Estrogens circulate at concentrations less than 20 pg/mL in men and postmenopausal women, presenting analytical challenges. Quantitation by immunoassay is unreliable at these low concentrations. Liquid chromatography tandem mass spectrometry (LC–MS/MS) offers greater specificity and sometimes greater sensitivity, but ionization of estrogens is inefficient. Introduction of charged moieties may enhance ionization, but many such derivatives of estrogens generate non-specific product ions originating from the “reagent” group. Therefore an approach generating derivatives with product ions specific to individual estrogens was sought.p-toluenesulfonate (FMP-TS). Electrospray in positive mode with multiple reaction monitoring using a QTrap 5500 mass spectrometer was used to quantify “FMP” derivatives of estrogens, following LC separation.Estrogens were extracted from human plasma and serum using solid phase extraction and derivatized using 2-fluoro-1-methylpyridinium-m/z 362→238 and m/z 364→128, respectively). The limits of detection and quantitation were 0.2 pg on-column and the method was linear from 1–400 pg/sample. Measures of intra- and inter-assay variability, precision and accuracy were acceptable (<20%). The derivatives were stable over 24 h at 10 °C (7–9% degradation). Using this approach, E1 and E2, respectively were detected in human plasma and serum: pre-menopausal female serum (0.5 mL) 135–473, 193–722 pmol/L; male plasma (1 mL) 25–111, 60–180 pmol/L and post-menopausal female plasma (2 mL), 22–78, 29–50 pmol/L.Transitions for the FMP derivatives of estrone (E1) and estradiol (E2) were compound specific (Thus FMP derivatization, in conjunction with LC–MS/MS, is suitable for quantitative analysis of estrogens in low abundance in plasma and serum, offering advantages in specificity over immunoassay and existing MS techniques.  •Quantitative analysis of low amounts of estrone and estradiol in plasma and serum.•Quantitation across physiological range in men and pre- and post-menopausal women.•Methylpyridinium ether derivatives improve analytical specificity and sensitivity. Concentrations are commonly <50 pg/mL (<184 pmol/L) in men Liquid chromatography–mass spectrometry (LC–MS) provides an alternative analytical method of high specificity and indeed is already replacing immunoassays in the analysis of other steroids routinely monitored in clinical practice, e.g. testosterone and cortisol While ultra-sensitive methods have been developed using state-of-the art tandem MS N-methyl-nicotinic acid N-hydroxysuccinimide ester Previous derivatization reagents employed include dansyl chloride in vivo tracers or internal standards. Therefore a derivative generating analyte-specific precursor and product ions is desirable to improve specificity p-toluenesulfonate (FMP-TS) (Some of these problems can be averted by chromatographic separation but confounding interference can still arise from unknown estrogen metabolites or from stable isotope labeled estrogens used as (FMP-TS) , which hWe aimed to establish an analytical approach using a methylpyridinium derivative of estrogens to detect these hormones in low abundance in human plasma and, in particular, to evaluate if the approach could quantify circulating steroids in post-menopausal females. A highly sensitive method was validated using ion-exchange solid phase extraction, in conjunction with LC–MS/MS.22.1p-toluenesulfonate (FMP-TS) were from Sigma-Aldrich, Inc. . 3,4-[13C]2-estrone (13C2E1), 2,4,16,16-[2H]4-estrone (95-97%) (d4E1), 3,4-[13C]2-estradiol (13C2E2) and 2,4,16,16-[2H]4-estradiol (d4E2) were from Cambridge Isotope laboratories Inc. . HPLC grade glass distilled solvents  were from Fisher Scientific UK Limited .Estrone (E1), 17β-estradiol (E2), 17α-estradiol (17αE2), formic acid , triethylamine  and 2-fluoro-1-methylpyridinium-2.2Structural elucidation of derivatives of steroids in solutions of high concentration was performed on a LC triple quadrupole mass spectrometer, a TSQ Quantum Discovery MS  and operated using Xcalibur software version 2.0. Confirmation of accurate mass was performed by direct infusion into a 12 T SolariX dual source Fourier Transform Ion Cyclotron Resonance MS , operated with SolariX control v1.5.0 (build 42.8) software. High-sensitivity quantification was performed on a QTrap 5500  coupled to an Acquity™ Ultra Performance LC , operated using Analyst software v1.5.1.2.3Male and female human plasma and post-menopausal female human serum for method development and validation were from TCS Biosciences , stored at −20 °C. This was prepared from human blood from healthy donors in approved blood collection centers. Plasma was collected