O,O,O-orthoesters serve as bridgeheads. In contrast to their classic analogues, the compounds described herein are constitutionally dynamic in the presence of acid and can be induced to release their guest via irreversible deconstruction of the cage. These properties open up a wide range of application opportunities, from systems chemistry to molecular sensing and drug delivery.The discovery of coronands and cryptands, organic compounds that can accommodate metal ions in a preorganized two- or three-dimensional environment, was a milestone in supramolecular chemistry, leading to countless applications from organic synthesis to metallurgy and medicine. These compounds are typically prepared via multistep organic synthesis and one of their characteristic features is the high stability of their covalent framework. Here we report the use of a dynamic covalent exchange reaction for the one-pot template synthesis of a new class of coronates and cryptates, in which acid-labile  Cryptands and related molecules are macrocyclic polyethers capable of strongly binding cations. Here, the authors use orthoester exchange for the dynamic one-pot synthesis of crypates, which can bind cations and, given their constitutionally dynamic nature, can also be decomposed to release their guest. Past progress in supramolecular chemistry has been driven chiefly by the development of new macrocyclic molecules3In the last decade, rationally designed three-dimensional cage compounds679101112151618202122232426282930Here we describe how orthoester exchange, a previously ignored dynamic covalent reactionO,N,P-orthoesters)33O,O,O-orthoesters with simple alcohols from a DCC perspectiveInspired by reports on dynamic ‘scaffolding ligands' ((1) and diethylene glycol (2), with catalyst trifluoroacetic acid (TFA) and a stoichiometric metal template (1H nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry revealed that complex mixtures, containing the desired cryptate among other exchange products, had formed. Careful optimization of the reaction conditions phenyl]borate (BArF−))36373839Na+⊂o-Me2-1.1.1]BArF−  as the predominant reaction product .To test these hypotheses, we treated a chloroform solution of two bulk chemicals, trimethyl orthoacetate template . Analysi1H NMR spectra presented in 1 and 2 initially generates a remarkable diversity of different exchange products  and it products .1 can lead to the slow formation of methyl acetate (MeOAc) as a side product . This result indicates that there is a thermodynamic bias for the formation of the final cryptate in the presence of sodium template. Second, the fact that we observe only one type of ester (MeOAc) suggests that exchange products incorporating one or more diethylene glycol chains (generated much more rapidly than MeOAc) are kinetically stabilized against hydrolysis, presumably due to (chelate) binding of sodium.It should be noted that during the exchange process, water has to be excluded from the reaction mixture, which is not trivial to achieve, because even rigorously dried MS tend to slowly release residual water. As a consequence, hydrolysis of  product . From a  product . In addiNa+⊂o-Me2-1.1.1]BArF−. For example, when we mixed [Na+⊂o-Me2-1.1.1]BArF− with trimethyl orthoacetate (1) in water-saturated chloroform, only the simple orthoester 1 was found to hydrolyse, whereas cryptate [Na+⊂o-Me2-1.1.1]BArF− remained stable for 7 days Na+⊂o-Me2-1.1.1]BArF− hydrolyses readily in the presence of excess water and acid; This kinetic stabilization due to metal bindingr 7 days . In the r 7 days . These oNa+⊂o-Me2-1.1.1]BArF− by NMR spectroscopy and mass spectrometry Na+⊂o-Me2-1.1.1]BArF− in chloroform. As shown in +⊂2.2.1]BArF− and orthoester cryptand o-Me2-1.1.1, indicating that 2.2.1 has a significantly higher binding constant under these conditions. The reaction outcome also confirms that sodium can exit from the orthoester cage and the observed 1H NMR spectra demonstrate that in this experiment sodium ion exchange is slow between the two competing cryptands. Following such a titration, we treated a 1:1 mixture of [Na+⊂2.2.1]BArF− and o-Me2-1.1.1 with catalytic TFA, resulting in the complete conversion of o-Me2-1.1.1 into orthoester products featuring eight-membered rings Na+⊂o-Me2-1.1.1]BArF−. As evident from the series of 1H NMR spectra (o-Me2-1.1.1 is about one order of magnitude higher than that of 15-crown-5 (see Na+⊂o-Me2-1.1.1]BArF− and cryptand o-Me2-1.1.1 (vide infra for preparation method), we were able to determine an exchange rate of 0.6 s−1 for sodium exchange between degenerate orthoester cryptands (NMR exchange spectroscopy (EXSY); In a second competition experiment, we titrated weaker complexation agent 15-crown-5  having displaced two molecules of methanol (3). However, when such a dynamic mixture was treated with one equivalent of Sodium tetrakisborate (NaBArF), the dynamic system responded by forming cryptate [Na+⊂o-Me2-1.1.1]BArF− quantitatively and in a relatively short time 2-16-crown-6 had formed as a mixture of syn and anti diastereomers . Using mass spectrometry, 23Na and 7Li NMR spectroscopy and, most notably, atom absorption and emission spectroscopy, we were able to confirm that these cage compounds were indeed the sodium cryptates [Na+⊂o-Me2-1.1.1]X− , sodium (1.2 Å) and potassium (1.6 Å)To our initial surprise, the crown ethers originating from the lithium and potassium salts eventually transformed into the corresponding sodium cryptates [1.1.1]X− , and thaNa+⊂o-Et2-1.1.1]BArF− , could be prepared without difficulty, following a similar procedure to that used for cryptate [Na+⊂o-Me2-1.1.1]BArF−. Using trimethyl orthoformate as the starting material  led to the formation of remarkably stable and pure crown ether complexes [Na+⊂o-H2-(OMe)2-16-crown-6], which did not further react to the corresponding cryptates under our standard conditions. A notable limitation of the self-assembly reaction concerns the counteranion of the sodium template. Thus far, we were able to prepare cryptate [Na+⊂o-Me2-1.1.1]X− with three different tetraarylborate anions borate), while simpler anions such as PF6− or BF4− have failed, suggesting that a truly ‘non-coordinating'We conducted preliminary studies on the scope of the described orthoester cryptates and coronates. A cryptate derived from orthoester trimethyl orthopropanoate, [Na+⊂o-Me2-1.1.1]BArF− to chloride, we discovered a convenient method for preparing cryptand o-Me2-1.1.1 (Na+⊂o-Me2-1.1.1]BArF− in CDCl3 was treated with anion exchange resin Lewatit MP-64 (Cl form), we observed the clean formation of o-Me2-1.1.1, for which the precipitation of NaCl is presumably the driving force. With pristine o-Me2-1.1.1 in our hands, we were able to prepare cryptate [Li+⊂o-Me2-1.1.1]TPFPB− by exposing the cryptand to a solution of the lithium salt  appears to not enter the cage, but presumably ‘nests' on the crown-ether-type faces of the cryptand. Further experiments to such ends are ongoing in our laboratory.In an attempt to exchange the counteranion in pristine BArF− was found to be surprisingly stable against water in neutral solution, but susceptible to hydrolysis in the presence of water and acid. We believe that this property will make orthoester cages useful for the traceless delivery of cations into biological systemskobs=0.6 s−1) and the binding constant for Na+ lies between the classic complexation agents 15-crown-5 and 2.2.1. Besides their interesting structural and dynamic properties, orthoester cryptates offer preparative advantages over their classic analogues: their synthesis relies on a one-pot, dynamic covalent ring-closing reaction, and substituted cages o-R2-1.1.1 are accessible simply by using different orthoesters as starting materials. We are currently working towards further diversifying the target structures, as well as increasing the dynamic system's complexity .We have shown that a dynamic system based on two strikingly simple organic starting materials converges to three distinct types of exchange products under the influence of dry MS: (i) in the absence of a template, a simple exchange product featuring an eight-membered ring is formed; (ii) in the presence of a sodium template, an unprecedented dynamic orthoester cryptate is formed, in which nine oxygen donors are bound to the metal ion; (iii) 3 and the mixture was dried over MS  for 3 days. TFA  was dissolved in CDCl3 .NaBArF  and diethylene glycol  were dissolved in 14 ml CDCl1H NMR spectroscopy. After 5 days, the solvent was removed under reduced pressure and [Na+⊂o-Me2-1.1.1]BArF− was obtained as a colourless solid (67% yield). Characterization data: M.p. 124 °C –128 °C. 1H NMR : δ=7.68 , 7.51 , 3.79–3.77 , 3.50–3.48 , 1.43 p.p.m. . 13C NMR : δ=162.8, 162.3, 161.8, 161.3, 135.1, 129.7, 129.4, 129.1, 128.9, 128.8, 126.2, 123.5, 120.8, 117.7, 113.0, 69.2, 62.0, 17.7 p.p.m. 11B NMR : δ=−6.7 p.p.m. 19F NMR : δ=−62.1 p.p.m. 23Na NMR : δ=−5.9 p.p.m. HRMS (ESI+): m/z=389.1794 [M+Na]+ . For further experimental details and characterization data, see To 6 ml of stock solution were added fresh MS  and the reaction mixture was left to stand at room temperature. After 16 h, 1 mol% TFA (10 μl) was added from stock solution, the mixture was shaken and trimethyl orthoacetate  was added. Every 24 h, 1 mol% TFA was added to keep the exchange reaction active . The reaction progress was monitored regularly by −2 mbar). All solvents were dried over MS for at least 24 h. All orthoester exchange reactions  were carried out under nitrogen. After the acid was quenched , most orthoester complexes described herein were found to be unusually stable against water and could be handled on the benchtop without further precautions , under CCDC number 1038394. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.How to cite this article: Brachvogel, R.-C. et al. Self-assembly of dynamic orthoester cryptates. Nat. Commun. 6:7129 doi: 10.1038/ncomms8129 (2015).Supplementary Figures 1-47, Supplementary Tables 1-2, Supplementary Note 1, Supplementary Methods and Supplementary ReferencesCrystallographic Information File for


To maximize power adaptations, resistance training (RT) should be performed at maximal power output. In sports where more than one training bout is necessary in a day, subsequent RT may be limited by muscle glycogen, resulting in lower power output. High molecular weight (HMW) carbohydrate (CHO) solutions have been shown to result in greater glycogen re-synthesis rates, and greater work output during a subsequent cycling time trial compared to a low molecular weight (LMW) CHO solution. However, the effect of a HMW CHO on RT power output following exhaustive exercise is unknown.2 max (37.4 ± 4.3 ml·kg·min-1) were initially assessed in order to prescribe exercise intensities during experimental trials. In a double-blind, placebo-controlled, randomized cross over design consisting of three testing sessions separated by one week, subjects completed a glycogen depleting exercise bout on a cycle ergometer. Immediately post-exercise, subjects ingested a placebo (PLA), or a LMW or HMW CHO solution (10%) providing 1.2 kg· bw-1 CHO, assigned randomly. Two hours post-ingestion, subjects performed 5 sets of 10 repetitions back squat (75% 1RM) "as explosively as possible". If subjects paused for more than 2 seconds or were unable to complete a rep, resistance was lowered by 13.6 kg. Kinematic and kinetic measurements were sampled at 1000 Hz via force plate and two linear position transducers.Sixteen resistance trained men  participated in this study. One-repetition maximum (1RM) back squat , and VO-1; Set 5, 0.62 ± 0.03 m·s-1) compared to PLA , but not LMW , with no difference between LMW and PLA . HMW conferred a likely beneficial effect in Sets 4 and 5 , compared to PLA; while ingestion of LMW conferred only a possibly beneficial effect (68.7%) and likely beneficial effect (83.9%) in Sets 4 and 5, respectively.Average power following ingestion did not differ between CHO solutions until Set 4 (p = 0.108) and Set 5 (p = 0.083). Average power collapsed across the latter Sets was greater following ingestion of the HMW solution  compared to PLA , but not compared to ingestion of LMW . No difference was observed between LMW and PLA . The difference in average power was driven by velocity, as similar trends were observed in Set 4 and 5 . Average velocity was higher following ingestion of HMW (Set 4, 0.63 ± 0.03 m·s-1 CHO may allow athletes to sustain power output in a subsequent resistance training session when time between training sessions is limited.These data suggest post-exercise ingestion of a HMW CHO solution providing 1.2 kg· bw


