik3r1(assay Mm01282781_m1), Akt1(assay Mm01331624_m1), Ins1(assay Mm01259683_g1), Ins2(assay Mm00731595_gH) and Gcgr(assay Mm00433546_m1). Hprt(assay Mm01318743_m1) was assessed as endogenous control.2O and 1 µl cDNA (diluted or undiluted as above). Thermal FAST cycle programme was run at 95°C for 20 s; followed by 40 cycles at 95°C for 3 s and at 60°C for 30 s. Reactions were set up in duplicate for each sample and target gene expression levels were normalized to Hprt expression. All assays were performed in 96-well format plates (P/N 4366932) covered with optical adhesive cover . The TaqMan assay efficiencies were calculated to be in the range 89–108%. The 2−ΔΔct method was applied to calculate the relative gene expression In brief, 10 µl reactions were prepared consisting of 5 µl 2× TaqMan ®FAST Universal Master Mix , 0.5 µl 20×TaqMan Assay mix/probe , 3.5 µl AcuGENE Hpost hoc tests (when appropriate) was used to detect statistically significant differences when more than two groups were compared. P<0.05 was considered to be significant.Data is expressed as mean ± SEM of three or four independent experiments. Data analysis and graphs were performed using GraphPad Prism 4.0 . Statistical significance between two groups was evaluated by a two-tailed Student's unpaired T-test. One-way ANOVA followed by Dunnett's or Tukey


SpineCor and rigid braces both have both results testifying to their effectiveness in Adolescent Idiopathic Scoliosis (AIS) treatment: an RCT recently compared the two, showing the superiority of rigid braces. In a previous study, we found similar short- term results in curves between 20° and 30° Cobb. The objective of this study was to compare the short-term results of the Spinecor vs SPoRT brace for AIS in this selected population. Study design: retrospective controlled study. Population: Rigid Brace Groups (RBGs) 20 patients , age 13±1, Cobb 24±5°, ATR 8±3°, TRACE score 7, Risser 0-3. Spinecor Group (SG): 41 patients  age 13±1, Cobb angle 24±5°, ATR 8±3°, TRACE score 6, Risser 0-3. Both groups were treated with a full-time brace . Clinical and radiological evaluations were performed at the beginning and end of treatment. Main outcome measures: Cobb angle (changes > ±5), ATR, TRACE (changes ≥3). Statistics: Chi square, t-test. Considering patients with more than 5° of Cobb angle change, 40% improved, 45% remained stable and 15% worsened in the RBG vs. 22%, 34% and 44% (p<0.05), respectively. No differences were found for ATR. For TRACE, there were no differences among groups: in RBG, 50% improved and 50% were stable, vs. 65%, 31% and 5% worsened (p>0.05). Both treatments showed to be effective in improving the aesthetics in AIS. For the other parameters, the SPoRT brace seemed to be more effective than the SpineCor to avoid curve progression, since the number of worsened patients was much higher for the SG. The main limits of the study were the retrospective design and the small population, so further studies are required.


Purpose. Laparoscopic cholecystectomy has been accepted as an alternative to laparotomy, but there is still controversy regarding the effects of pneumoperitoneum on splanchnic and hepatic perfusion. We assessed the effects of different insufflation pressures on liver functions by using indocyanine green elimination tests (ICG-PDR). Methods. We analyzed 43 patients who were scheduled for laparoscopic cholecystectomy. The patients were randomly allocated to two groups. In Group I, the operation was performed using 10 mmHg pressure pneumoperitoneum. In Group II, 14 mmHg pressure pneumoperitoneum was used. The ICG-PDR measurements were made after induction (ICG-PDR 1) and after the end of the operation (ICG-PDR 2). Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin levels were all recorded preoperatively, 1 hour, and postoperative 24 hours after surgery. Results. The ICG-PDR 1 values for Groups I and II were as follows: 26.78 ± 4.2%per min versus 26.01 ± 2.4%per min (P > 0.05). ICG-PDR 2 values were found to be 25.63 ± 2.1% per min in Group I versus 19.06 ± 2.2% per min in Group II (P < 0.05). There was a statistically significant decrease between baseline and postoperative ICG-PDR values in Group II compared to Group I (P < 0.05). Statistically, there was an increase between baseline and postoperative 1st-hour serum AST and ALT level in Group II (P < 0.05) compared to Group I. No statistical differences were detected on postoperative 24st-hour serum AST and ALT levels and all the time bilirubin between groups (P > 0.05). Conclusion. In conclusion, the results show that 14 mmHg pressure pneumoperitoneum decreased the blood flow to the liver and increased postoperative 1st-hour serum AST and ALT levels. We think that 10 mmHg pressure pneumoperitoneum is superior to 14 mmHg pressure pneumoperitoneum in laparoscopic cholecystectomy. Laparoscopic cholecystectomy is widely used for treatment of benign gallbladder diseases. Today more than 90% of cholecystectomies are performed laparoscopically –3.A transient increase in intra-abdominal pressure (IAP), such as pneumoperitoneum during laparoscopic surgery, causes only minimal adverse effects –6. SeverPlasma disappearance rate of indocyanine green (ICG-PDR) is a dynamic test for the assessment of liver function, and this has been suggested as a marker of global hepatosplanchnic blood flow , 14. TheThe aim of the present study was to examine the effects of different insufflation pressures on liver function as assessed with LiMON on patients scheduled for laparoscopic cholecystectomy.The Regional Committee on Medical Research Ethics approved the study. Informed, written consent was obtained from each patient. Forty-three ASA physical status I or II patients undergoing elective laparoscopic cholecystectomy were included in the study. Patients with liver failure, coagulopathy, and known allergy to medication drugs were excluded from the study.