into anticoagulant (citrate phosphate dextrose adenine), whereas serum was collected without anticoagulant and allowed to clot naturally.For method application, serum was collected from 27 pre-menopausal women undergoing investigations for menstrual disorders (24–52 years old); these patient samples were anonymised prior to analysis and ethical approval was thus not required for this method development study. Further plasma samples were obtained from subjects participating in experimental medicine studies for which local ethical approval had been obtained; 20 post-menopausal women (58–60 years old) and 48 men (21–85 years old) men 2.4Estrogens and internal standards  were dissolved in acetone (5 mL) and stored at −20 °C. Working solutions (1 pg/mL to 100 µg/mL) were prepared by serial dilution on the day of use.2.5FMP-TS ) was freshly prepared prior to reaction and added to the standard/extract. The mixture was vortexed (10 s) then incubated . Mobile phase (water: methanol 65:35), containing FA  was added to quench the reaction.2.6m/z 100 to m/z 50 above the mass of the molecular ion under analysis. Conditions for multiple reactions monitoring (MRM) were optimized by autotuning during infusion of solutions of the E1 and E2 derivatives (50 ng/mL). Collision energy was optimized to achieve suitable fragmentation for each transition.For product ion characterization by tandem MS at nominal mass, molecular ions of derivatives were isolated by Q1 and subjected to collision-induced dissociation (CID) in Q2 . Q3 was operated in scanning mode from m/z 200–1500) were isolated for 20 s prior to CID experiments at 37 eV.For structural elucidation by FTICR-MS, ions of accurate mass . Optimized conditions achieving maximal sensitivity for detection of quantifier and qualifier ions are given in MRM conditions for estrogen FMP derivatives were tuned by infusion using a QTrap 5500 mass spectrometer, operated in positive ion electrospray ionization (ESI) mode. Estrogen FMP derivatives (1 µg/mL) were diluted 1:10 in mobile phase (water: methanol 65:35), containing FA (0.1%). Precursor ions for the derivatives were selected; 2.818 column . An isocratic solvent system of water: methanol (65:35), containing FA  was diverted to waste for the initial 2 minutes and maintained for a further 6 min followed by a rapid gradient executed over 2 min until final conditions of water: methanol (5:95), containing FA (0.1%) (0.4 mL/min) were achieved. The column and autosampler temperatures were 25 °C and 10 °C, respectively. Injection volume was usually 20 µL in partial loop mode with needle overfill.Chromatographic conditions were optimized using an Acquity UPLC® BEH C2.9Aliquots of pre-menopausal female serum (0.5 mL), post-menopausal female plasma (2 mL) and male plasma (1 mL) were subject to centrifugation for 20 minutes  and sediment removed. The sample volumes were adjusted to 2 mL with water and internal standards (200 pg) added. Solid-phase extraction using Oasis® MCX  extraction cartridges was performed under gravity. Prior to loading the sample (2 mL), the cartridges were conditioned with methanol (2 mL), followed by water (2 mL). The sample was loaded and allowed to pass through the cartridges and the eluate discarded. Next, the cartridges were washed with FA  and again the eluate discarded. Finally, the steroids were eluted in methanol (2 mL). Extracts were reduced to dryness under oxygen-free nitrogen (40 °C) and the residues were derivatized as above.2.102.10.1Reaction conditions were optimized first using aqueous extracts and then extracts of plasma. Conditions evaluated were incubation temperature (25–70 °C), time (5–60 min), reaction volume (25–250 µL) and FMP-TS concentration (0.1–10 mg/mL).2.10.2n=6). Mean peak areas of derivatives following extraction in pre-spiked samples were divided by those in post-spiked samples and expressed as a percentage.Recoveries of IS from water and plasma were assessed by comparison of signal intensities, following extraction from samples pre- and post-spiked with IS . Mean peak areas of derivatives of steroids from post-spiked extracted plasma was divided by mean peak areas of unextracted standards and expressed as a percentage.Ion suppression of signal of estrogenic derivatives in the presence of extracts of plasma was evaluated by comparing signal intensity of IS (200 pg) post-spiked into extracted plasma with that of aqueous IS solutions of the same concentration  were analysed by LC–MS/MS and the Signal/Noise (SNR) calculated from peak areas of steroids and adjacent background noise (over the same time window as the peak width). The LOD was assigned as a SNR ~3.2.10.6r, was >0.99. Weightings