Mitochondria have recently emerged as novel targets for cancer therapy due to its important roles in fundamental cellular function. Discovery of new chemotherapeutic agents that allow for simultaneous treatment and visualization of cancer is urgent. Herein, we demonstrate a novel bifunctional mitochondria-targeted anticancer agent (FPB), exhibiting both imaging capability and anticancer activity. It can selectively accumulate in mitochondria and induce cell apoptosis. Notably, it results in much higher toxicity toward cancer cells owing to much higher uptake by cancer cells. These features make it highly attractive in cancer imaging and treatment. ΔΨm)7ΔΨm between carcinoma and normal cells is sufficient to account for 10-fold greater accumulation of DLCs in mitochondria910111314151618Mitochondria play a crucial role in energy producing, programmed cell death regulation, reactive oxygen species (ROS) generation and calcium homeostasis maintenance. Mitochondrial targeted drugs, namely mitocansCurrently, extensive research has focused on the discovery of new multifunctional anticancer agents which allows for simultaneous monitoring, treatment and direct visualization of cancer202224FPB, by conjugating F16, a DLC compound, and boron-dipyrromethene (BODIPY), a widely used fluorescent dye, with a phenylethynyl linker 4, THF and triethylamine gives C3 in 90% yield. BODIPY-I is synthesized according to our previous work3)4, THF and triethylamine affords C4 in 33% yield. Methylation of C4 with iodomethane affords FPB in 88% yield. For comparison, C3 is also methylated to get F16-Ph-Ace as a reference compound -2,5-diphenyltetrazolium bromide] method. Three normal cell lines, human gastric mucosal epithelial cell lines (GES-1), human embryonic kidney 293 cell lines (HEK293) and mouse fibroblast cell lines (NIH/3T3), and three cancer cell lines, gastric cancer cell lines (SGC-7901), pulmonary adenocarcinoma cell lines (A549) and human breast adenocarcinoma cell lines (MCF-7) were used as target cells. The cytotoxicity data of FPB toward the six different cells are provided in IC50 values were calculated as shown in FPB shows different toxic effects towards carcinoma and normal cell lines. All carcinoma cell lines  are sensitive to FPB with the IC50 in the range of 2.49 μM to 6.20 μM, while two normal cell lines (GES-1 and HEK293) are less sensitive to FPB with the IC50 of 16.4 μM and 12.2 μM. Especially for SGC-7901 and GES-1, the human gastric carcinoma cells and corresponding normal cells, the selective factor (ratio of IC50) is about 6.58. GES-1 and HEK293 cells are not strictly normal cells as they are transformed with SV40 or Ad5 virus, so we further tested the toxicity of FPB on the normal fibroblast cells. The results indicated that the toxicity of FPB on 3T3 cells is lower compared with GES-1 and HEK293. The phase contrast images of SGC-7901 cells were also taken before and after incubating with 5 μM FPB for 48 h. As shown in FPB were obviously smaller than that without FPB. The reduced cell size and abnormal cellular morphology indicated that the cells were dead or in the late stage of apoptosis. This result is consistent with the MTT experiment. These results demonstrate that FPB can act as an anticancer agent of broad spectrum and exhibits good selectivity to cancerous cell lines over normal cell lines.The cytotoxicity of IC50 = 15.1 μM) and BODIPY-I exhibits no obvious toxicity on SGC-7901 cell lines (IC50 > 100 μM). Surprisingly, conjugation of F16-Ph-Ace and nontoxic BODIPY-I exhibits better anticancer activity efficiency . The results suggest that the BODIPY part of FPB might play a specific role for its toxicity.We further investigated the cytotoxicity of BODIPY-I and F16-Ph-Ace for a structure-activity relationship study. As shown in FPB on cell proliferation were measured by MTT cell proliferation assay using SGC-7901 cells and 3T3 cells. The effect of F16 was also studied for comparison. As shown in FPB or F16 upon 5 days of exposure while 3T3 cells proliferation was not affected under the same conditions. Compared with F16, FPB shows a little weaker efficiency on inhibition of cancer cell proliferation at low concentration. At a concentration of 2 μM, the difference is approximately 10%. Based on these results, we can conclude that FPB has a similar selectivity and anti-proliferative activity as F16.Information on cell proliferation is one of the most the important indicators in the evaluation of drug effect and physiological research. The MTT cell proliferation assay has already been demonstrated to be a rapid, versatile, quantitative, and highly reproducible colorimetric assay for determining viable cell number in proliferation28FPB in SGC-7901 cells, we performed a colocalization experiment using Nikon A1 Confocal laser scanning microscope. The cells were first incubated with FPB in the culture medium for 24 h, and then stained with Mito Tracker Red, a commercial mitochondrial dye. FPB display intriguing fluorescent property in SGC-7901 cells and could be directly visualized in living cells. From a contrasting merged image of FPB and Mito Tracker Red staining, we can observe that the FPB fluorescence perfectly overlapped with the mitochondria staining  and the fluorescence quantum yield is 20.2% (using fluorescein in ethanol as the reference). The remarkable optical properties allowed the direct visualization of FPB in cells. The results strongly demonstrate the excellent mitochondrial targeting and subcellular imaging capability of FPB in living carcinoma cells.To access the intracellular distribution of staining , which isolution . FPB exhFPB for SGC-7901 and GES-1 cells with flow cytometry. Cultured cell lines were incubated with 0.5 μM FPB for 24 h, and then we studied the uptake content of FPB by its intrinsic fluorescence. As shown in FPB uptake capability of SGC-7901 cells is significantly greater than that of GES-1 cells. These results suggest that FPB could selectively accumulate in cancer cells. The preferential accumulation of FPB in cancer cell lines accounted for higher toxicity to cancer cells than to normal cells.We then investigated the cellular uptake of FPB, we performed apoptosis assay of SGC-7901 cells after incubation with FPB for 48 h by flow cytometry. Cells were stained with propidium (PI) dye for nucleic acid staining. Because the apoptotic cells are characterized by DNA fragmentation at the internucleosomal section and consequently loss of nuclear DNA content, they display a broad hypodiploid (sub-G1) peak after stained with PI and analysed with flow cytometerFPB, the percentage of apoptotic cells is over 50%  which accumulates in the mitochondrial matrix in proportion to ΔΨmFPB on SGC-7901 cells after incubation for 48 h is shown in FPB could induce dissipation of ΔΨm in a dose-dependent manner. As apoptosis is often accompanied by a decrease in ΔΨm, this result further confirms that FPB could induce cell apoptosis.Next, we analysed physiological alterations of mitochondria in cancer cells treated by 2O2) and superoxide anion (O2−), are generated as a by-product of respiration and play an important role as a messenger in cellular signal transduction. ROS are expected to be more abundant during apoptosis and in response to mitochondrial dysfunctionFPB treated cancer cells using a fluorescent probe, dihydroethidium (DHE). The non-fluorescent DHE could penetrate cells freely and interact with O2− to form the membrane-impermeant ethidium cation, which becomes fluorescent upon intercalating DNA, thus the fluorescence intensity of ethidium-DNA can indicate the level of intracellular ROSFPB in SGC-7901 cells. The significant increase in O2− level with FPB treatment indicated that the mitochondria in cancer cells are damaged or in serious disorders.Reactive oxygen species (ROS), such as hydrogen peroxide : δ = 7.03–7.19 , 7.42–7.52 , 7.69–7.74 , 8.03 , 8.45 , 11.50 ; HRMS: Exact mass calculated for C15H12N2 (M+) 221.1073; Found: 221.1079.A mixture of pyridnecarboxaldehyde , N-butyl phosphorus , gramine , and 5.0 mL acetonitrile was stirred at 60 °C for 24 h. The mixture was concentrated under vacuum, and the oil product was purified by silica chromatography with hexanes/EtOAc (1:1 V/V) to give C1 . 2CO3 , CuI , piperidine-2-carboxylic acid  in dry DMF (5 mL) and stirred at 110 °C for 24 h. The product was separated by silica chromatography with hexanes/EtOAc (1:1 V/V) and recrystallized with EtOAc/H2O to give C2 . C2 is a yellow powder and soluble in water. 1H NMR : δ = 7.15–7.28 , 7.42–7.62 , 7.72–7.78 , 7.95 , 8.05 , 8.15 , 8.50 ; HRMS: Exact mass calculated for C21H15BrN2 (M+) 337.0473; Found: 337.0470.The 1-bromo-4-iodobenzene  was added to a suspension of C1 , K3)4  and CuI  was added to 6 mL THF/Et3N  and stirred at 45 °C overnight. The product was purified by silica chromatography with hexanes/EtOAc (1:1 V/V) to give yellow oily C3 . 1H NMR : δ = 4.31 , 7.15–7.25 , 7.55–7.78 , 8.10–8.14 , 8.50 , 8.15 , 8.50 ; HRMS: Exact mass calculated for C23H16N2 (M+) 321.1386; Found: 321.1390.A mixture of C2 , trimethyl silyl acetylene , Pd was added. After stirring for 30 min, triethylamine (20.0 mL) and BF3•OEt2 (20.0 mL) were added and stirred overnight. The organic product was purified by column chromatography on silica gel using hexane/DCM  to obtain an orange crystalline solid . 1H NMR : δ = 1.41 , 2.25 , 5.99 , 7.04 , 7.84 .p-Iodobenzaldehyde , and 2,4-dimethylpyrrole  were dissolved in 250 mL of dry CH1H NMR : δ = 1.42 , 2.47 , 6.22 , 7.23–7.35 , 7.48 , 7.59 , 7.68–7.86 , 8.18 , 8.33 , 8.52 .C4 was prepared from C3  and BODIPY-I  as the previous synthesis procedure for C3 in 33% yield (65 mg). 3I (10 μL) were added to 3 mL dry DMF at room temperature and stirred overnight. DMF was removed and the residues were recrystallized in CHCl3 and n-hexane to obtain C5 . 1H NMR : δ = 1.43 , 2.48 , 4.23 , 6.23 , 7.41 , 7.57–7.45 , 7.71 , 7.80 , 7.88 , 8.21 , 8.35–8.26 , 8.36 , 8.78 ; HRMS: Exact mass calculated for C42H36BF2N4+(M+) 657.3003; Found: 657.3068.C4  and CH3I (10 μL) as the previous synthesis procedure for C5 with a yield of 92% (68 mg). 1H NMR : δ = 4.22 , 4.38 , 7.37–7.50 , 7.64–7.74 , 8.18–8.30 , 8.76 ; HRMS: Exact mass calculated for C24H19N2 (M+) 335.1542; Found: 335.1549.F16-Ph-Ace was prepared from C3  and CHΦF) were measured according to the equation:ŋ are the integrated fluorescence area , the absorbance at the excitation wavelength (501 nm), and the relative index of the solvent, respectively. Fluorescein in ethanol was used as the reference with a value for ΦF = 0.92Fluorescence quantum yields  were added to each well followed by incubation for 4 h. The supernatant was abandoned and 150 μL DMSO was added into each well. The plate was then incubated at 40 °C and shaken for 10 min. The absorbance value at 570 nm at each well was recorded by a microplate reader. The cell viability rate (VR) was calculated according to the following equation:FPB drug. The survival curves plotted as a function of concentration of FPB and cell viability rate (%). The IC50 values were determined as the concentration that cause 50% inhibition of cell viability and were analyzed by the statistic software Statistical Product and Service Solutions (SPSS Version 13.0).Six kinds of cells were cultured in DMEM medium containing 10% fetal bovine serum  in a humidified atmosphere containing 5% COFPB (5 μM) for 48 h. Phase contrast bright-field images were taken using fluorescence microscopy.The SGC-7901 cells suspension were plated in 35 mm dishes and incubated in 1 mL DMEM medium for 24 h. The cells were washed once with PBS and then grown in 1 mL DMEM medium with and without FPB or F16 was added to the cells and incubated for 5 days. The ability of viable cells was determined by MTT method as described above.The SGC-7901 cells and NIH/3T3 cells were seeded onto 96-well plates and incubated for 24 h. On the following day, different concentration of FPB (0.5 μM) for 24 h. Mitotracker Red (0.5 μM) was used to stain the cells for 15 min. The cells were then examined by confocal laser scanning microscopy.SGC-7901 cells were seeded onto 35 mm dishes and incubated for 24 h for attachment. Cells were then treated with FPB (0.5 μM) for 24 h, the cells were washed 3 times with PBS, digested with trypsin, resuspended in PBS and then analyzed with BD ACCURI C6 flow cytometer by detecting the fluorescence of FPB.SGC-7901 cells and GES-1 cells suspension were seeded onto 6-well plates and adhered to the walls overnight. After incubation with FPB  for 48 h. Then the cells were harvested, centrifuged, and suspended in PBS, and fixed by 70% (v/v) cold ethanol solution at 4 °C overnight. Then the cells supernatant (ethanol solution) was removed. The cells were washed with PBS and then resuspended in DNA staining solution . After incubating with PI solution for 30 min at 4 °C, the cells were analyzed with BD ACCURI C6 flow cytometer to record red fluorescence of PI.SGC-7901 cells were incubated in DMEM medium with FPB treated SGC-7901 cells was obtained by flow cytometry, using the potentiometric probe tetramethylrhodamine methyl ester (TMRM). SGC-7901 cells were grown with FPB  for 48 h and resuspended in PBS. TMRM were added to a final concentration of 200 nM. After incubated for 20 min, the cells were analysed by flow cytometry.Changes in mitochondrial membrane potential of FPB  for 48 h and resuspended. To measure superoxide levels, the cells were incubated with 4 μM dihydroethidium for 15 min at room temperature, then immediately assessed by flow cytometry.SGC-7901 cells were grown with How to cite this article: He, H. et al. A novel bifunctional mitochondria-targeted anticancer agent with high selectivity for cancer cells. Sci. Rep.5, 13543; doi: 10.1038/srep13543 (2015).