μg/kg), propofol (2–2.5 mg/kg), as well as vecuronium (0.1 mg/kg) for muscle relaxation. Anesthesia was maintained with sevoflurane (2–2.5 vol%) and oxygen-in-air mixture (0.50 ratio). Ventilation was mechanically controlled to maintain normocapnia .The same senior surgeon (A. Sezer) operated all patients, and a similar anesthesia protocol was administered to all patients. Midazolam hydrochloride, used for premedication, was given 45 minutes before surgery at a dose of 0.7 mg/kg. Following 2 minutes of preoxygenation, anesthesia was induced using fentanyl citrate  were recorded three times: preinsufflation, 10 minutes after insufflation, and 10 minutes after desufflation. Anesthetic times and surgery times were also all recorded.ICG elimination tests were done as described by Sakka et al.  using thSerum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin levels were all recorded preoperatively, 1 hour, and postoperative 24 hours after surgery.Side effects, such as hypersensitivity, flushing, bronchospasm, and laryngospasm, were also all recorded.t-test for age, weight, hemodynamic values, and ICG-PDR. Mann Whitney U test for nonnormal distributed data for duration of anesthesia time and surgery time, AST, ALT, and bilirubin. Intragroup comparisons of normally distributed ISG-PDR paired sample t-test with Bonferroni correction. Nonnormal distribution of AST, ALT, and bilirubin of the Wilcoxon signed rank test with Bonferroni correction was used for intragroup comparisons. Sex, ASA data were compared using Chi-square test. Statistica 7.0  statistical software was used for statistical analysis. A P value < 0.05 was considered statistically significant.The numeric results were expressed as mean ± SD, and categorical results were expressed as a number. Differences between groups were assessed using Student's P > 0.05).Sex, age, weight of patients, duration of anesthesia time, duration of surgery time, and ASA scores are shown in P > 0.05)  .P > 0.05). ICG-PDR 2 values were found to be 25.63 ± 2.1% per min in Group I versus 19.06 ± 2.2% per min in Group II (P < 0.05). There was a statistically significant decrease between baseline and postoperative ICG-PDR values in Group II compared to Group I (P < 0.05). These data are shown in The ICG-PDR 1 values for Groups I and II were as follows: 26.78 ± 4.2% per min versus 26.01 ± 2.4% per min (P > 0.05), respectively. The postoperative 1st-hour serum AST level was as follows: 26.78 ± 7.2 U/L versus 34.06 ± 8.2 U/L (P < 0.05). Statistically, there was an increase between baseline and postoperative 1st-hour serum AST level in Group II (P < 0.05) compared to Group I (P > 0.05)  were 24.63 ± 8.1 U/L versus 22.01 ± 8.1 U/L in Groups I and II ( Group I . The pos > 0.05) .P > 0.05), respectively. The postoperative 1st-hour serum ALT level was as follows: 28.85 ± 10.4 U/L versus 38.52 ± 10.16 U/L (P < 0.05). Statistically, there was an increase between baseline and postoperative 1st-hour serum ALT level in Group II (P < 0.05) compared to Group I (P > 0.05)  were 24.5 ± 7.5 U/L versus 24.91 ± 18.9 U/L in Groups I and II ( Group I . The pos > 0.05) .P > 0.05). The postoperative 1st-hour serum bilirubin activities were as follows: 0.73 ± 0.39 mg/dL versus 0.75 ± 0.62 mg/dL, and the postoperative 24th-hour serum bilirubin activities were 0.71 ± 0.28 mg/dL versus 0.73 ± 0.52 mg/dL. No statistically significant difference was detected at any time (P > 0.05)  were 0.67 ± 0.34 mg/dL versus 0.69 ± 0.57 in Groups I and II ( > 0.05) .No side effects were recorded during the study period.2 into the abdominal cavity. To provide good exposure of the surgical field, generally 10 to 15 mmHg pressure ranges are used during pneumoperitoneum [Worldwide, laparoscopic cholecystectomy is most often performed by pumping COritoneum , 12, 18.2 on splanchnic vessels. The authors found that intra-abdominal pressure of more than 16 mmHg is associated with a decrease in splanchnic perfusion.The effects of pneumoperitoneum on splanchnic and hepatic perfusion are not clearly understood. Junghans et al.  demonstr2 pneumoperitoneum with an IAP of 12 mmHg is associated with an increase in hepatic perfusion in healthy adults. The authors do not have a definite explanation for these findings. Another study, done by Sato et al. [Several previous studies reported the effects of pneumoperitoneum on splanchnic and liver perfusion , 22. MeiDynamic liver function tests, such as ICG-PDR, should provide better direct measurement of liver function. Thus, this has been suggested as a marker of global hepato-splanchnic blood flow, and the normal range is 18% to 25% per minute , 23, 24.2 pressure. In our study, we found an increase between baseline and postoperative 1st-hour serum AST and ALT levels in Group II compared to Group I. Also, an increase was detected between the preoperative and postoperative 1st-hour serum bilirubin values, but no statistical difference was detected. We believe that the main reason for this was high insufflation pressure.Morino et al.  evaluateIn conclusion, the results show that 14 mmHg pressure pneumoperitoneum decreased the blood flow to the liver and increased postoperative 1st-hour serum AST and ALT levels. We think that 10 mmHg pressure pneumoperitoneum is superior to 14 mmHg pressure pneumoperitoneum in laparoscopic cholecystectomy.