of 1, 1/x and 1/x2 were compared.Blank samples and aliquots containing estrogens  and internal standards (200 pg) were analysed by LC–MS/MS. Calibration curves were plotted as the peak area ratio (standard/IS) versus amount of estrogens. Calibration lines of best fit were acceptable if the regression coefficient, 2.10.7n=6) of estrogens and internal standards were prepared as above and analysed. The LOQ was calculated as that amount affording precision and accuracy of ~20% or less.Replicate aliquots . The precision was calculated as the Relative Standard Deviation (RSD) (standard deviation/mean×100), and % accuracy was the Relative Mean Error (RME) ×100).Injector reproducibility was tested by injecting the same extracts of male and female plasma 6 times on the same day. The intra- and inter-assay precision and accuracy were assessed using 6 samples  prepared on the same day and different days, respectively, alongside a standard curve (2.10.9Stability following storage in the auto-sampler (10 °C) was evaluated by reinjection of a calibration curve and plasma sample after 24 h. The effects of short-term storage in the freezer (−20 and −80 °C) on sample integrity were assessed by reinjection after 24 h and 48 h and 28 days storage.2.11Estradiol and estrone were quantified in human serum or plasma from pre and post-menopausal females and males using the validated approach.33.13.1.1m/z 362 and 364, respectively. However, after CID experiments, product ion spectra of E1 FMP and E2 FMP differed from each other and showed an analyte-specific fragmentation pattern as reported previously m/z 110) representing the derivatization group for both steroids did not dominate . The formation of m/z 252 was proposed to be a charge-remote concerted mechanism involving the elimination of CO as neutral loss before methyl migration from the 13C to the C16 position in the steroid ring, followed by loss of [C6H10]+ via a four-center concerted mechanism after α-cleavage of the C14-C15 bond. This may result in potential isomeric structures through the double bond at C9. The product ion at m/z 238 was proposed to form by loss of CH2  from C9, again creating two possible isomeric structures. FTICR-MS analysis confirmed the structural formulae of the product ions of m/z 252.13821 and 238.12260 within 0.3 and 0.2 ppm of the theoretical masses . Similarly for 13C2-E1, masses of the product ions (m/z 254.15090 and 240.13509) were within 0.2 and 0.4 ppm of the theoretical masses .Stable-isotopically labeled estrogens were used to elucidate potential fragmentation pathways and the nature of both quantifier and qualifier ions was proposed, based on a mechanism including α–β cleavages, retro-Diels-Alder, methyl migration and hydrogen shift (McLafferty +1 reaction) m/z 364 generated two product ions of significant abundance but with lower mass, at m/z 128 and 110, which underwent a mass increment to m/z 130 and 111, respectively for d4-FMP-E2. 13C2-FMP-E2 in contrast generated product ions of m/z 130 and 110. The product ion at m/z 110 was assumed to originate from the derivatization tag, proposed to form by β-cleavage of the ether bond at C4 followed by a proton migration via a (four or six) center concerted mechanism from either C2 or C4 of the steroid moiety. Therefore in analysis of d4-FMP-E2, an A-ring deuterium may migrate, generating a product ion of m/z 111. In contrast, the product ion at m/z 128 was proposed to form from the steroid moiety, incorporating the A-ring (and hence two deuteriums or two 13C atoms). The mechanism may be a charge-remote concerted mechanism causing dehydration of the secondary alcohol at C17 with further β-cleavage of the ether bond at C4 followed by a retro-Diels-Alder concerted mechanism, resulting in two possible conjugated isomers. Due to the lower masses of the product ions, further confirmation could not be obtained by FTICR-MS. The differences in behavior between the E1-FMP and E2-FMP fragmentation may relate to the unique stabilization of the conjugated system of the E1 related fragment at m/z 252, after ketone release and conformational rearrangement.Conversely, CID experiments carried out at 55 eV on the FMP-E2 precursor ion at m/z 128 as the quantifier ion for E2- FMP, as opposed to m/z 110, allowed distinction between analyte and A-ring isotopically-labeled estrogens, used either as internal standards or in vivo tracers. Phase 1 metabolism of estrogens via oxidation occurs in the A-ring, to form hydroxy and methoxy estrogens on the 2 and 4 positions Accordingly, distinct quantifier ions, derived from the steroidal structure, were selected  for E1 a3.1.2Reaction conditions to generate FMP