IAV possessing a monobasic cleavage site in the haemagglutinin (HA) protein replicates poorly in TMPRSS2 knockout mice owing to insufficient HA cleavage. In the present study, human isolates of influenza B virus (IBV) strains and a mouse-adapted IBV strain were analysed. The data showed that IBV successfully underwent HA cleavage in TMPRSS2 knockout mice, and that the mouse-adapted strain was fully pathogenic to these mice. The present data demonstrate a clear difference between IAV and IBV in their molecular mechanisms for spreading in vivo.Influenza A and B viruses show clear differences in their host specificity and pandemic potential. Recent studies have revealed that the host protease TMPRSS2 plays an essential role for proteolytic activation of H1, H3, and H7 subtype strains of influenza A virus (IAV)  Orthomyxoviridae, as influenza A virus (IAV)1242Influenza B virus (IBV) is classified into the same family, TMPRSS2 gene knockout (TMPRSS2 KO) mice have revealed that IAV strains possessing a monobasic cleavage site in HA primarily undergo proteolytic activation by TMPRSS2, a type II transmembrane serine protease (TTSP), in vivo8989in vivo replication of IBV was examined.The HA proteins of both IAV and IBV are synthesized as inactive precursors, and their proteolytic cleavage is a prerequisite for the HA conformational changes essential for virus infectivity. Recent studies using 1 and HA2) were clearly detected in HeLa/mTM2 and HeLa/hTM2 cells, but not in the parental HeLa cells, infected with either B/Aichi/99[V] or MA-B/Ibaraki/85[V] (Human TMPRSS2 (hTMPRSS2) has been shown to cleave IBV HAki/85[V] . The cleki/85[V] . These dP > 0.05), with only the data for the lung homogenates of B/Yamagata/88[Y] and B/Shiga/98[Y] at 2 days p.i. showing significant differences (P < 0.05) . When challenged with B/Yamagata/88[Y] strain, both WT and TMPRSS2 KO mice showed very mild body weight loss . Neither < 0.05) . Histopa < 0.05) . The imm < 0.05) . Althoug < 0.05) . Most imP < 0.05). The extent of activation in vivo of the progeny viruses was analysed. Even in KO mice, the infectivity titres were not restored significantly by in vitro trypsin treatment  were analysed by SDS-PAGE and immunoblotting. Generally, the HA signal intensities were greater in WT mice than in KO mice (1 and HA2) in both WT and KO mice (0 and the cleaved HA subunits (HA1 and HA2) were similar in WT and TMPRSS2 KO mice  and have a complete C57BL/6 genetic background. WT C57BL/6 mice were purchased from Japan SLC.TMPRSS2 KO mice were reported previouslyFour IBV human isolates, two from the Victoria lineage, B/Aichi20/99 (B/Aichi/99[V]) and B/Ibaraki/2/85 (B/Ibaraki/85[V]), and two from the Yamagata lineage, B/Yamagata/16/88 (B/Yamagata/88[Y]) and B/Shiga/T30/98 (B/Shiga/98[Y]), and a mouse-adapted (MA)-B/Ibaraki/85[V], were used. The human strains were isolated in MDCK cells or embryonated eggs. The adaptation process of the MA-B/Ibaraki/85[V] strain was reported previously3, 1.6 × 104, and 1.3 × 104 PFU of B/Aichi/99[V], B/Yamagata/88[Y], and B/Shiga/98[Y], respectively. For mock infection, the same volume (20 μL) of PBS was used. The body weight and clinical signs were monitored daily. For histopathological analysis, WT and TMPRSS2 KO mice were euthanized and autopsied at 2 days p.i. (n = 3) or 6 days p.i. (n = 3). The viral antigens of IBV were detected using a mouse antiserum against IBV NP (Lot. N179). This antiserum was obtained from BALB/c mice immunized intraperitoneally with inactivated IBV virions (Victoria lineage) using the Sigma adjuvant system (Sigma-Aldrich). The inflammation levels in individual mice were scored as follows: 0, no apparent changes; 1, minimal changes or bronchiolitis; 2, bronchiolitis and/or slight alveolitis; 3, mild alveolitis with neutrophils, monocytes/macrophages, or lymphocytes; 4, moderate alveolitis. The cut-off in body weight loss for euthanasia was 25%. Lung lavage fluids and lung homogenates were collected at 2, 4, and 6 days p.i. and subjected to a standard plaque assay.WT and TMPRSS2 KO mice (6–7-week-old) were infected intranasally with 4.6 × 103, 2.0 × 104, and 2.0 × 105 PFU of MA-B/Ibaraki/85[V]. These doses of MA-B/Ibaraki/85[V] correspond to 30, 300, and 3,000 mouse lethal dose 50 (MLD50) for WT mice. The body weight and clinical signs were monitored daily. For histopathological analysis, similar mice were also infected intranasally with 6.7 × 103 PFU of MA-B/Ibaraki/85[V] and autopsied at 2 days p.i. (n = 3) or 6 days p.i. (n = 3).WT and TMPRSS2 KO mice (6–7-week-old) were infected intranasally with 2.0 × 10n = 3) were infected with 2.0 × 103 PFU of MA-B/Ibaraki/85[V] and lung lavage fluids were collected at 2, 4, and 6 days p.i. Monolayers of MDCK cells were infected with serially diluted lung lavage fluid and lung homogenate samples for 1 hour at 4 °C, washed twice with PBS, overlaid with MEM/1% agarose, and incubated for 24 hours at 37 °C to allow the viruses to enter the cells. Trypsin was omitted to avoid HA cleavage before virus entry. At 24 hours p.i., the cell monolayers were additionally overlaid with MEM/1% agarose supplemented with 4.0 μg/mL of trypsin to allow plaque formation. To determine the infectious titres including viruses that had not been activated in vivo but possessed an infectious potential, virus samples were treated with 2.0 μg/mL of trypsin, and subjected to a standard plaque assay.WT and TMPRSS2 KO mice (3 PFU of MA-B/Ibaraki/85[V] (n = 3) or mock-infected (n = 1). Lung lavage fluids were collected at 2 days p.i. HeLa/mTMPRSS2, HeLa/hTMPRSS2, and parental HeLa cells were infected with B/Yamagata/88[Y], B/Shiga/98[Y], MA-B/Ibaraki/85[V], and parental B/Ibaraki/85[V] at a multiplicity of infection (MOI) of 1.0, and collected at 12 and 24 hours p.i. The lung lavage fluids and cells were lysed with lysis buffer to make a final solution containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 4 mM EDTA, 0.1% sodium deoxycholate, 1% Nonidet P-40, and 0.1% SDS. The polypeptides were separated by SDS-PAGE in 10–20% polyacrylamide gels , and blotted onto PVDF membranes . Rabbit antisera raised against IBV HA  and NP  were used for detection of HA and NP, respectively.WT and TMPRSS2 KO mice were infected with 6.7 × 10How to cite this article: Sakai, K. et al. TMPRSS2 Independency for Haemagglutinin Cleavage In Vivo Differentiates Influenza B Virus from Influenza A Virus. Sci. Rep.6, 29430; doi: 10.1038/srep29430 (2016).