Pleurotus), that is, P. flabellatus, P. florida, P. ostreatus and P. sajor-caju were used for decolorization of direct blue 14 (DB14). Among all four species of Pleurotus, P. flabellatus showed the fastest decolorization in petri plates on different concentration, that is, 200 mg/L, 400 mg/L, and 600 mg/L. All these four species were also evaluated for extracellular ligninolytic enzymes (laccase and manganese peroxidase) production and it was observed that the twelve days old culture of P. flabellatus showed the maximum enzymatic activity, that is, 915.7 U/mL and 769.2 U/mL of laccase and manganese peroxidase, respectively. Other three Pleurotus species took more time for dye decolorization and exhibited less enzymatic activities. The rate of decolorization of DB14 dye solution (20 mg/L) by crude enzymes isolated from P. flabellatus was very fast, and it was observed that up to 90.39% dye solution was decolorized in 6 hrs of incubation.In the present investigation, four species of white rot fungi ( Azo dyes are the largest class of synthetic dyes used for textile dyeing, paper printing, and other industrial applications. During the dyeing process, 5–20% of the used dyestuffs are released into the processed water , 2. It wPleurotus species for dye decolorization.White rot fungi degrade lignin because they secrete oxidoreductases including lignin peroxidase  propene-1,3-diol:hydrogen-peroxide-Lip EC 1.1.1.14), manganese peroxidase (Mn(II):hydrogen-peroxide oxidoreductase EC 1.11.1.13), and laccase (benzenediol:oxygen reductase EC 1.10.3.2). These enzymes oxidise in a nonspecific way both phenolic and nonphenolic lignin derivatives and thus are promising candidates for the degradation of environmental pollutants, for example, phenols, anilines, dyes, lignocelluloses . BiodegrPleurotus species, that is, P. flabellatus, P. florida, P. ostreatus, and P. sajor-caju were obtained from Directorate of Mushroom Research, Solan , India. Throughout the study, cultures were maintained on MEA  medium at 28°C and subcultured at the regular interval of three weeks. The pure cultures of Pleurotus spp.  were inoculated on MEA plate containing 200 mg/L, 400 mg/L, and 600 mg/L of DB14. These plates were incubated on 28 ± 2°C. DB14 (direct blue 14) was used as reference dye for group of azo dye. Inoculums of four 2 agar pieces from actively growing fungus on malt extract agar plate. The medium for enzyme production contained 2% wheat bran and 2.5% malt extract, and the pH was adjusted to 6.0 by using NaOH or HCl. Incubation was carried out at 28°C in biological oxygen demand BOD incubator in cotton-plugged 250 mL Erlenmeyer flasks containing 50 mL of media. Flasks were inoculated with 1 cmSamples of substrate were collected at regular interval of 3 days and extracted in phosphate buffer (pH 6.0) for ligninolytic enzymes. Filtrate of extraction was used for enzyme assay.via the oxidation of o-methoxyphenol catechol monomethylether (guaiacol) as substrate. The reaction mixture contained 1 mL of 1 mM guaiacol in 0.1 M sodium phosphate buffer (pH 6.0) and 1 mL of crude enzyme solution was incubated at 30°C for 10 min. The oxidation was followed by the increase in absorbance at 495 nm. One activity unit was defined as 1 μmol of guaiacol oxidised per minute [Laccase activity was determined r minute .4, 0.1 mL of 1 mM H2O2, 0.25 mL of 1 mM guaiacol and 0.3 mL of crude enzymes. The oxidation of substrate at 30°C was followed spectrophotometrically at (A465) [Manganese peroxidase (MnP) activity was determined using guaiacol as substrate. The reaction mixture contained 0.2 mL of 0.5 M Na-tartrate buffer (pH 5.0), 0.1 mL of 1 mM MnSO (A465) . −1), 0.2 mL 0.5 M sodium acetate buffer pH 3.5, 0.2 mL crude enzyme, 0.2 mL of deionised water, 0.1 M H2O2, 0.1 M MnSO4 or 5.5 mM ABTS solutions, at 25 ± 2°C [Decolorization of the azo dye direct blue 14 was monitored in a mixture containing 1.4 mL DB14 solution 20 mg L mg L−1, λ = 595 nm. The rate of decolorization was expressed as the percentage decrease in absorbance at the peak wavelength. Control tests were conducted with crude enzyme replaced by deionised water. Experiments were performed in triplicate, and results were expressed as the mean values.Visible spectra were recorded with a UV-visible (Elico-SL191) spectrophotometer at Pleurotus selected in the present work showed different rate of decolorization of DB14. Among the four species of Pleurotus, P. flabellatus showed the fastest decolorization of DB14 followed by P. florida, P. sajor-caju, and P. ostreatus on 200 mg/L of DB14  in 12 days. Subsequently, P. ostreatus and P. florida showed maximum laccase activity, that is, 436.3 U/mL and 353.3 U/mL in 9 days and 21 days, respectively  [Streptomyces spp. and Phanerochaete chrysosporium [The laccase  and MnP adicals) , 27. Accadicals) , becauseosporium . The breosporium . Pleurotus flabellatus can be used in bioremediation of dye-contaminated ecosystems. This is because of the presence of powerful enzymatic machinery which can effectively degrade the recalcitrant and toxic dyes. Out of four species investigated, P. flabellatus showed the maximum enzymatic activity. Extracellular enzymes extracted from 12 days old culture of P. flabellatus decolorized the DB14 to maximum extent. For better enzyme production the wheat bran can be used as supplement in liquid media. Decolorized effluent after enzymatic treatment can be reused by industries and also in agriculture. The present investigation suggests that the white rot fungus