derivatives were optimized first using aqueous extracts and then extracts of male and female plasma and final conditions reported. The efficiency of reaction was improved by increasing temperature from 25 to 40°C and time from 5 to 15 min, but further improvement was not seen at higher temperatures or with longer incubations. Reassuringly the intensity of signal was not adversely affected upon prolonged incubation, proving robustness of the protocol within day-to-day practice, but was improved by increasing the volume and concentration of FMP-TS from 25 to 50 µL and from 0.1 to 5 mg/mL, respectively3.2Baseline resolution between estrogen FMP derivatives within reasonable analytical time was achieved using a C18 UHPLC column. Typical retention times for E1, E2 and 17αE2 were 4, 4.5 and 7.5 minutes, respectively A. At ear3.3t-butyl ether 13C2E1 98±10% and 13C2E2 96±5%), but significant ion suppression was identified in the plasma extracts . Previous problems with ion suppression of derivatized estrogens by phospholipids have been reported 13C2E1 97±12% and 13C2E 90±6%), with minimum ion suppression .Previous methods to extract estrogens from biological matrices had often employed solvent extraction, e.g. with methyl 3.43.4.113C2 E1 1.1 and 13C2 E2 1.4), comparable to those in aqueous solutions, showing differences less than 20% . Interferences were not observed in plasma at the retention times of the internal standards (Baseline chromatographic separation of estrogens (E1 and E2) and endogenous isomers in plasma (17αE2) was achieved A. In platandards D–G.3.4.213C2 labeled estrogens were selected as internal standards, since loss of deuterium may happen during derivatization, particularly from the A-ring. At the outset of this work only estrogens with two labeled carbons were available, but subsequently those with three have become commercially available and may offer enhanced mass distinction of isotopologues in future studies. Linear standard curves of estrogen FMP derivatives were generated in the range (1–400 pg/sample) with a mean r value of 0.9953. Weighting of 1/x2 was applied, yielding a mean intercept of 0.0017. This range is similar to that determined using other sensitive derivatives developed recently 3.4.3The LOD and LOQs of FMP derivatives of both estrogens were 1 pg/sample (~0.2 pg on column)  at the LOQ of 1 pg/sample  and <15%3.4.5The FMP derivatives demonstrated suitable stability upon storage in an auto-sampler (10 °C) over 24 h, with limited degradation measured for derivatized E1  and E2  in extracts of samples from male and female subjects, respectively. Derivatives were stable upon short-term storage in the freezer  for 24 h; reduction from original response E1 , E2  in male and E1 , E2  in female. However, degradation of the derivative was significant after 48 h at −20°C; derivatized E1 , E2  in male and female extracts, respectively. At −80°C, stability of derivatives was acceptable after storage for 28 day; reduction from original response for E1 , E2  in male and female plasma, respectively.3.5The method was applied to samples from pre- and post-menopausal females and males and compared to reported biological concentrations . In clinIn pre-menopausal female serum 0.5 mL of serum was required to ensure reliable detection throughout the menstrual cycle. In males, E1 and E2 concentrations were quantified using 1 mL plasma, whereas with plasma from post-menopausal female 2 mL was required to ensure steroids were detected in all subjects. Using these volumes, all concentrations fell within the quantitation limits of the assay of (1–400 pg/sample) for E1 and E2 and quantitiation was achieved within the range of the standard curves.These volumes and LOQs compared favorably with previous literature. Volumes of serum or plasma from human and bovine in previous MS methods 4In summary, derivatization to form FMP ethers of phenolic estrogens, in conjunction with LC–MS/MS, is suitable for quantitative analysis of E2 and E1 in low abundance in biological fluids. This approach allows robust measurement across typical physiological ranges found in pre- and post-menopausal women and men, contrasting favorably with immunoassays (for which imprecise measurements are often recorded in the lower physiological range of concentrations Use of FMP-TS reagent offers improved specificity over previously reported derivatization approaches e.g. dansylation, but care should be taken to store derivatized samples at −80 °C if not being analysed within 24 h. Under these conditions all indices of assay quality were acceptable. The derivatization reaction may be applicable to other naturally occurring phenolic steroids.