Genome analyses reveal that one of the human isolates contains both Shiga toxin-encoding genes ( E. coli (STEC) species are important emerging pathogens due to the rise of STEC-related illnesses worldwide  in humans and has been implicated in outbreaks . Previou5–de novo assembly of the contigs. The assembly results for each isolate are as follows: H1, 292 contigs, average coverage of 23×, contig N50 of 40,255 bp, and genome size of 5,068,406 bp; H2, 133 contigs, average coverage of 32×, contig N50 of 140,146 bp, and genome size of 5,039,473 bp; P, 99 contigs, average coverage of 36×, contig N50 of 166,484 bp, and genome size of 4,982,843 bp. The G+C contents for isolates range from 46.6% to 51.5%.Genomic DNA was extracted from overnight culture using a DNAeasy blood and tissue kit . The genomes were sequenced on the Ion PGM (Thermofisher), and MIRA (v4.0.5)  was usedstx1 and stx2, whereas H2 and P only have stx2. H1 and H2 contain hemolysin gene, hlyA. All three isolates contain the antibiotic resistance gene, ampC. It is widely documented that this serogroup does not harbor intimin (eae)  and 98.1% similarity to an enterotoxigenic E. coli strain E24377A (CU928145). Comparative analysis of whole genome sequences from these three isolates could provide insights into attributes of potentially re-emerging STEC strains and further reveal the influence of genomic differences on virulence. Observations from the comparison will be the focus of a future publication.The contigs were annotated using the CLC Genomic WorkBench Genome Finishing Module 1.3 . H1 contains Shiga-toxin encoding genes, in (eae) –16, and JNNI00000000 (H1), JNNJ00000000 (H2), and JNNL00000000 (P).GenBank accession numbers for the sequences are


Its radiochemical characteristics, biodistribution, and SPECT imaging were evaluated in nude mice bearing human HepG2 hepatoma. Nude mice bearing HepG2 were randomly divided into 5 groups with 5 mice in each group and injected with ~7.4 MBq 99mTc-NGR. The SPECT images were acquired in 1, 4, 8, and 12 h postinjection via caudal vein. The metabolism of tracers was determined in major organs at different time points, which demonstrated rapid, significant tumor uptake and slow tumor washout. The control group mice were blocked by coinjecting unlabelled NGR (20 mg/kg). Tumor uptake was (2.52 ± 0.83%) ID/g at 1 h, with the highest uptake of (3.26 ± 0.63%) ID/g at 8 h. In comparison, the uptake of the blocked control group was (1.65 ± 0.61%) ID/g at 1 h after injection. The SPECT static images and the tumor/muscle (T/NT) value were obtained. The highest T/NT value was 7.58 ± 1.92 at 8 h. The xenografted tumor became visible at 1 h and the clearest image of the tumor was observed at 8 h. In conclusion, 99mTc-NGR can be efficiently prepared and it exhibited good properties for the potential SPECT imaging agent of tumor.A peptide containing Asn-Gly-Arg(NGR) sequence was synthesized and directly labeled with  Meanwhile, CD13 receptor mediated binding to tumor vasculature is specific but not to the other CD13 rich tissues, which was proved by in vivo studies +). After purification, the specific activity of 99mTc-labeled tracers was determined to be about 3.08~6.17 MBq/nmol. The labeling yield of the product was 95 ± 0.35% and the radiochemical purity was greater than 98%. The in vitro stability of 99mTc-NGR in PBS (pH 7.4) at 37°C was shown in 99mTc-NGR peptide remained intact in mice serum.NGR peptide was well prepared . The ana99mTc-NGR bound to HepG2 tumor cells directly. During the first 15 min, about 0.49 ± 0.05% of 99mTc-NGR uptake in HepG2 cells were determined. After 2 h incubation, the peptide uptake in HepG2 cells reached the maximum of 1.52 ± 0.13%  in 99mTc-NGR, the tumor uptake was determined to be 2.52 ± 0.83, 3.03 ± 0.71, 3.26 ± 0.63, and 2.81 ± 0.25% ID/g at 1, 4, 8, and 12 h, respectively. 99mTc-NGR exhibited 7.93 ± 2.13% ID/g kidney uptake and 4.07 ± 0.76% ID/g liver uptake at 1 h pi. The nonspecific uptake in the muscle was at a very low level. 99mTc-NGR exhibited high tumor uptake at the early time point (Tissue distribution data for me point , indicat99mTc-NGR nonblocking group, 4.07 ± 0.76% ID/g in liver and 7.93 ± 2.13% ID/g in kidney were decreased to 3.27 ± 0.16% ID/g and 5.03 ± 0.97% ID/g by blocking, respectively.A decrease of radioactivity was observed in all dissected tissues and organs similar to SPECT imaging results in blocking group , with thIn this study, we synthesized a novel NGR peptide and investigated its biological targeting specificity, which turned out to be a promising tumor molecular imaging probe for clinical practice.99mTc has favorable chemical and physical properties and can be produced from the generator directly [99mTc-labeled tracer also showed good stability (directly . CD13 retability  and affitability . The resThe development of radiolabeled peptides for diagnostic and therapeutic applications has expanded exponentially in the last decades , 13, 14.99mTc-NGR in nude mice bearing HepG2 hepatoma showed notable uptake in tumor and dominant renal and hepatic clearance. But the unspecific binding on the other tissues was decreasing and the tumor to nontumor ratio was consequently increasing. The receptor specificity of 99mTc-NGR was further confirmed by effective inhibition of tumor uptake in the presence of excess nonlabeled NGR peptide in biodistribution study (99mTc-NGR uptake in liver and gastrointestinal tract was lower than previous results, the practice in detecting tumor and metastases in the abdominal area is inapplicable. Since the excretion of the probe was mainly renal, fast blood depuration should be another favorable feature.SPECT scans of on study . Althoug99mTc-NGR was a promising synthetic strategy for SPECT imaging in terms of in vitro and in vivo properties. Our future work will continually focus on more optimal approach to reduce liver and gastrointestinal tract uptake by modifying the peptide structure and keep the specificity binding. Additionally, a thorough comparison between various NGR peptides is warranted to screen the better radiotracers.In brief, our data demonstrated that synthesizing novel 99mTc for SPECT imaging of tumor CD13 receptor. 99mTc-NGR exhibited good properties in terms of binding affinity, cellular uptake, tumor uptake and retention, and pharmacokinetics. 99mTc-NGR peptide is a potential SPECT agent for imaging and early diagnosis of tumor.NGR peptide was successfully labeled with the generator-produced


Eriosema laurentii De Wild. (Leguminosae) is a plant used in Cameroon against infertility and gynecological or menopausal complaints. In our previous report, a methanol extract of its aerial parts was shown to exhibit estrogenic and aryl hydrocarbon receptor agonistic activities in vitro and to prevent menopausal symptoms in ovariectomized Wistar rats.In order to determine the major estrogen receptor α (ERα) agonists in the extract, an activity-guided fractionation was performed using the ERα yeast screen. To check whether the ERα active fractions/compounds also accounted for the aryl hydrocarbon receptor (AhR) agonistic activity of the crude methanol extract, they were further tested on the AhR yeast screen.This study led to the identification of 2′-hydroxygenistein, lupinalbin A and genistein as major estrogenic principles of the extract. 2′-hydroxygenistein and lupinalbin A were, for the first time, also shown to possess an AhR agonistic activity, whereas genistein was not active in this assay. In addition, it was possible to deduce structure-activity relationships.Eriosema laurentii.These results suggest that the identified compounds are the major active principles responsible for the estrogenic and AhR agonistic activities of the crude methanol extract of the aerial parts of  Glycine max L.) and red clover (Trifolium pratense L.) on menopause-related health problems have frequently been ascribed to their high isoflavone content +; +ESIMS2 (287.0 →) m/z 271.0 (8), 268.9 (8), 259.0 (27), 244.9 (35), 230.9 (19), 216.9 (100), 202.9 (11), 189.0 (16), 175.0 (34), 161.0 (23), 153.0 (95), 149.0 (34), 137.0 (11); -ESIMS m/z 284.9 [M-H]-; -ESIMS2 (284.9 →) m/z 240.9 (6), 216.9 (100), 198.9 (8), 174.9 (6).Lupinalbin A: +ESIMS m/z 285.0 [M + H]+; +ESIMS2 (285.0 →) m/z 256.9 (100), 228.9 (23), 212.9 (10), 185.0 (12), 173.0 (6); +ESIMS3 (285.0 → 256.9 →) m/z 228.9 (100), 173.2 (7); -ESIMS m/z 282.9 [M-H]-; -ESIMS2 (282.9 →) m/z 265.8 (8), 264.8 (17), 254.9 (100), 238.9 (24), 236.9 (7), 226.9 (10), 214.9 (8), 210.9 (8), 172.9 (7); -ESIMS3 (282.9 → 254.9 →) m/z 236.8 (6), 226.9 (100), 210.9 (14), 182.9 (10).Genistein: +ESIMS m/z 271.0 [M + H]+; +ESIMS2 (271.0 →) m/z 253.0 (9), 242.9 (12), 215.0 (25), 153.0 (100), 149.0 (11), 147.1 (37), 145.0 (8).The chemical structures of these 3 major compounds are depicted in Figure For MS identification of other compounds see Table 2HPO4 · 2H2O), potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO4 · 7H2O), sodium carbonate (Na2CO3) and ammonium sulfate ((NH4)2SO4) were obtained from Merck . Sodium dihydrogen phosphate dihydrate (NaH2PO4 · 2H2O), N-lauroylsarcosine (Sarkosyl), o-nitrophenol-β-galactopyranoside (ONPG), DL-dithiothreitol (DTT), dimethylsulfoxide (DMSO), D-(+) glucose, estradiol, 5α-dihydroxytestosterone, genistein, progesterone, and β-naphthoflavone were purchased from Sigma-Aldrich . Yeast nitrogen base was obtained from Difco , amino acids from Serva Feinbiochemica , dropout medium without tryptophan (DO-trp) from Sigma-Aldrich, and dropout supplement media without tryptophan and uracile (CSM-trp-ura) from MP Biomedicals.Disodium hydrogen phosphate dihydrate  genes integrated in chromosome III. Assay performance and data evaluation have been described previously [The estrogenic and aryl hydrocarbon receptor agonistic activities were assessed using recombinant eviously , 15.600 = 0.4) and incubated at 30°C for 5 h and 17 h for ERα and AhR, respectively. After incubation cells were disintegrated by adding 150 μL 2 mM Z-sarcosyl-buffer, the OD600 measured and the microtiter plate incubated at 30°C for 20 min for the complete disintegration. In each well, 50 μL of o-nitrophenyl β-D-galactopyranoside (4 mg/mL in lacZ-buffer) were added and the plate was incubated at 37°C till the development of yellow color. Afterwards, the reaction was stopped by adding 50 μL 1M Na2CO3, the total reaction time noted and the absorption measured at 405 nm (reference wavelength 620 nm).Briefly, 1 μL of sample was added in 100 μL of yeast culture (ODt-test. Results were considered significant when p ≤ 0.05.Data are expressed as means of three independent duplicate experiments ± standard deviation (SD) and analyzed using Student’s E. laurentii, an activity-guided fractionation was performed as described in more detail above  of the methanol extract from the aerial parts of E. laurentii was the only fraction that exhibited estrogenic activity. The three subfractions from CMAL  also induced a dose-dependent and significant β-galactosidase activity in estrogen receptor α yeast assay (yERα) from 1 μg/mL (p ≤ 0.05) compared with DMSO  starting from 0.1 μg/mL. Its magnitude of activity at 1 μg/mL equals that of estradiol at 0.5-1 nM. Moreover, the fraction 7 of Ci (F7) was about a factor of ten more potent than fraction Ci  agonistic activity of the crude methanol extract, they were additionally tested on the AhR yeast screen. According to our results, the tested fractions also showed AhR agonistic properties  as the reference compound β-naphthoflavone at 20 