Statistically significant differences in frequency of LOH/MSI alterations in particular chromosomal regions have been found for 1p31.2, 7q32.2 and 16q22.1 . We documented statistically significant association between Fractional Allele Loss (FAL) index and advanced tumor stage (p < 0.05). We suggest that genomic instability of LOH/MSI type is a frequent event in prostate carcinogenesis and assessed as FAL index has clinical value for the molecular staging of prostate cancer in (TRUS)-guided prostate biopsy material.The biological behavior of prostate cancer is uncertain, and therefore, search for molecular prognostic markers associated with disease progression seems to be essential. We performed microsatellite allelotyping of DNA isolated from primary prostate tumors biopsies . We evaluated the frequency of allelic imbalance (AI), including loss of heterozygosity and/or microsatellite imbalance (LOH/MSI) as well as the association of these DNA alterations with clinicopathological variables. We assessed the significance of LOH/MSI alterations in selected imprinted and non-imprinted chromosomal regions (IR and NIR) in PCa. A total of 50 biopsies of prostate tumor (containing >75 % tumor cells) were histologically examined confirming prostate carcinoma. Microsatellite allelotyping using 16 microsatellite markers linked to the following chromosomal regions: 1p31.2, 3p21.3–25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1 was performed. The incidence of LOH/MSI alterations in prostate tumor cells was the highest for chromosomal regions 7q32.2 and 16q22.1 , followed by 1p31.2 and 3p21.3–25.3 . Statistically significant increase in LOH/MSI alterations has been observed for markers: D1S2137 (1p region;  Tp53), as well as mutator gene defects  [loci have been reported [Prostate cancer is one of the most common non-skin cancer affecting older men >65 years) worldwide, and among US men, it is the second leading cause of cancer death . However years woTp53 alterations, or histopathological characteristics [Recently, chromosomal instability involving alterations in chromosomal segregation and/or structure via deletion, duplication, as well as microsatellite instability (MSI) and/or loss of heterozygosity (LOH) has been described as a distinct type of genetic instability, characteristic for prostate cancer , 8. Manyeristics . Howevereristics .loci including suppressor genes located in imprinting regions (IRs) and non-Imprinting regions (NIRs) of human genome. Finally, we evaluated the clinical value of LOH/MSI incidence for the molecular staging of prostate cancer.The aim of our study was to answer the question whether LOH/MSI represents the important mechanism in prostate tumorigenesis. Our additional goal was to identify molecular markers associated with disease progression. In order to investigate whether an allelic imbalance (AI) is associated with clinicopathological variables of patients with prostate cancer, we investigated the incidence of LOH and MSI in sporadic adenocarcinomas in different stage of clinical progression, focusing on selected The studied biological material was received from 2nd Department of Urology, Medical University of Lodz, Poland, between October 2009 and December 2011 and comprised of 50 prostate cancer biopsy specimens. All samples were obtained from the peripheral zone of prostate gland in patients who underwent transrectal ultrasound (TRUS)-guided prostate biopsy. All tissues were selected and evaluated by the independent pathologists who determined Gleason grading and differentiation status. None of the recruited patients received preoperative chemo- or radiotherapy. The pathological evaluation report was obtained for each patient. Clinical and demographic characteristics of study subjects are presented in Table ® solution  and stored at −70 °C. Simultaneously, blood samples (2 ml) from each patient were collected on EDTA and frozen. Informed consent was obtained from patients, and this study was approved by the Ethical Committee of the Medical University of Lodz (RNN/59/09/KE).Immediately after (TRUS)-guided prostate biopsy, the obtained samples were placed in RNAlaterIsolation of genomic DNA from prostatic biopsies and blood samples—which served as a reference DNA—was performed using QIAamp DNA Investigator Kit , according to the manufacturer’s protocol. Quality and quantity of each DNA sample was spectrophotometrically assessed, measuring absorbance at wave length of 260/280 nm . DNA with a 260/280 nm ratio in range 1.8–2.0 was considered as high quality.http://www.ncbi.nlm.nih.gov/genome/sts/sts) with supplementary mapping information, if necessary, provided in Cooperative Human Linkage Centre Database (http://www.chlc.org), the Genome Database (http://www.gdb.org). Each forward primer was labeled at 3′end with fluorescent dye: 6-FAM, NED, PET, or VIC.Sixteen primer pairs were used to perform microsatellite analysis, based on amplification of polymorphic microsatellite repeats: (T)n, (CA)n, (TTA)n, and (TCTA)n in paired DNA samples, i.e., obtained from prostatic tumor cells and blood  from the same patient. These markers linked to the following chromosomal regions: 1p31.2, 3p21.3–25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1. In LOH/MSI analysis, 9 microsatellite markers mapped to known IRs, and 7 to NIRs of human genome, were used. Nucleotide sequences of microsatellite markers used in the study and their chromosomal localization are presented in Tables ® 360 buffer , 360 GC Enhancer , 5 U/μl AmpliTaq Gold® 360 DNA Polymerase , 25 mM MgCl2, 10 mM dNTPs , 30–40 ng DNA, 0.5 μM each primer and nuclease-free water. Each microsatellite marker was amplified at its own specific annealing temperature to optimalize the PCRs.Amplification reactions with microsatellite markers were performed in a Personal Thermocycler , in a total volume of 25 μl, including 10× AmpliTaq