The application of hydroxyethyl starch (HES) for volume resuscitation is controversially discussed and clinical studies have suggested adverse effects of HES substitution, leading to increased patient mortality. Although, the intestine is of high clinical relevance and plays a crucial role in sepsis and inflammation, information about the effects of HES on intestinal function and barrier integrity is very scarce. We therefore evaluated the effects of clinically relevant concentrations of HES on intestinal function and barrier integrity employing an isolated perfused model of the mouse small intestine.An isolated perfused model of the mouse small intestine was established and intestines were vascularly perfused with a modified Krebs-Henseleit buffer containing 3% Albumin (N=7) or 3% HES . Intestinal metabolic function , edema formation (wet-to-dry weight ratio), morphology , fluid shifts within the vascular, lymphatic and luminal compartments, as well as endothelial and epithelial barrier permeability (FITC-dextran translocation) were evaluated in both groups.Compared to the Albumin group, HES perfusion did not significantly change the wet-to-dry weight ratio and lactate-to-pyruvate ratio. However, perfusing the small intestine with 3% HES resulted in a significant loss of vascular fluid (p<0.01), an increased fluid accumulation in the intestinal lumen (p<0.001), an enhanced translocation of FITC-dextran from the vascular to the luminal compartment (p<0.001) and a significantly impaired intestinal galactose uptake (p<0.001). Morphologically, these findings were associated with an aggregation of intracellular vacuoles within the intestinal epithelial cells and enlarged intercellular spaces.A vascular perfusion with 3% HES impairs the endothelial and epithelial barrier integrity as well as metabolic function of the small intestine. A common therapy for the treatment of hypovolemia is the application of crystalloid and colloidal solutions for fluid resuscitation . Among oThe intestine is a typical barrier organ with a large inner surface area and one of its major function is to maintain a selective barrier between the organism and the environment . Under pTo gain insight into the possible effects of HES on intestinal function and barrier integrity, we evaluated the HES mediated cellular effects employing a newly established isolated perfused model of the mouse small intestine showing that the vascular perfusion with clinically relevant concentrations of HES impairs the endothelial and epithelial barrier integrity as well as metabolic function of the intestine.ad libitum for at least 24 hours before surgery. This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experiments were approved by the local authority .Female C57/BL6 mice  were used for all experiments. Animals were housed with standard diet and water To evaluate the intestinal effects of a vascularly perfused HES solution, two experimental settings were established. The first group  received a 135 min vascular perfusion with Albumin (3%) containing buffer. The second group  received a vascular Albumin (3%) perfusion for 60 min (equilibration phase) followed by a HES 130/0.4  perfusion for 75 min. Perfusions were continuously applied without intermittent stops. When establishing the mouse model of the isolated perfused intestine, various control experiments were performed to investigate the physiological and metabolic stability of the perfused intestines during the ex-vivo experiment. Employing the described experimental setup, mouse intestines are physiologically and metabolically stable for up to 135 minutes. This was the main reason, why all experiments were performed for a maximum time period of 135 minutes including an equilibration phase of 60 minutes. To exclude influences of the perfusion time on the observed effects, the results obtained at a respective time point in the HES perfusion group were compared to the respective time point in the Albumin perfusion group (inter group comparison) as well as to the “internal” HES control which consisted of 60 min equilibration with Albumin prior to the HES perfusion . Additionally, the vascular lactate concentration was evaluated using a blood gas analyser  and the lactate-to-pyruvate ratio was calculated.FITC-dextran  was added into the vascular perfusate in a concentration of 40 mg/l to determine the endothelial and epithelial permeability for macromolecules. Under physiological conditions the endothelial and also the epithelial barrier is impermeable for the 150 kDa FITC-dextran .t30 (19.36 ± 6.94) vs. HES t105 (8.74 ± 2.21); HES t120 (8.23 ± 2.75); HES t135 (7.28 ± 1.99); p < 0.05 for HES t105 and HES t120, p < 0.01 for HES t135; intra group comparison. Albumin t30 (17.34 ± 4.94) vs. Albumin t105 (9.42 ± 1.42); Albumin t120 (8.74 ± 2.41); Albumin t135 (9.11 ± 2.46); p < 0.05 for all; intra group comparison].As a parameter for the metabolic status the vascular lactate-to-pyruvate ratio (LPR) was determined and all values were within the physiological range of aerobic metabolism. There were no statistically significant differences between the time response characteristics of the LPR in the HES perfusion group and the time response characteristics of LPR in the Albumin perfusion group  was evaluated before (W/d1) and after (W/d2) the perfusion with HES and Albumin, respectively. No statistically significant differences were detected between the HES perfusion group and the Albumin perfusion group at the start or end of perfusion  vs. HES t105–120 (2.28 ± 0.51 ml ·15 min-1); HES t120–135 (3.04 ± 0.48 ml · 15 min-1); p < 0.001 for all; intra group comparison; t90–105 ; Albumin t105–120 (0.96 ± 0.08 ml · 15 min-1); Albumin t120–135 (1.01 ± 0.13 ml · 15 min-1) vs. HES t90–105 (1.66 ± 0.47 ml · 15 min-1); HES t105–120 (2.28 ± 0.51 ml · 15 min-1); HES t120–135 (3.04 ± 0.48 ml · 15 min-1); p < 0.001 for all; inter group comparison; Vascular HES perfusion resulted in an alteration of fluid distribution and significantly increased luminal flow [HES t45 (2.04 ± 1.73 03BCg· ml-1); HES t60 (1.57 ± 1.37 μg· ml-1); HES t75 (1.14 ± 1.41 μg· ml-1); HES t90 (2.70 ± 3.39 μg· ml-1) vs. HES t135 (16.71 ± 5.17 μg · ml-1); p < 0.05 for t45 and t90, p < 0.01 for t60, p < 0.001 