nM (=EC50). Fraction Bu (from W-CMAL) as well induced a significant and dose-dependent activity of AhR already starting from 1 μg/mL. Its activity at 10-25 μg/mL equaled that of β-naphtho-flavone at 20 nM  β-galactosidase activity starting from 0.5 μg/mL. At 10-50 μg/mL, the magnitude of activity of Ci was the same as β-naphthoflavone at its EC50-value. Sub-fraction F7 from Ci had the same effect already at 0.05-0.1 μg/mL. A growing body of evidence indicates that AhR/ERα interactions greatly impact the gene expression and proliferative programs of estrogen-sensitive cancers cells. Research addressing this crosstalk showed repressive effects of ligands of AhR on ERα in the estrogen-induced proliferative responses in endometrium and breast cancer cells [p-dioxin (TCDD) than after treatment with TCDD alone [E. laurentii activated the AhR and thus, active constituents may be considered as exogenous ligands of this receptor. Given these repressive mechanisms of AhR on ERα signaling pathways and due to the coexistence of both receptors in estrogen-sensitive tissues including breast, uterus and ovary our results suggest that the effect of E. laurentii on AhR could be of physiologic and anti-tumorigenic relevance. However, besides these anti-estrogenic effects of AhR, data in literature are quite conflicting regarding the implication of AhR in cancer promotion or prevention. It has also been related to cancer initiation or progression via the expression of CYP 450 enzymes (CYP1A1/2 and CYP1B1). The expression of CYP1A1 is part of negative feedback mechanisms regulating the AhR; by metabolization and agonist depletion, CYP1A1 prevents a constitutive activation of the AhR pathway. If this mechanism is impaired the AhR pathway is dysfunctional and toxic responses are the consequence [vs. CYP1B1 (detoxication/procarcinogen activation) appears to be an indispensable precondition for tumor prevention. However, the underlying mechanisms that modulate this balance still remain unclear depending on the AhR agonists, cell context as well as co-regulators present in different cell types. Therefore, although several constituents of E. laurentii are AhR agonists, a final conclusion about their contribution to cancer prevention or progression is not possible, yet.In order to check whether the ERα-active fractions/compounds identified in the methanol extract of s Figure . As in ts Figure A. In comM Figure B. SimilaM Figure C. Fractier cells –19. In rer cells , 9. ThisDD alone . In addiDD alone  as well DD alone . AccordiDD alone , 25. Oursequence . Uno et sequence . Nowadaysequence , 29 whersequence . In resp50 value of lupinalbin A was 1.34 μM while the one of 2′-hydroxygenistein was not determined because a saturation of the receptor was not achieved. To the best of our knowledge, these compounds have not been previously associated with any AhR activity. According to our results, genistein was not active, which is in agreement to previous data obtained with the same yeast system by Medjakovic and Jungbauer [As far as the isolated compounds are concerned, 2′-hydroxygenistein and lupinalbin A were found to be active in the AhR yeast assay Figure . The EC5Eriosema laurentii lead to the identification of components with estrogenic and aryl hydrocarbon receptor agonistic activities. Genistein, 2′-hydroxygenistein and lupinalbin A, which were identified in or isolated from the most active fractions, were found to be full agonists of ERα. Furthermore, we provide the first report of an agonistic activity of lupinalbin A and 2′-hydroxygenistein towards the AhR, whereas genistein remained without effect at the AhR. The results enabled the deduction of structure-activity relationships for AhR agonistic activity between these compounds for the first time as well. Thus, the data presented in this study confirmed that these phytoconstituents contribute to the estrogenic and aryl hydrocarbon receptor agonistic activities of the extract of Eriosema laurentii and might partly account to the beneficial effects of the methanol extract in vivo.The activity-guided fractionation of the crude methanol extract of aerial parts of


Objective: Several phytochemical agents have been known to exhibit a neuroprotective effect. Among them, curcumin, linalool, rutin, safranal, and thymoquinone were widely investigated and neuroprotective activity of each of them was shown by several studies. This work was planned to investigate whether different combinations of them could induce better neuroprotective effect against glucose/serum deprivation (GSD)-induced cytotoxicity.Materials and Methods: PC12 cells were cultivated for 8 h in GSD condition in both the absence and presence of curcumin, linalool, rutin, safranal, thymoquinone, or combinations of them. At the end of the experiment, the cell viability was determined using MTT assay. Results: The cells cultured in GSD condition showed a significant decrease in viability (28±1%) as compared with those cultured in standard condition (100±2%). In the presence of curcumin (10 µg/ml), linalool (16 µg/ml), rutin (200 µg/ml), safranal (50 µg/ml), and thymoquinone (1 µg/ml), the cell viability increased to 69±3.4% (p<0.001), 44±1.4% (p<0.01), 64±0.5% (p<0.001), 49±2% (p<0.001), and 70±3.2% (p<0.001), respectively. When different combinations of the agents were tested, the best cytoprotective activity was obtained from safranal + curcumin + thymoquinone .Conclusions: The present study demonstrated that a combination of safranal + curcumin + thymoquinone can block GSD-induced cell death and has the potential to be considered for management of cerebral ischemia and neurodegenerative diseases. The beneficial effect of linalool on nervous disorders is well documented -induced neuronal damage has also been reported by Mousavi and colleague (2010a)Thymoquinone is the natural main constituent of the volatile oil in Cell lines and chemicalsPC12, a rat pheochromocytoma-derived cell line, was obtained from Pasteur Institute . High glucose (4.5 g/L) Dulbecco’s Modified Eagles Medium (DMEM), glucose-free DMEM and fetal bovine serum were purchased from Gibco . Penicillin, streptomycin, and 3--2,5-Diphenyl-2H-tetrazolium bromide (MTT) were obtained from Sigma (USA).Cell culture and treatment4 cells/ml) seeded in 96-well plate. Twenty-four hours later, the standard medium was replaced by glucose- and serum-free DMEM containing curcumin (1-50 µg/ml), linalool (4-32 µg/ml), rutin (50-400 µg/ml), safranal (50-400 µg/ml), thymoquinone (1-50 µg/ml), or combinations of them. These concentrations were chosen according to our previous works on linalool, rutin, and safranal (unpublished studies) as well as literature reports on neuroprotective effects of curcumin and thymoquinone . The cells at sub-confluent stage were harvested using trypsin and aliquots of 100 µl of cell suspension . When the cells were exposed to 1, 5, 10, and 50 µg/ml of curcumin, percentage of cell viability increased from 28±1% (untreated cells) to 48±2.2% (p<0.001), 54±2.3% (p<0.001), 69±3.4% (p<0.001), and 78±3.4% (p<0.01), respectively , respectively. Thymoquinone at concentration of 50 µg/ml not only failed to increase cell survival, but also further decreased the viability .As shown in Effect of combined agents on cell viability The effect of all these combinations were statistically significant (p<0.001) compared with untreated cells (20±2%).When three agents were mixed, percentage of cell surviving increased from 20±2% (untreated cells) to 87±2% (p<0.001) and 97±5% (p<0.001) at these combinations rutin+curcumin+thymoquinone and safranal+curcumin+thymoquinone combinations, respectively. The combinations of four agents were also tested on the cell viability. Except for linalool+rutin+safranal+thymoquinone, all other combinations decreased (p<0.001) the GSD-induced cell death significantly.The mixture of all agents, curcumin+linalool+rutin+safranal+thymoqui-none, was also able to increase cell viability under GSD condition .In this study, glucose and serum limitation-induced damage in PC12 cells were used to partially model the pathological process of cerebral ischemia in an attempt to search a phytochemical compound with potent neuroprotective effect. Our results showed that all the tested agents  are able to inhibit neurotoxic effect of GSD condition. These results are in agreement with previous studies demonstrated neuroprotective effects of curcumin  not only failed to enhance cell viability, but also abolished the cytoprotective activities of each individual agent. Although it is difficult to draw a conclusion from such complex finds, this effect may be due to possible interaction between thymoquinone and other agents when curcumin is absent.in vivo studies, this combination has the potential to be used as a new therapeutics for cerebral ischemia and neurodegenerative diseases.In conclusion, our study revealed that curcumin and thymoquinone have the best neuroprotective effects among the tested phytochemicals; a combination of safranal+curcumin+thymoquinone can block GSD-induced cell death. Therefore, confirming these results in


Since then, RIT with β-emitters has shown efficacy in treating hematologic cancer. In this paper, we investigate the therapeutic efficacy of RIT in the 5T33 murine MM model using a new anti-CD138 monoclonal antibody labeled either with 213Bi for α-RIT or 177Lu for β-RIT.Radioimmunotherapy (RIT) has emerged as a potential treatment option for multiple myeloma (MM). In humans, a dosimetry study recently showed the relevance of RIT using an antibody targeting the CD138 antigen. The therapeutic efficacy of RIT using an anti-CD138 antibody coupled to 177Lu-radiolabeled 9E7.4 mAb was performed in KalwRij C57/BL6 mice 10 days after i.v. engraftment with 5T33 MM cells. Animal survival and toxicological parameters were assessed to define the optimal activity.A new monoclonal anti-CD138 antibody, 9E7.4, was generated by immunizing a rat with a murine CD138-derived peptide. Antibody specificity was validated by flow cytometry, biodistribution, and α-RIT studies. Then, a β-RIT dose-escalation assay with the 213Bi-labeled 9E7.4 anti-CD138 mAb increased median survival to 80 days compared to 37 days for the untreated control and effected cure in 45% of animals. β-RIT performed with 18.5 MBq of 177Lu-labeled 9E7.4 mAb was well tolerated and significantly increased mouse survival (54 vs. 37 days in the control group); however, no mice were cured with this treatment.α-RIT performed with 3.7 MBq of This study revealed the advantages of α-RIT in the treatment of MM in a preclinical model where β-RIT shows almost no efficacy. Multiple myeloma (MM) represents 1% of all cases of cancer and 10% of hematological malignancies , 3. Depe– emitters, such as 131I, 90Y, 177Lu, and a number of α-emitters such as 213Bi, 211At, or 225Ra . This range is similar to that of 131I (2.9 mm), but shorter than that of 90Y (11 mm). This characteristic may reduce non-specific radiation damage to surrounding healthy tissues. In addition, its low ɣ-emission (208 keV) is an advantage in terms of radioprotection.In the past two decades, radioimmunotherapy (RIT) has emerged as a novel strategy in the anti-cancer arsenal. This therapeutic approach consists in specifically targeting tumor cells using an antibody coupled with a radionuclide. The radionuclide can then kill tumor cells by direct cytotoxicity as well as through a bystander effect –9. Amongor 225Ra –15. The or 225Ra . Indeed,leukemia , 19 and leukemia , 21. 