GoldPCR conditions were as follows: initial denaturation at 95 °C for 10 min, then 30 cycles of amplification with denaturation at 95 °C for 45 s, primer annealing for 30 s at temperature specific for each marker, i.e., in the range of 47–50 °C , 51–59 °C , followed by elongation step at 72 °C for 1 min.loci with LOH or MSI to the total number of informative loci examined.Afterward, PCR product (0.5 μl) was mixed with 0.25 μl GS500-LIZ Size Standard  and Hi-Di™ Formamide  up to the final volume of 10 μl. The obtained mixture was denatured for 5 min at 95 °C, then cooled on ice for 3 min, and separated by capillary electrophoresis in 3130xl Genetic Analyzer  using GeneMapper Software v 4.0, according to the manufacturer’s protocol. Study samples were considered informative, i.e., heterozygous, when two distinct alleles were visible in the reference leukocyte DNA sample (from blood of each patient). Evaluation of LOH/MSI was performed by calculating the ratio of the fluorescence intensity of the alleles, originating from blood sample  to the fluorescence intensity of the alleles originating from prostatic biopsy . For each informative tumor–normal DNA pair (paired T and N samples), an Allelic Imbalance Ratio (AIR) was calculated, based on the maximum allele peak heights (fluorescence intensity), as follows: normal allele 1: normal allele 2/tumor allele 1: tumor allele 2 (N1:N2/T1:T2) according to protocol [U test, Kruskal–Wallis test) were applied. Statistical significance was determined at the level of p < 0.05. For calculations, Statistica for Windows v. 10  program was applied.Since values determined in experiments did not show normal distribution (Kolmogorov–Smirnov test), the nonparametrical statistical tests  microsatellite markers. The obtained results indicate that in all (50) studied prostate adenocarcinoma biopsies, LOH/MSI frequency was present in the range of 6.80–31.25 % (mean 20.18 % ± 0.08), depending on the chromosomal region. Representative examples of LOH/MSI in prostate adenocarcinoma cells are shown in Fig. Using the panel of 16 microsatellite markers, LOH/MSI analysis was performed in DNA from all (50) patients. The normativity of the markers was assessed to be in the range of 52–88 % (mean 74 % ± 0.12). All the studied DNA (50 samples) derived from prostate adenocarcinoma biopsies were informative at least for two studied loci) LOH/MSI alteration has been observed for D16S3025 marker, spanning the chromosomal region 16q22.1, followed by D1S2137 marker , covering the chromosomal region 1p31.2. Additionally, for D16S496 marker—localized also at 16q22.1—the frequency of LOH in the studied DNA samples was 14.70 % (5/34 informative loci), and 12.50 % (5/40 informative loci) for D1S368 (1p31.2). Genetic instabilities of LOH/MSI type were also observed for D3S3615, D7S2544, D7S530, and D9S974 markers with similar frequencies, which were as follows: 13.20, 12.20, 15.80, and 15.90 %, respectively. The lowest incidence of LOH/MSI (<10 %) was observed for markers: D3S3611, D7S2519, D9S1604, D11S4088, D11S1318, D12S1041, and D12S1727. The LOH/MSI alteration has not been found for D3S1317 marker. Statistically significant increase in LOH/MSI alterations has been observed for markers: D1S2137 , D9S974 , and D16S3025 .We evaluated the frequency of LOH/MSI alteration for each marker used in the study, separately. The most frequent , the highest frequency of LOH/MSI was observed at 7q32.2 (31.25 %), 16q22.1 (26.60 %), and 1p31.2 (26.50 %) and lower in 9p21.3 (17.80 %), 3p21.3–25.3 (17.40 %) and 11p15.5 (14.90 %). The lowest frequency of LOH/MSI was found at 12q23.2 (6.80 %) chromosomal region  has not confirmed the significant difference in LOH/MSI frequency in IR versus NIR (p > 0.05). The frequency of genetic instability (LOH/MSI) in the studied prostate adenocarcinoma group was also assessed as FAL index, ranging from 0.00 to 0.93 (mean 0.10 ± 0.17). FAL indexes were evaluated only for those prostate adenocarcinoma samples, for which LOH/MSI was present at least in one microsatellite locus. The results are shown in Table Statistical analysis  samples separately in relation to clinical features of patients: total PSA value (TPSA), total/free PSA value (T/FPSA), PSA density PSAD), prostate volume, and patient’s age at time of diagnosis as well as histopathological characteristics of tumors . We observed that patients with FAL index ≥0.4 had diagnosed advanced tumors  according to TNM system . Especially, in case of one studied patient—with PSAT 1,489 ng/ml; PSAF 198.2; F/T PSA 0.13 and tumor stage T3bNxM1b—FAL index value of 0.93 was recognized  as well as between FAL index values and Gleason score . Moreover, no significant association between FAL index values and total PSA value, F/T PSA value, and prostate volume  was observed.There was no statistically significant correlation between FAL index levels and patients’ age, PSAD, TRUS image, DRE results  genes  [MEST) in the initiation of breast and lung neoplasms [Interestingly, despite several studies evaluating LOH/MSI on 7q (7q31.1-32 region), no important gene entified , 14. On 4, CPA5) , which aeoplasms . Therefolocus has been observed as next important region with significant (~30 %) frequency of LOH/MSI in the studied adenocarcinoma group. Our observation was confirmed by others [DCC gene) revealed AI in different types of human cancer including prostate [In our data, 16q 21.1 y others . Accordiprostate , 19. Ourloci. However, it should be stressed that in our study statistically significant correlation between total FAL index and advanced tumor stage has been documented. We assume that small numbers of tumors included in our study might influence the