for t75; intra group comparison; t105 (0.49 ± 0.26 μg· ml-1); Albumin t120 (0.47 ± 0.27 μg· ml-1); Albumin t135 (0.60 ± 0.37μg· ml-1) vs. HES t105 (7.03 ± 6.41 μg· ml-1); HES t120 (12.24 ± 5.91 μg· ml-1); HES t135 (16.71 ± 5.17 μg· ml-1); p < 0.001 for all; inter group comparison; t45, HES/Albumint60) can be explained by inter-individual variations of the intestines used in the respective perfusion experiments. The respective intestines were however not excluded from the study as all other physiologic and metabolic parameters were unaltered and within the expected limits.The endothelial and epithelial barrier integrity was estimated by determining the translocation of vascularly applied FITC-dextran into the luminal compartment. No changes in the luminal FITC-dextran concentration were detected in the Albumin perfusion group  vs. Albumin t105 (0.72 ± 0.21); Albumin t120 (0.68 ± 0.23); Albumin t135 (0.67 ± 0.28); p < 0.01 for t105, p < 0.001 for t120 and t135; intra group comparison; t60 (set to 1) vs. HES t75 (0.96 ± 0.21); HES t90 (0.59 ± 0.076); HES t105 (0.32 ± 0.04); HES t120 (0.17 ± 0.06); HES t135 (0.08 ± 0.03); p < 0.001 for all; intra group comparison; t105 (0.72 ± 0.21); Albumin t120 (0.68 ± 0.23); Albumin t135 (0.67 ± 0.28) vs. HES t105 (0.32 ± 0.04); HES t120 (0.17 ± 0.06); HES t135 (0.08 ± 0.03); p < 0.001 for all; inter group comparison; To validate the resorptive capacity and metabolic function of the isolated intestine during HES and Albumin perfusion, the luminal galactose uptake was measured. (LPR) Vascular perfusion with Albumin led to a time-dependent reduction of galactose uptake [Albumin The application of hydroxyethyl starch (HES) for volume resuscitation in critically ill patients is controversially discussed and several recent clinical studies have suggested adverse effects of HES substitution, leading to increased patient mortality , 3, 18. Using a model of the isolated perfused mouse small intestine in combination with Krebs-Henseleit based perfusion media, we analysed the intestinal effects of a vascular perfusion with 3% HES in relation to a control perfusion with 3% Albumin. The employed system is a further development of our previously described model of the perfused rat intestine  and was One of our main results is a significantly altered fluid shift from the intestinal vasculature into the lumen of the small intestine during HES perfusion, indicative of an increased endothelial and epithelial barrier permeability. Our flow-rate data showed an increased luminal flow while the venous flow was reduced under perfusion with HES, suggesting a loss of vascular fluid into the intestinal lumen due to negative effects of HES on endothelial and epithelial barrier integrity. These results were confirmed by our FITC-dextran measurements, where FITC-dextran of 150 kDa was supplemented to the vascular perfusion buffer. While in the Albumin perfusion group only very low concentrations of FITC-dextran appeared in the luminal effluents, HES perfusion resulted in a dramatic increase of luminal FITC-dextran levels, confirming the hypothesis that HES impairs the endothelial and epithelial barrier function in the small intestine. Our results are also in accordance with data published by Gao et al. who employed a rat model of hemorrhagic shock and detected an initially higher intestinal bacterial translocation to the liver after HES resuscitation compared to Ringer’s solution . A studyAlthough the clinical effects of a perfusion with HES are well described, the detailed mechanisms of HES on epithelial cells are still mainly unknown. Our electron microscopic experiments revealed numerous of intracellular vacuoles and enlarged intercellular spaces in the HES perfusion group, while these signs were absent after Albumin perfusion. Several authors detected similar structures in cells and classified them as HES containing vesicles that might be derived from lysosomes –28. OtheIn the intestine, the transfer of galactose across the luminal epithelium occurs by a sodium/galactose co-transporter (SGLT1), while the galactose transfer across the basolateral cell membrane is achieved by an uniporter (GLUT2) . Our galThe employed model of the isolated perfused small intestine offers the unique possibility to investigate organ-specific effects of a vascular perfusion with HES, while at the same time interfering systemical effects of HES are excluded. However, the following points should be considered when interpreting the results of our study. (I) A single pass application of a cell free vascular perfusate was performed in our study. Therefore, the model does not account for possible effects of systemic immune cells and humoral factors on the described HES and Albumin mediated mechanisms. (II) The perfusate used in our system was deficient in plasma proteins including coagulation factors, which may have an influence on the endothelial barrier function. (III) Possible effects mediated by the splanchnic nerves are not reflected in the perfused model of the small intestine, as the innervation was disrupted during organ preparation. However, the autonomously working enteric nervous system is still functioning as can be seen by the regular peristaltic movements of the perfused intestine in our model .Taken together, we show that a vascular perfusion with clinically relevant concentrations of HES impairs the endothelial and epithelial barrier integrity as well as metabolic function of the small intestine. Our ex-vivo experiments extend recent studies that suggested negative effects of HES during fluid resuscitation and we propose the small intestine as a potential target for HES mediated adverse effects.S1 FigVenous flow was measured in 15 minutes intervals (t30–45 = 1). Areas shaded in grey indicate the equilibration phase. Bars denote the mean ± SD. Albumin (N = 7), HES (N = 7). **, p < 0.01; ***, p < 0.001.(TIF)Click here for additional data file.S2 FigLymphatic FITC-dextran concentrations were evaluated every 15 minutes. Areas shaded in grey indicate the equilibration phase. Bars denote the mean ± SD. Albumin (N = 7), HES (N = 7). *, p < 0.05.(TIF)Click here for additional data file.S1 MovieReal-time movie; scale bar = 2cm.(MP4)Click here for additional data file.