177Our group has recently proven RIT to be effective in treating MM in humans  and α-RI131I-CD138 mAb  and confirmed that it could effectively treat MM tumors using α-RIT. Then, we studied the tissue distribution of 177Lu-9E7.4 mAb and went on to perform dose-escalation and toxicity studies. Finally, we administered 177Lu-9E7.4 at different times during disease progression.Based on our study in MM patients with D138 mAb , and thend 177Lu , 19, we 177LuCl3 was purchased from Perkin Elmer (France) in 0.05 N HCl and 225Ac/213Bi generator was obtained from the Institute for Transuranium Elements . All buffers were prepared in H2O milliQ  and Trace SELECT chemicals, when available. Rat IgG2a,κ anti-mouse CD138 antibody (clone 281-2) (ref.553712) was purchased from BD Pharmingen (France).The bifunctional chelators S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) and 2-(4-isothiocyanato-benzyl)-cyclohexyl-diethyletriaminepenta-acetic (SCN-CHX-A″-DTPA) were purchased from Macrocyclics (USA). ® CRL-1581™) and the CTLL-2 T lymphocyte cell line (ATCC® TIB-214™) were purchased from ATCC. All cell lines were cultured in RPMI 1640 medium  containing 2 mM l-glutamine, 100 U/mL penicillin/100 μg/mL streptomycin and 10% heat-inactivated fetal calf serum . Cells were incubated at 37°C, 5% CO2 in a humidity-saturated incubator.The 5T33 murine MM cell line was kindly provided by Dr. Harvey Turner  with permission from Dr. J. Radl  , 32. TheFemale C57BL/KalwRij mice were purchased from Harlan CPB and housed under conventional conditions at the UTE animal facility . Experiments were approved by the local veterinary committee (License No. CEEA.2013.2). Mice were 8–12 weeks old at the time of experiments.The 9E7.4 mAb was produced by immunization of a rat with a 40-amino-acid peptide  derived from the murine CD138 protein (aa 90–130) (GenBank: CAA80254.1). The isotype of 9E7.4 immunoglobulin was determined using a Rat Isotyping Kit  according to the manufacturer’s instructions.The specificity and affinity of 9E7.4 mAb for CD138 were determined by flow cytometry . Staining was detected indirectly with a secondary antibody, anti-rat IgG/PE (Jackson-ImmunoResearch Laboratories).4OAc, pH 7 (DOTA-9E7.4 mAb) or for PBS (CHX-A″-DTPA-9E7.4 mAb) by several centrifugal filtration steps using a 30-kDa cutoff Centricon concentrator . Integrity of the immunoconjugate was verified by size-exclusion HPLC. The conjugated 9E7.4 mAb was filtered through a 0.2-μm filter before storage at 4°C. The number of chelators per antibody was estimated at between 1 and 4 for p-SCN-Bn-DOTA and 2 for SCN-CHX-A″-DTPA by an 111In dilution assay (A reaction solution containing a 20M excess of p-SCN-Bn-DOTA or SCN-CHX-A″-DTPA over the 9E7.4 mAb in 0.3M carbonate buffer pH 8.7 was incubated overnight (16 h) at room temperature (RT). Free chelator was eliminated and the conjugation buffer was exchanged for 0.25M NHon assay  and/or m177Lu-chloride (370 MBq) was added to a solution containing DOTA-9E7.4 mAb, prepared in 0.25M NH4OAc pH 7 and 0.05M ascorbic acid buffer pH 5.5, and was adjusted to pH 5.5 with 0.5M NH4OAc. The reaction mixture was incubated at 42 ± 1°C for 3 h. Complexation was then stopped by adding a 10M excess of EDTA and mixing for 15 min at RT. Labeling of anti-CD138 9E7.4 mAb with 213Bi was performed as described in Chérel et al.  to tumor-free C57BL/KalwRij (n = 3 in each group). Mice were sacrificed 5 min, 4, and 24 h later. To study the tissue distribution of 177Lu-9E7.4 mAb, 3 μg were i.v. injected into tumor-free C57BL/KalwRij (n = 3 in each group). Mice were sacrificed 5 min, 2, 8, 24, 48, 96, or 168 h later. For all experiments, tissues were collected and radioactivity was counted using a ɣ-counter . To account for physical decay of radioactivity, a triplicate standard dose was counted alongside the samples, and results were expressed as a percentage of injected dose per gram of tissue.To determine the biodistribution of anti-mouse CD138 mAb, 3 μg of 6 5T33 MM cells. α-RIT was then performed on day 10 and β-RIT on day 10, 15, or 20. Animal survival was monitored over time. Mice were sacrificed when paralysis was detected and/or their weight dropped by 20%. In some cases, tumor masses were detected. For α-RIT, a single dose of 3.7 MBq 213Bi-9E7.4 mAb (n = 20) was injected i.v., in line with the previously described protocol (n = 14), 7.4 (n = 5), 11.1 (n = 5), 14.8 (n = 5), 18.5 (n = 15), 27.75 (n = 11), or 37 MBq (n = 7) 177Lu-9E7.4 mAb at a specific activity of 1480 ± 74 MBq/mg of antibody.Mice were engrafted with 1 × 10protocol . For β-RTo assess hematologic toxicity, blood was collected from the retro-orbital sinus for leukocyte, erythrocyte, and thrombocyte counting on an automated hematology analyzer . Plasma was isolated from blood samples, pooled, and then used to monitor bone marrow, liver, and kidney toxicity based on the following biomarkers: Flt3-L – measured at different time points using DuoSet ELISA kits , according to the supplier’s instructions – ASAT, ALAT, urea, and creatinin – concentrations of these markers were monitored using a Roche Cobas Analyzer  with the appropriate reagents .® software (GraphPad Software).Data are presented as mean ± SEM. Statistical significance was determined using the Mann–Whitney test. Animal survival data were analyzed using Kaplan–Meier survival curves combined with the log-rank test. All statistical analyses were performed using GraphPad Prismin vitro competition assay indicated that these two anti-CD138 mAbs bind to different epitopes on CD138 (data not shown). The binding affinity for mAbs 281-2 and 9E7.4 on 5T33 MM cells was comparable: 1 × 10−10 and 2.3 × 10−10M, respectively (data not shown). However, maximal binding of the 281-2 mAb was at least twofold more intense than 9E7.4 mAb binding  was calculated . This specificity was confirmed by flow cytometry on different cell lines and compared to that of 281-2 mAb, a commercial anti-mouse CD138 mAb, which we had previously used  . This level of uptake was due to specific binding, as confirmed by comparison with a 177Lu-isotype control (data not shown). Uptake of 177Lu-9E7.4 mAb was also high in the spleen (27.6 ± 8.2% ID/g tissue between 2 and 48 h) and in the intestines (18.8 ± 9.1% ID/g tissue at 24 and 48 h)  and results in long-term survival for 45% of animals. In contrast, we showed that single-dose β-RIT performed in the same conditions was not sufficient to cure mice developing MM.125I-radioiodinated 281-2 and 9E7.4 mAbs were very similar. The only differences observed were a lower uptake in the liver and a higher uptake in the spleen with both 125I and 177Lu-radiolabeled 9E7.4 mAb compared to 281-2 mAb. These differences might be explained as mentioned above and will have to be further investigated. However, the difference in liver uptake could be correlated to the lower binding level of 9E7.4 mAb to CD138 compared to 281-2 mAb. Whatever the explanation, our results with 177Lu-9E7.4 mAb suggest that the therapeutic effect of β-RIT is not enough to offset its toxicity. Thus, accumulation of 177Lu-9E7.4 mAb appears to be deleterious in the bone marrow and liver. Many other studies performed with β-emitters have reported dose-limiting toxicity due to hematologic and bone marrow toxicities , suggests that the drop in thrombocyte levels is probably due to destruction of their precursors in the bone marrow. This hypothesis is supported by the increase in plasma Flt3-L noted for these two groups. Based on the uremia, there was no apparent kidney toxicity in these two groups, although creatinemia indicated a certain level of toxicity at 27.75 MBq. However, this experiment was only performed once and will have to be confirmed. Finally, at these activity levels, no hepatic toxicity was observed, even though it is important to note that in the 27.75 MBq group, one mouse had to be sacrificed soon after treatment as it exhibited the same signs of toxicity as animals in the 37 MBq group. All these results combine to indicate that the potential efficacy of the treatment at 27.75 MBq is counterbalanced by its toxicity. This suggests that 18.5 MBq is not only the maximum tolerated dose, but also that this is the only activity, administered as a single dose, allowing a certain efficacy of the treatment. However, in the group which received 18.5 MBq, despite the lower toxicity and statistical improvement in median survival compared to the control group, no cure was effected. Mice in this group were euthanized because of paralysis and an increase in plasma paraprotein, with one exception (data not shown). Therefore, even if the treatment was associated with some toxicity, animals mainly died due to tumor burden.This lack of efficacy cannot be ascribed to the new anti-CD138 mAb since α-RIT was effective and tissue distributions of xicities , 41, 42.177Lu emits β-ionizing radiation in a range of 2 mm within tissues, and the literature indicates that these β-particles are more efficient in hematologic cancers with a high-tumor burden (r burden . In our r burden . Therefo213Bi-281-2 mAb killed the human MM cell line U266 more efficiently than 131I-281-2 mAb (213Bi) vs. β (177Lu) ionizing radiation at the cellular level.The difference between α-RIT and β-RIT in our MM model could be due to a difference in the radiosensitivity of 5T33 MM cells. Our group has already demonstrated that 131I-B-B4 mAb in a previous dosimetric study (177Lu than with 90Y in an acute myeloid leukemia syngeneic mouse model. These authors hypothesized that the lower dose-rate and longer half-life of 177Lu could be responsible for the effect observed and suggested that dose fractionation is better adapted to the physical properties of this radionuclide. Therefore, further studies with the 177Lu-9E7.4 mAb in the immunocompetent MM mouse model will focus on assessing fractionated β-RIT treatment. In conclusion, this preclinical study indicates that, with a single dose, α-RIT is more efficient than β-RIT in treating MM in this mouse model.Finally, although our study does not demonstrate any advantage of using β-RIT to treat MM, some therapeutic efficacy was observed with ic study . In the ic study . Dose fric study –46 and aic study . Finallyic study  demonstrConception and design: NF, SG, CM, JB, LF, AM, FB, AF-C, JG, and MC. Method development: NF, SG, CM, JB, LF, AM, FB, AF-C, EB-C, FD, JG, and MC. Data acquisition: NF, SG, CM, EB-C, JG, and MC. Data analysis and interpretation: NF, SG, CM, JB, LF, AM, FB, AF-C, EB-C, FD, JG, and MC. Writing, review, and/or revision of the manuscript: NF, SG, JB, LF, AM, FB, AF-C, EB-C, FD, JG, and MC. Study supervision: JB, JG, and MC.