above-mentioned observation.Moreover, we did not confirm any correlations between clinicopathological parameters and frequency of LOH/MSI in any studied chromosomal regions, or in any separately examined locus, where statistically significant increase in LOH/MSI alteration has been observed (for marker D1S2137). According to earlier study focused on 1p region in prostate cancer susceptibility, only one locus known as CRAB has been recognized on 1p36 [ARHI and GADD45A genes) have not been used in PCa study yet. Molecular downregulation of ARHI (including LOH) is proved to be linked with several cancer types in human, such as ovarian, breast, hepatocellular and thyroid cancer [loci in prostate carcinogenesis is innovative and should be continued as a new project.Very important aspect of our study seems to be associated with 1p31.2  on 1p36 . To our d cancer , 21, 22,RASSF1A locus (3p21.3). Our observation is important with regard to RASSF1A biological function as TSG in which inactivation has been implicated in wide variety of sporadic human cancers as a negative regulator of cell proliferation and apoptosis.In our study, LOH/MSI alteration was observed also in 3p21.3–25.3 region. We documented—as the first research team—frequent and statistically different LOH in CDKN2A/p16 locus) confirmed in our study provides the evidence supporting the thesis about the presence of 9p21 gene cluster associated with increased tumorigenicity [It should be stressed that statistically significant increase in AI in 9p21.3 region , CDKN1C (11p15.5), and SLC5A8 (12q23.2). In our opinion, especially the CDKN1C gene, negative regulator of cell proliferation alterated in many tumors may be important in prostate carcinogenesis [As so far, there are only few studies concerning 11p and/or 12q, as these regions have not been recognized as a “hot spot” for AI in prostate carcinoma . Our resogenesis , 27.Based on some earlier study, comparing the frequencies of AI with the clinicopathological features and prognosis in prostate cancer, the obtained results are highly controversial , 28.Reassuming, we identify major chromosomal regions  in which LOH/MSI presence is connected with prostate cancer size (T in TNM staging). We suggest that total LOH/MSI alteration assessed as FAL index from biopsies may have clinical value for the molecular staging of prostate cancer in transrectal ultrasound (TRUS)-guided prostate biopsy material and seem to be promising.


Each molecule has essentially the same feature of non-coplanar aromatic rings whereby the two 4-isopropoxybenzoyl groups are twisted in a perpendicular manner to the naphthalene ring and oriented in the same direction (syn-orientation). The benzene rings of the aroyl groups make dihedral angles of 16.13 (7) and 25.31 (7)° in the two molecules. These benzene rings make dihedral angles of 88.38 (8) and 75.32 (7)° with the naphthalene ring system in one molecule, and 89.71 (7) and 82.11 (7)° in the other. In the crystal, molecules are linked via C—H⋯O hydrogen bonds, forming a three-dimensional network. In one independent molecule, the 2-propyl groups of both isopropoxy groups are disordered over two positions with site occupancies of 0.512 (3) and 0.488 (3).The title compound, C DOI: Click here for additional data file.10.1107/S1600536813004959/su2564Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813004959/su2564Isup3.cmlSupplementary material file. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Agrobacterium tumefaciens P4 is a quorum-sensing-signal-producing bacterium that has been isolated from the tobacco rhizosphere. This strain belongs to genomospecies 1 of the A. tumefaciens complex; it is avirulent on various putative host plants, devoid of the Ti plasmid, and contains a luxI homolog on the At plasmid. Agrobacterium tumefaciens strain P4 has been identified as an isolate that produces quorum-sensing (QS) signals of the N-acyl homoserine lactone (AHL) class (Datura stramonium and tomato plants) and defective for the plasmid Ti and, hence, for the traI gene that encodes the synthesis of the QS signal 3-oxo-octanoyl-homoserine lactone (3OC8-HSL) in agrobacteria. Using thin-layer chromatography, commercial 3OC8-HSL as a reference, and A. tumefaciens NT1(pZLR4) as an AHL biosensor strain  class . Howeverr strain , we confde novo genome assembly of A. tumefaciens strain P4. Two libraries were constructed using the TruSeq SBS version 3 sequencing kit: a shotgun (SG) paired-end library with a fragment size between 150 and 500 bp and a long jumping distance (LJD) mate-pair library with an average insert size of 7,765 bp. The two libraries were sequenced using a 2 × 100 bp paired-end read module of Illumina HiSeq 2000 by Eurofins Genomics (France). Sequences reads with low quality (<0.05), ambiguous nucleotides (n > 1), and a sequence length of <50 nucleotides were discarded prior to assembly. After trimming, we retained 49,531,690 paired-end reads  with an average length of 94.8 bp and 3,283,394 mate-paired reads  with an average length of 82.7 bp. Sequence assembly was carried out using the CLC Genomics Workbench version 5.5 , with a read length of 0.5 and a similarity of 0.8. Eighteen contigs were obtained with a length ranging from 2.4 kbp to 884 kbp, with an N50 value of 532,842 bp. The scaffolding was processed using SSPACE basic version 2.0 (in silico finishing of some gaps was carried out by mapping (read length of 0.9 and similarity of 0.95) the mate-pair reads on each of the 5-kbp contig ends. Next, the collected reads were used for de novo local assembling (read length of 0.5 and similarity of 0.8). The published sequence is composed of nine contigs (from 53.8 kbp to 1.63 Mbp) grouped in 3 scaffolds, with a coverage rate ranging from 853- to 965-fold.Here, we report the A. tumefaciens strain P4 genome consists of one circular chromosome containing 2,856,286 bp, one linear chromosome containing 2,052,829 bp, and one circular At plasmid containing 661,825 bp. The G+C contents are 58.8%, 58.6%, and 56.7% for the circular chromosome, linear chromosome, and At plasmid, respectively. A total of 5,379 putative coding sequences were predicted using the Rapid Annotations using Subsystems Technology (RAST) version 4.0 automated pipeline (luxI homolog on the At plasmid, the function of which remains to be investigated.The A. tumefaciens P4 genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession no. APJV00000000. The version described in this paper is the first version, APJV01000000.The