The mammalian INO80 remodelling complex facilitates homologous recombination (HR), but the mechanism by which it does this is unclear. Budding yeast INO80 can remove H2A.Z/H2B dimers from chromatin and replace them with H2A/H2B dimers. H2A.Z is actively incorporated at sites of damage in mammalian cells, raising the possibility that H2A.Z may need to be subsequently removed for resolution of repair. Here, we show that H2A.Z in human cells is indeed rapidly removed from chromatin flanking DNA damage by INO80. We also report that the histone chaperone ANP32E, which is implicated in removing H2AZ from chromatin, similarly promotes HR and appears to work on the same pathway as INO80 in these assays. Importantly, we demonstrate that the HR defect in cells depleted of INO80 or ANP32E can be rescued by H2A.Z co-depletion, suggesting that H2A.Z removal from chromatin is the primary function of INO80 and ANP32E in promoting homologous recombination. The INO80 family of chromatin remodelling enzymes is defined by an insertion in the ATPase domain and, in addition to INO80, includes yeast Swr1 and human p400 and SRCAP. Evidence suggests that members of this family are capable of histone exchange reactions . We found that the accumulation of H2A.Z in laser-irradiated chromatin is unaffected by depletion of INO80. In contrast, the removal of accumulated H2A.Z in damaged chromatin is significantly slower in cells depleted of INO80 when compared with control cells Fig and E, iThere is evidence that INO80 contributes to HR in mammalian cells. In order to investigate the contribution of INO80 and H2A.Z to this pathway, we restricted our analyses to G2 cells using CENP-F, whose expression is limited to late S and G2 phases of the cell cycle We next monitored the accumulation of RAD51 foci following irradiation and found a more substantial defect in INO80-depleted cells Fig. It seemWe hypothesised that the removal of H2A.Z from damaged chromatin by INO80 may be an important aspect of the ability of INO80 to promote RAD51 foci formation. To test this, we co-depleted H2A.Z and INO80 Fig and analVery recently, the histone chaperone ANP32E was shown to remove H2A.Z from chromatin in mammalian cells, both globally The reduction in RAD51 foci formation in cells lacking INO80 or ANP32E suggests that HR is impaired in these cells. However, the reduction is modest when compared with cells lacking core HR proteins, such as BRCA2. We therefore monitored sister chromatid exchanges (SCEs) as a measure of HR completion. To do this, we treated cells with mitomycin C (MMC) because this results in a very substantial increase in SCEs, giving us the best possibility of detecting defects in the pathway. Additionally, unlike IR, MMC-induced lesions are repaired predominantly by HR.We found that cells lacking either INO80 or ANP32E show reduced numbers of SCEs when compared with control cells Fig and B. DCollectively our data suggest that H2A.Z is removed from chromatin after DNA damage by ANP32E and INO80, and this removal—either from the chromatin flanking the DSB, or from the sister chromatid, or both—is required for HR. Very recently, Price and colleagues also found that H2A.Z is removed from chromatin at sites of DNA damage in an ANP32E-dependent manner 2 atmosphere. Cells were irradiated by exposure to a 137Cs source.A549, HeLa or U2OS cells were cultured in MEM or DMEM (Gibco), respectively, supplemented with 10% FCS, L-glutamine, penicillin and streptomycin (Gibco) at 37°C in a humidified 95% air and 5% CO5 of logarithmically growing cells per condition. Cells were harvested 24 h later, retransfected with siRNA and were then seeded and grown for 48 h. The siRNA oligonucleotide used for ANP32E was 5′-CGGCUUCCCAGCUUAAAUA-3′ (Dharmacon).siRNA-mediated knockdown was achieved using HiPerFect Transfection Reagent (Qiagen) following the manufacturer’s instructions. siRNA duplexes (Dharmacon SMARTpool) were transfected into 4 × 10The primary antibodies used were as follows: γH2AX  at 1:800 for