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mart1-9), also called sushi-ichi-related retrotransposon-homolog (SIRH) genes, are a class of Ty3/gypsy LTR-retroelements showing moderate homology to the sushi-ichi LTR-retrotransposon in pufferfish. Rtl1/Mart1 and Peg10/Mart2 expression in mouse placenta and demonstration of their functional roles during placental development exemplifies their importance in cellular processes. In this study, we analyzed all eleven mouse Mart genes from the blastocyst stage and throughout placentogenesis in order to gain information about their expression and regulation.LTR-retrotransposons became functional neogenes through evolution by acquiring promoter sequences, regulatory elements and sequence modification. Mammalian retrotransposon transcripts (in situ hybridization (ISH) and immunoblotting showed various expression patterns of the 11 mouse Mart genes through different placental stages. Zcchc5/Mart3, Zcchc16/ Mart4 and Rgag1/Mart9 expression was undetectable. Rtl1/Mart1, Peg10/Mart2, Rgag4/Mart5 – Cxx1a,b,c/Mart8b,c,a gene expression was very low at the blastocyst stage. Later placental stages showed an increase of expression for Rtl1/Mart1, Rgag4/Mart5 – Cxx1a,b,c/Mart8b,c,a, the latter up to 1,489 molecules/ng cDNA at E9.5. From our recently published findings Peg10/Mart2 was the most highly expressed Mart gene. ISH demonstrated sense and antisense transcript co-localization of Rgag4/Mart5 to Cxx1a,b,c/Mart8b,c,a in trophoblast subtypes at the junctional zone, with an accumulation of antisense transcripts in the nuclei. To validate these results, we developed a TAG-aided sense/antisense transcript detection (TASA-TD) method, which verified sense and antisense transcripts for Rtl1/Mart1, Rgag4/Mart5 – Cxx1a,b,c/Mart8b,c,a. Except for Rtl1/Mart1 and Cxx1a,b/Mart8b,c all other Mart genes showed a reduced amount of antisense transcripts. Northern blot and 5′ and 3′ RACE confirmed both sense and antisense transcripts for Ldoc1/Mart7 and Cxx1a,b,c/Mart8b,c,a. Immunoblotting demonstrated a single protein throughout all placental stages for Ldoc1/Mart7, but for Cxx1a,b,c/Mart8b,c,a a switch occurred from a 57 kDa protein at E10.5 and E14.5 to a 25 kDa protein at E16.5 and E18.5.Quantitative PCR, Mart genes support neo-functionalization of retrotransposons in mammalian genomes. Undetectable expression of Zcchc5/Mart3, Zcchc16/Mart4 and Rgag1/Mart9 indicate no role during mouse placentogenesis. Rgag4/Mart5 to Cxx1a,b,c/Mart8b,c,a gene expression support a role for differentiation from the ectoplacental cone. Mart antisense transcripts and protein alterations predict unique and complex molecular regulation in a time directed manner throughout mouse placentogenesis.RNA and protein detection of mouse The online version of this article (doi:10.1186/s12977-015-0138-8) contains supplementary material, which is available to authorized users. Functional sushi retrotransposons were not found in mammals, only sushi related neogenes. Mammalian retrotransposon transcripts (Mart) ) are derived from the gag genes of Ty3/gypsy LTR retroelements [Transposable elements are found from bacteria to humans and contribute to the dynamics of genomes, where they profoundly alter and affect structure and function. Mammals in particular have an abundant amount of transposable elements, which originated from retroelements  integrating into the genome over time. In the murine genome 40% of sequences account for transposable elements . Many prmodomain . Furtherfamilies . One of r fishes . The suselements  and idenelements .ERV gene family and the eleven mouse Mart genes with high sequence homology. Figure Mart genes [Mart genes (Mart 1–9) represent single gene copies, including three single gene copies of Cxx1/Mart8  in both mouse and human [sushi-ichi” and the mouse Mart genes with their main motives. Except for Rtl1/Mart1, Peg10/Mart2 and Ldoc1l/Mart6 all other Mart-genes are located on chromosome X. To date all Marts have newly recruited promoter sequences with no LTRs [Mart genes are not functional autonomous retrotransposons [Mart genes contain the CCHC-type Zn-finger (consensus: RKCYNCGKPGHMARDCPE), a motif which was shown in retroviral nucleocapsid proteins to help in viral genome packaging, but was also found in eukaryotic proteins involved in RNA or ssDNA binding [Cxx1a,b,c/Mart8b,c,a are flanked by regions of similar sequences indicating that their gene structure was formed by duplication events during evolution [Peg), Rtl1/Mart1 (Peg11) and Peg10/Mart2 are maternally imprinted. Mouse Rtl1/Mart1 is localized on chromosome 12, whereas Peg10/Mart2 is on chromosome 6. Additionally, the maternal chromosome 12 expresses an antisense transcript of Rtl1/Mart1 (Anti-Peg11) [Rtl1/Mart1 was shown to be expressed in the human and murine placenta [Peg10/Mart2, where a −1 frame shift resulted in two gag-/pol-like open reading frames (ORF) with an identical N-terminus  in the junctional zone and sinusoidal trophoblast giant cells (sTGC) of the labyrinth layer [Peg10/Mart2 knockout mice had smaller than normal placentas, absence of spongiotrophoblast markers and embryonic lethality until E10.5 [LDOC1/MART7 gene was found ubiquitously expressed in human tissues and down regulated in some cancer cell lines [One hypothesis states that through evolution frequent independent domestications occurred in ancestors of placentalia and that re-activation and gene duplications of these Metaviridae led to the highest number of conserved retroelement-derived domesticated genes ,13. Thisrt genes . All elend human . Figure  no LTRs . This gensposons ,14. Impo binding   . Rtl1/Maplacenta ,18. Knocnd ovary ,20. Mousplacenta ,22. For th layer . Peg10/Mil E10.5 . Lastly The mammalian placenta is formed during embryonic development by maternal and fetal cells. It is essential for embryo survival, to transport nutrients and waste products and is important for hormone production during gestation ,25. The Rtl1/Mart1 to Ldoc1l/Mart6 genes were found expressed in most organs tested during embryonic development [Rtl1/Mart1 and Peg10/Mart2, and recently for Ldoc1/Mart7 [Mart gene family, we performed gene expression quantification (qPCR) of the Mart family from mouse blastocysts (E4.5) and placentae stages E8.5 to E18.5 to determine their expression throughout placentogenesis. Cellular localization of expressed Mart genes was analyzed by In situ hybridization (ISH) at different stages , as well as protein expression of Ldoc1/Mart7 and Cxx1/Mart8 by immunoblotting . Both ISH and a first strand cDNA analysis showed the presence of antisense transcripts supporting RNA/protein regulation in the cell.Although mouse elopment , to datec1/Mart7 ,21,22,35Mart genes we performed absolute qPCR from two pooled samples of blastocysts (E4.5), one containing 10 and the other with 17 blastocysts. We then analyzed Rtl1/Mart1 and Zcchc5/Mart3 to Rgag1/Mart9 RNA expression with eleven different embryonic stages of placentae from E8.5 to E18.5. Furthermore we performed ISH with the four highly expressed Mart genes Rgag4/Mart5, Ldoc1l/Mart6, Ldoc1/Mart7 and Cxx1a,b,c/Mart8b,c,a, using placental tissues from an earlier stage E8.5 and a later stage E14.5/E15.5, where all trophoblast subtypes are formed. The expression of all Mart genes in blastocysts was either very low or un-detectable. For example, we detected mean values of 0.68 molecules/ng cDNA for Rtl1/Mart1 and 1.71 molecules/ng cDNA for Ldoc1/Mart7; whereas RNA amounts for Zcchc5/Mart3, Zcchc16/Mart4 and Rgag1/Mart9 were un-detectable .In order to determine the expression level of the mouse e Figure A. Regardanalyzed . Rtl1/MaRgag4/Mart5 and Ldoc1l/Mart6 were the lowest expressed genes among the Marts  to E12.5 (mean: 737.16 molecules/ng cDNA), which was followed by a significant decrease until E18.5  and E18.5  compared to Ldoc1/Mart7 , could identify Cxx1c/Mart8a antisense and Cxx1a,b/Mart8b,c sense transcripts  with gene specific primers, but with no distinction between sense and antisense transcripts. Results showed no differences in Cxx1c/Mart8a and Cxx1a,b/Mart8b,c expression between the different placental stages, which was comparable to our qPCR findings . To circumvent this problem we developed a new technique called “TAG-aided sense/antisense transcript detection” (TASA-TD), involving a specific TAG-sequence not present in the mouse genome, which was located at the 5′ ends of each annealing sense or antisense primer  was previously demonstrated to have an antisense transcript, thus represented a positive control [Rtl1/Mart1 sense and antisense transcripts (1: 1.05), whereas Rgag4/Mart5, Ldoc1l/Mart6 and Ldoc1/Mart7 showed 3.5-4.5-fold less antisense than sense transcripts at E14.5. Interestingly, Cxx1c/Mart8a antisense transcript was 2.1-fold less, but in contrast Cxx1a,b/Mart8b,c antisense transcripts were 1.5-fold higher expressed than the sense transcripts. To further confirm sense and antisense transcripts, we performed Northern blot analyses and 5′ and 3′ RACE to determine Cxx1/Mart8 and Ldoc1/Mart7 transcript sizes. Due to the fact that Cxx1a,b,c/Mart8b,c,a are highly homologues to each other and Cxx1a/Mart8b and Cxx1b/Mart8c are transcribed complementary to Cxx1c/Mart8a, it was not possible to discriminate the Cxx1/Mart8 sense and antisense transcripts separately using probes  and ~1.0 kb (3′ RACE) antisense transcript totaling ~3.0 kb  identified 51% DNA nt identity (672 of 1298 nt) and 24% amino acid identity (98 of 406 amino acids) [Rtl1/Mart1, Peg10/Mart2 and Zcchc5/Mart3 [Mart genes, most likely by recombination events including gene duplications, deletions or coding sequence modification. Thus, these processes contributed to their overall gene/protein sizes. For example, protein size variations span from 113 amino acids for Cxx1a,b,c/Mart8b,c,a to 1364 amino acids for Rgag1/Mart9 [Mart genes occurred, their establishment in the genome must have evolved by acquiring new promoter sequences in order to develop into neofunctional genes [Mart genes acquired promoters and regulatory elements as well as other structural changes throughout evolution leading to their essential roles in the development of the placenta in mammals, e.g. for Rtl1/Mart1 and Peg10/Mart2 [The overall structure and phylogenetic analysis shown in Figure ications . For exao acids) . Pol-likc5/Mart3 . Furtheral genes ,39. Diff10/Mart2 ,22.Mart genes in mice, and the determination that Rtl1/Mart1, Peg10/Mart2 and recently Ldoc1/Mart7 play a functional role during placentogenesis, we focused our study on the expression patterns of Marts not yet described in mouse placental development. Our results showed that not all Mart genes were expressed during placentogenesis. Specifically, Zcchc5/Mart3, Zcchc16/Mart4 and Rgag1/Mart9 were not expressed at the blastocyst (E4.5) stage nor at later stages of placentation, thus we conclude that these three genes play no essential role in placental developmental although a functional role during embryogenesis is possible. On the other hand we found very low expression for the other Mart genes in the blastocyst in contrast to higher levels of expression at E8.5 and the following stages, supporting potential functional roles in placental development.The existence of 11 Rtl1/Mart1 and Peg10/Mart2 were expressed in mouse placenta with an essential role for embryo survival [Mart members in this report with our previous study of Peg10/Mart2 and other developmental genes [Peg10/Mart2 was the highest expressed retrotransposon gene with a maximum of 188,917.13 molecules/ng cDNA at E16.5 [Rgag4/Mart5 and Ldoc1l/Mart6 genes were more constantly expressed at lower levels (between 1.82 and 20.18 molecules/ng cDNA) throughout development  and -beta (Tpbpβ) which also showed a comparable expression pattern between ST and GlyT [Rgag4/Mart5 and Ldoc1l/Mart6 intensely localized to the cytosol of pTGCs in the junctional zone, which points to further specialized functions of these Marts  and delayed parturition (1–4 days later) [Ldoc1/Mart7 localization by ISH along with decreased numbers of ST in the placentae of the knockout mice confirmed our localization results along with the functional importance of the gene in pTGC, GlyT and especially ST. In addition to Cxx1a,b,c/Mart8b,c,a expression in the junctional zone, positive expression was also noted in GlyT islands in the labyrinth layer. Similar to Rgag4/Mart5 and Ldoc1l/Mart6 strong