Background. Bone loss in ankylosing spondylitis may be related to inflammation. Data from previous studies on circulating levels of sRANKL, OPG, MMP3, and TIMP is inconsistent; thus this study is planned to look at this aspect in Asian Indian patients. Methods. Cross-sectional study included patients with ankylosing spondylitis and age- and gender-similar controls. Serum levels of sRANKL, OPG, MMP-3, and TIMP-1 were measured by ELISA. Results. Included 85 patients (M : F = 82 : 3) having mean age (±SD) 33.0 ± 10.0 years and disease duration 11.3 ± 7.3 years. BASDAI, BASFI, BASMI, and ESR were 4.0 ± 2.2, 3.9 ± 2.8, 3.0 ± 2.8, and 59.2 ± 31.2, respectively. Patients had higher mean (±SD) OPG level . However, there was no difference in sRANKL . Serum MMP-3  and TIMP-1  levels were higher in patients; however, there was no difference in MMP-3/TIMP-1 ratio. Conclusion. Circulating levels of OPG were higher; however, there was no difference in sRANKL in Asian Indian ankylosing spondylitis patients. Although both MMP-3 and TIMP-1 were raised, their ratio was not different from that of controls. RANKL and cytokines lead to osteoclast and other inflammatory cell activation and release of bone and cartilage degrading enzymes like cathepsin K and matrix metalloproteinases (MMPs) [Patients with ankylosing spondylitis have been shown to have low bone mineral density at spine and propensity for vertebral fractures. The loss of bone mineral density has been shown to be more marked in late than early disease . The bonleukin-6 . These ms (MMPs) , 6. Theis (MMPs) . There iThis cross-sectional study was carried out in a North Indian university hospital between April 2010 and February 2011. Institutional ethical clearance was taken and all patients gave written consent.Consecutive patients with ankylosing spondylitis, attending the outpatient rheumatology clinic, and who gave consent were recruited. Patients fulfilled 1984 modified New York Criteria for Ankylosing Spondylitis [Disease activity was assessed using the “Bath Ankylosing Spondylitis Disease Activity Index” (BASDAI). This consists of 6 questions on 5 major symptoms of spinal pain, joint pain, entheseal pain, fatigue, and early morning stiffness . FunctioA radiograph of lumbosacral spine to detect syndesmophytes was done. Most were tested for presence of the gene HLA-B27 using polymerase chain reaction. Erythrocyte sedimentation rate (Westergren method) was noted. Serum was separated and stored at −80°C. Soluble receptor activator of NF-Kappa B (sRANKL) was determined by sandwich ELISA . Osteoprotegerin was determined using sandwich ELISA  with range 62.5–4000 pg/mL. MMP-3 and TIMP-1 were estimated by ELISA using a commercial kit . The MMP-3 kit detected both active and pro-MMP-3  with range of 31.25–2000 pg/mL.t-test was used to compare means and correlation was done by Pearsons correlation.Analysis was done using SPSS version 15. Student's The study included 85 patients (M : F = 82 : 3) of ankylosing spondylitis with mean age (±SD) 33.0 ± 10.0 years. Mean duration of disease was 11.3 ± 7.3 years and time since diagnosis was 3.8 ± 5.1 years. The means (±SD) of BASDAI, BASFI, BASMI, and ESR were 4.0 ± 2.2, 3.9 ± 2.8, 3.0 ± 2.8, and 59.2 ± 31.2 mm, respectively. Syndesmophytes were present in 36 (42%) patients and HLA-B27 positive in 95% (59 of 63 tested). r = −0.05), MMP-3 (r = −0.04), or TIMP-1 . There was also no correlation between ESR and OPG (r = −0.12) or MMP-3 . However, TIMP-1 correlated with ESR .Patients had higher levels of osteoprotegerin (OPG) than controls. However, sRANKL were similar in both patients and controls, being detectable in only a quarter of both . Even onThis study found higher levels of osteoprotegerin (OPG) in patients of ankylosing spondylitis than controls; however, sRANKL was detected in only a minority in both. Matrix metalloproteinase 3 (MMP-3) and tissue inhibitor of metalloproteinase 1 (TIMP-1) were both elevated in patients; however, there was no difference in the MMP-3/TIMP-1 ratio. This study found higher levels of OPG in ankylosing spondylitis patients compared to controls. This is similar to what most of the previous studies have found –14. HoweThis study found the values of sRANKL to be similar in patients and controls. This is different from previous studies, most of which found higher levels in patients compared to controls , 15. TheThis study found higher levels of both MMP-3 and TIMP-1 in ankylosing spondylitis. High levels of MMP-3 have been shown in serum of ankylosing spondylitis patients compared to controls , 24. ImmTo conclude, this study did not find elevated circulating levels of sRANKL or elevation in the MMP-3/TIMP ratio in ankylosing spondylitis patients. However, a study of bone density and radiological damage with these markers may provide a better understanding of the role of these markers in bone loss in ankylosing spondylitis.


Melophlus. The structure of the compound was elucidated on the basis of spectroscopic analysis . Aurantoside K did not show any significant activity in antimalarial, antibacterial, or HCT-116 cytotoxicity assays, but exhibited a wide spectrum of antifungal activity against wild type Candida albicans, amphotericin-resistant C. albicans, Cryptococcus neoformans, Aspergillus niger, Penicillium sp., Rhizopus sporangia and Sordaria sp.A new tetramic acid glycoside, aurantoside K, was isolated from a marine sponge belonging to the genus  Melophlus  are promising sources of structurally unusual and diverse secondary metabolites. Meophlus spp. sponges have been reported to produce compounds primarily from three classes, specifically tetramic acid derivatives +); calcd. for C33H44ClN2O15, 743.2425; ∆ = 0.4 ppm, (−ESILCMS m/z 741.43 [M − H]−), which was consistent with the NMR data. The 1H NMR spectra (1 was nearly superimposable on aurantoside I +) and a fragment at m/z 627 corresponding to loss of 130 Da that was assigned to loss of 5-deoxy-2-O-methylarabinofuranose. Compound 1 (m/z = 743 [M + H]+) also showed a distinct fragment ion at m/z 627 however, the difference was 116 daltons, further indicating that the sugar unit which is cleaved off is indeed 14 amu smaller than the sugar III of aurantoside I (2). Moreover, the psuedomolecular ion of compound 1 at m/z 743 produced prominent fragment ions at m/z 627 [M + H − sugar III]+, 495 [M + H − sugar II + III]+ and at 363 [M + H − sugar I + sugar II + III]+, which were generated due to the cleavage of the three glycosidic bonds, confirming the trisaccharide series. Although the 1H NMR signals were complicated by broad and overlapping signals, two distinct anomeric protons were identified from the 1H–1H COSY spectrum (H 4.79 ppm) was assigned to H-1" of the arabinopyranose structure (sugar II) while the other anomeric proton H-1"', (δH 4.93 ppm) was assigned to the 2"'-des-O-methyl derivative of 5-deoxypentofuranose unit (sugar III) with the signal at δH 1.22 ppm due to the methyl group of sugar III. Absence of an O-methyl signal in the NMR spectra and an upfield shift of C-2"' (δC 67.6 ppm) were consistent with replacement of the 2"' OMe group by a hydroxyl group when compared to the known aurantosides 27D −127° : UV (MeOH) λmax (log ε) 434 (3.95), 265 (2.41) nm; NMR data, see m/z 743.2428 [M + H]+ .A. niger, Penicillium sp., R. sporangia and Sordaria sp. The plates were observed for zones of inhibition after 7–10 days of incubation at room temperature. The compound was also assayed against Cr. neoformans ATCC 32045 which was inoculated on PDA agar plates, and zones of inhibition were recorded after 24 h of incubation at 27 °C. In all cases, for the controls containing only the respective amount of solvent, no growth inhibition was observed. The procedure was the same as previously reported [Susceptibility discs (6 mm in diameter) were impregnated with 100 µg of the pure compound dissolved in MeOH and placed on potato dextrose agar (PDA) plates inoculated with the test fungal pathogens reported .P. falciparum parasites  were cultured in human O+ erythrocytes as previously described [1 was diluted in complete medium and 40 µL transferred to 96-well assay plates. To this 80 µL of complete media with 3D7 infected erythrocytes were dispensed in order to obtain a 2.5% hematocrit and 0.5% parasitemia in the assay. Uninfected erythrocytes were dispensed into the background wells at the same final hematocrit. Plates were incubated for 72 h in a low oxygen environment  in a modular incubation chamber. The plates were sealed and placed in a −80 °C freezer overnight then thawed, and 120 µL of lysis buffer  was dispensed into each well and incubated at 37 °C in the dark for 6 h. The plates were read with a Molecular Devices SpectraMAX Gemini EM at ex: 495 nm, em: 525 nm with 515 nm cut-off.Antimalarial activity was determined with a SYBR Green based parasite proliferation assay, adapted from Smilkstein  and Bennescribed . Compoun1 was evaluated against human colon cancer cell lines, HCT-116. In vitro cytotoxicity was tested with the -5-(3-carboxylmethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) MTS dye conversion assay as described previously [Compound eviously .C. albicans ATCC 32354 and amphotericin resistant C. albicans ATCC 90873, were inoculated separately in each well. The antifungal agent nystatin and the solvent MeOH were included in the bioassay as positive and negative controls, respectively. The treated cultures were incubated for 24 h at 37 °C. Replicates were maintained for each treatment. The MIC was defined as the lowest concentration of the purified compound/nystatin showing no visible growth after the incubation time. In the case of antibacterial assays were performed using methicillin-resistant S. aureus  and vancomycin-resistant E. faecium  as test pathogens. Vancomycin and chloramphenicol were used as positive controls for MRSA and VREF, respectively, and MeOH was used as negative control. The isolated pure compound was dissolved in MeOH at 10 mg/mL concentration and diluted further to give required concentrations (µg/mL) such as 500, 250, 125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.48, 0.24, 0.12, 0.06, 0.03 and 0.01. The diluted solutions (100 µL) were added to separate wells on a 96 wells plate. An inoculum of 100 µL from 24 h old culture of each test human pathogens, wild type 1) from a marine sponge Melophlus sp. Aurantoside K (1) was evaluated in different bioassays that displayed promising antifungal activity against a wide spectrum of fungal pathogens. However, it showed no appreciable activity against human cancer cell lines, the malaria parasite P. falciparum, or any bacteria tested in our assays.In conclusion, we described a new tetramic acid glycoside, aurantoside K (