nuclear expression in the pTGCs was found for Cxx1c/Mart8a antisense and Cxx1a,b/Mart8b,c sense at E8.5 using ISH. Due to the high homology and gene orientation of the Cxx1a,b,c/Mart8b,c,a gene cluster, RNA probes cross-hybridized thus we could not identify single independent transcripts using ISH. Therefore for verification of the antisense and sense Mart transcripts by ISH, we developed a novel method TASA-TD to specifically identify and quantify sense and antisense transcripts in tissues. Based upon a specific first strand cDNA synthesis of isolated RNA and using a non-murine TAG-sequence, we detected sense and antisense transcripts of Rtl1/Mart1, Rgag4/Mart5, Ldoc1l/Mart6, Ldoc1/Mart7, Cxx1c/Mart8a and Cxx1a,b/Mart8b,c in E14.5 placenta. If antisense transcripts regulate transcription and esp. translation of sense transcripts, Rtl1/Mart1 and Cxx1a,b,c/Mart8b,c,a are more highly regulated than Rgag4/Mart5, Ldoc1l/Mart6 and Ldoc1/Mart7 due to their higher ratios of antisense: sense transcripts. Furthermore, using Northern blot analysis of Cxx1a,b,c/Mart8b,c,a and 5′ and 3′ RACE methodology of Ldoc1/Mart7 determined sense and antisense transcript sizes corroborating both TASA-TD results and ISH expression.s later) . The latRgag4/Mart5 to Cxx1a,b,c/Mart8b,c,a genes are mainly restricted to trophoblast cells in the junctional zone. This was distinctly different to Rtl1/Mart1 and Peg10/Mart2, where Rtl1/Mart1 only localized in fetal capillaries of the labyrinth [Peg10/Mart2, in addition to expression in the junctional zone, localized specifically to sTGCs of the labyrinth layer, supporting functional roles at the fetal-maternal blood barrier [Peg10/Mart2 with the fusogenic ERV genes, Syncytin-A and Syncytin-B, which were restricted to SCT-I and –II in the labyrinth, demonstrated no functional role of Peg10/Mart2 in cell fusion [Marts expressed in different trophoblast cells have no functional roles in cell fusion [Thus, we can conclude that expression of the abyrinth  and Peg1 barrier ,22. Furtl fusion . Therefol fusion .Rtl1/Mart1 (Peg11) maternally expressed antisense transcripts code for miRNAs and target the Rtl1/Mart1 transcript [Caenorhabditis elegans and was directly linked with transposon antisense RNA during development [β-site APP-cleaving enzyme 1 gene (BACE1) encodes for an enzyme which has an important role in progression of Alzheimer’s disease [There are multiple mechanisms by which antisense transcripts regulate sense transcription leading to changes in gene/protein expression. These include, direct inhibition of sense transcription via antisense hybridization to RNA; induction of DNA methylation for gene silencing by antisense mediation; blockage of RNA splicing, processing, stability and miRNA binding sites via antisense/sense hybrids; DICER targeting and processing of antisense/sense hybrids to siRNAs; induction of RNA editing or changes of secondary structures through antisense/sense hybrids and finally, inhibition of translation due to cytoplasmic antisense/sense hybrids -47. The anscript . Therefoelopment . Antisenelopment . For exa disease .in vivo has to be further analyzed.Verification of protein supports regulation beyond the cellular functions of RNA. Comparing Ldoc1/Mart7 and Cxx1/Mart8 proteins we found regulatory differences throughout placental development. By E18.5 the ~40 kDa Ldoc1/Mart7 protein was decreased by 2.6-fold compared to E10.5, which corroborated our qPCR data. On the other hand, Cxx1a,b,c/Mart8b,c,a showed a unique developmental regulation such that a ~57 kDa protein was detected in earlier stages of placentation but in later stages a ~25 kDa protein was found. It should be noted that the calculated protein size for Ldoc1/Mart7 is 17.54 kDa (IEP: 4.08) and for Cxx1a,b,c/Mart8b,c,a 13.6 kDa (IEP: 9.02). However, the observed protein sizes with repeated SDS-PAGE/ Immunoblots were ~40 kDa for Ldoc1/Mart7 and for Cxx1/Mart8 ~ 25 and ~57 kDa , which resulted in receptor inhibition. Co-expression of both proteins in cell-lines led to morphological changes [MART genes, like RTL1/MART1, PEG10/MART2, LDOC1L/MART6, LDOC1/MART7 and RGAG1/MART9 may play a role in tumorigenesis [Although, protein expression of Rtl1/Mart1 and Peg10/Mart2 was demonstrated as essential for placental development ,22,52, f changes . Okabe e changes . In cont changes . Therefoigenesis ,62.Mart gene and protein expression occur throughout mouse placental development speaks for essential functions during placentogenesis.Taken together, our findings that Mart genes derived from the gag genes of Ty3/gypsy LTR retroelements showed different expression patterns in mouse placentation. Rgag4/Mart5 to Cxx1a,b,c/Mart8b,c,a gene expression in the mouse placenta demonstrated specific localization in trophoblast lineages, which evolved from cells of the ectoplacental cone. Presence of antisense transcripts and alterations in protein expression at different developmental stages points to complex regulatory mechanisms of sense transcript and protein expression for the Marts. We predict that due to the similar homology between mouse Mart and human MART genes their functional roles could be analogous during placental development.Our results confirm the hypothesis of neofunctionalization of retroelements in mammals throughout evolution and the conservation of their cellular functions for placental development and ultimately, offspring survival. Eleven mouse Pregnant C57BL/6 and C3H mice were provided from the Institute of Biochemistry of Friedrich-Alexander-University Erlangen-Nürnberg. Experiments were performed in strict accordance with the protocol, which was approved by the Committee on the Ethics of Animal Experiments of the University of Erlangen-Nürnberg (Permit Number: TS-00/12- Biochemistry II). Placentae for RNA isolation and frozen sections were prepared according to previously published methods . BlastocMart genes was performed according to Dereeper et al. [http://www.phylogeny.fr/. For this analysis the entire codogenic sequence of every mouse Mart gene was used according to NCBI references: Rtl1/Mart1 (NM_184109.1), Peg10/Mart2 (NM_001040611.1), Zcchc3/Mart3 (NM_199468.1), Zcchc16/Mart4 (NM_001033795.4); Rgag4/Mart5 (NM_001278534.1), Ldoc1l/Mart6 (NM_177630.3), Ldoc1/Mart7 (NM_001018087.1), Cxx1a/Mart8b (NM_024170.2), Cxx1b/Mart8c (NM_001018063.1), Cxx1c/Mart8a (NM_028375.3) and Rgag1/Mart9 (NM_001040434.2).A multiple alignment, phylogenetic reconstruction and graphical representation for all 11 mouse r et al.  using htParaffin embedded mouse placenta of E15.5 were cut into 2 μm tissue sections using a microtome , de-paraffinized, rehydrated and treated with 1% periodic acid (wt/vol)  in water for 5 min at room temperature according to Tunster et al. .Total RNA from mouse snap frozen placentae was extracted using peqGOLD TriFast (PEQLAB Technologies) according to manufactures’ protocol. After precipitation the extracted RNA was solubilized in 0.1% DEPC-treated water and then pre-treated with 40 U DNaseI  for 60 min at 37°C. RNA from 10 and 17 pooled blastocysts (E4.5) was extracted using the Absolute RNA Nanoprep Kit  according to the company’s protocol then cDNA was synthesized using the High Capacity cDNA Kit (Applied Biosystems (ABI), Germany) in a thermal cycler  for 2 h at 37°C.Mart gene the copy numbers were calculated and used as an external standard to generate a standard curve with a cycle threshold (Ct) value against the log of amount of standard. Expression values were calculated as molecules per ng total cDNA using a standard curve of each cloned Mart gene determined by real time PCR. The efficiencies (γ) of the qPCR were between −3.52 to −3.17, the limit of detection (t) and the correlation coefficient (R2) is documented in Additional file For qPCR, gene fragments of interest were amplified with specific primers . All probes were then pretreated with 40 U DNaseI  for 60 min at 37°C. Tissue preparation and ISH of mouse placentae of different embryonic stages were performed according to published methods [For the synthesis of the specific digoxigenin (DIG)-labeled sense and antisense RNA probes of  methods . A contrCxx1a,b,c/Mart8b,c,a expression was analyzed via PCR with specific primers  was denatured in a buffer with: 1 × MOPS, 50% formamide, 20% formaldehyde for 10 min at 65°C and electrophoresed in a 1.2% agarose gel containing 2.2 M formaldehyde and 1 × MOPS. The transfer was done overnight by capillary blot methodology onto a nylon membrane in the presence of 20 × SSC. After transfer, the RNA was fixed at 80°C for 2 h. Prehybridization was done with ULTRAhyb® Ultrasensitive Hybridization Buffer (Ambion/Applied Biosystems) at 68°C for 1 h. To detect both transcripts, blots were hybridized separately with antisense or sense Cxx1a/Mart8b DIG-labeled nucleotide probes at a concentration of 100 ng/ml overnight at 68°C. After stringency washes with 2 × SSC/ 0.1% SDS, 1 × SSC/ 0.1% SDS and 0.5 × SSC/ 0.1% SDS the membranes were incubated with a Blocking Buffer (Sigma) for 30 min. Detection of RNA-probe hybrids was performed with anti-digoxigenin-AP, Fab-fragments  and a chemi-luminescence reaction with AP-Juice , and then visualized with X-ray films.Ldoc1/Mart7 sense and antisense transcripts was performed with the 5′/3′ RACE Kit, 2nd Generation (Roche) according to the manufactures protocol. Briefly, 200 ng placental RNA (E14.5) was used for the Ldoc1/Mart7 sense transcript with a gene specific (GS) sense (s) primer (GS-s-cDNA) for the 5′RACE first strand cDNA synthesis. After purification of the cDNA with the High Pure PCR product purification kit (Roche), a homo-polymeric d(A)-tail was ligated to the 3′end of the first strand cDNA using a terminal transferase and dATP. PCR amplification of poly-d(A)-tailed cDNA was performed with an oligo-d(T)-anchor primer and a GS-s-BR-PCR primer. For the 3′RACE of Ldoc1/ Mart7 sense transcript, 500 ng placental RNA (E14.5) was transcribed with an oligo-d(T)-anchor primer in the first strand cDNA. PCR amplification was done with a GS-s-TF primer and a BR anchor primer -tailing was performed at the 3′end of placental RNA (E14.5) with a poly(A)-polymerase (New England Biolabs). For the first strand cDNA synthesis 500 ng of the poly(A)-tailed RNA was used with an oligo-d(T)-anchor primer and gene specific amplification followed with a GS-as-TF primer and an BR anchor primer .We developed the following new approach called the TAG-aided sense/antisense transcript detection (TASA-TD) method in order to identify and quantify sense and antisense transcripts. For sense and antisense RNA transcript analysis RNA from E14.5 placenta was isolated as described above. An overview of the technique is represented in Figure β-actin and Ldoc1/Mart7, 400 ng RNA for Rgag4/Mart5 and Ldoc1l/Mart6 and 100 ng for Rtl1/Mart1 and Cxx1c/Mart8a and Cxx1a,b/Mart8b,c was used. Furthermore 1 μM GSP-TAG, 0.5 mM dNTP, 5 mM MgCl2, 10 mM DTT, 40 U RNaseOUT, 100 U SuperScriptIII® RT  and 240 ng Actinomycin D  were added for a 20 μl reaction. Importantly, a RT with very low intrinsic RNase H activity (for cleavage of RNA from RNA/DNA duplexes) and Actinomycin D was necessary to prevent RT of second strand cDNA and thus antisense artifacts [β-actin which was shown to have no antisense transcript [Primer sequences are shown in Additional file rtifacts . RNA andanscript . PCR reahttp://imagej.nih.gov).Mart7 and Mart8 protein expression was analyzed in lysates from mouse placentae of stage E10.5, E14.5, E16.5 and E18.5 according to Strick et al. . Fifteen