IF, RPA  at 1:100 for IF, RAD51  at 1:200 and CENP-F  for IF, INO80  at 1:2,000 for WB, H2A.Z  at 1:1,000 for WB, ANP32E  at 1:1,000 for WB, KAP1  at 1:1,000 for WB and KU80  at 1:1,000 for WB.The secondary antibodies used were as follows: FITC  at 1:100 for IF, Cy3  at 1:200 for IF, AlexaFluor 488  at 1:400 for IF, Goat Anti-Rabbit Immunoglobulin/HRP  at 1:2,000 for WB, Rabbit Anti-Mouse Immunoglobulins/HRP  at 1:2,000 for WB.A549 cells plated on glass slides were fixed for 10 min with fixative (2% (w/v) PFA, 3% (w/v) sucrose, 1× PBS) and permeabilised for 3 min with 0.2% Triton X-100 in PBS. When staining for RPA/RAD51, pre-extraction was performed by treatment with 0.2% Triton X-100 in PBS for 0.5–1 min prior to PFA fixation. Cells were rinsed with PBS and incubated with primary antibody diluted in PBS + 2% (w/v) BSA for 1 h at room temperature (RT). Cells were washed three times, incubated with secondary antibody (diluted in PBS + 2% (w/v) BSA) for 30 min at RT in the dark, incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and washed three times with PBS. Slides were mounted using Vectashield and visualised/analysed using a Nikon-e400 microscope and imaged using an Applied Precision® Delta Vision® RT Olympus IX70 deconvolution microscope and softWoRx® Suite software. For γH2AX, RPA, and RAD51 foci quantification, a minimum of 30 cells per experiment was scored blindly and error bars represent the SD between three experiments.For analysis in FigAnalysis of SCEs was carried out as described previously Exponentially growing human U2OS cells were plated onto 35-mm glass-bottom dishes (MatTek) and transfected with the pTGFP-H2AZ construct, pEGFP-RUVBL2 (Origene) or pEGFP-H2B using NanoJuice according to the manufacturer’s protocol. The cells were allowed to express the construct for 24 h and were then incubated with 10 μg/ml Hoechst 3458 for 30 min at 37°C before irradiation. The microscope system used was an Intelligent Imaging Innovations spinning disc confocal with a Yokogawa CSU-X1 on an Olympus IX-71. GFP-positive cells were irradiated with 405-nm ultraviolet laser set at power of 7:1,000 for either H2AZ or H2B and at 30:1,000 for RUVBL2 and channelled through a 60× objective. Images were captured at 10-s intervals following laser damage for a total time of 5 min for H2AZ or H2B and 30 min for RUVBL2. Images generated were acquired on a Photometrics Evolve 512 × 512 EMCCD using Slidebook 6 software. In protein recruitment experiments, signal intensity was quantified along the laser path, using Slidebook 6 software, in a minimum of 10 cells and error bars represent the SD between three independent experiments.


In general, 3,5-diiodothyronine  increases the resting metabolic rate and oxygen consumption, exerting short-term beneficial metabolic effects on rats subjected to a high-fat diet. Our aim was to evaluate the effects of chronic 3,5-T2 administration on the hypothalamus–pituitary–thyroid axis, body mass gain, adipose tissue mass, and body oxygen consumption in Wistar rats from 3 to 6 months of age. The rats were treated daily with 3,5-T2  for 90 days between the ages of 3 and 6 months. The administration of 3,5-T2 suppressed thyroid function, reducing not only thyroid iodide uptake but also thyroperoxidase, NADPH oxidase 4 (NOX4), and thyroid type 1 iodothyronine deiodinase (D1 (DIO1)) activities and expression levels, whereas the expression of the TSH receptor and dual oxidase (DUOX) were increased. Serum TSH, 3,3′,5-triiodothyronine, and thyroxine were reduced in a 3,5-T2 dose-dependent manner, whereas oxygen consumption increased in these animals, indicating the direct action of 3,5-T2 on this physiological variable. Type 2 deiodinase activity increased in both the hypothalamus and the pituitary, and D1 activities in the liver and kidney were also increased in groups treated with 3,5-T2. Moreover, after 3 months of 3,5-T2 administration, body mass and retroperitoneal fat pad mass were significantly reduced, whereas the heart rate and mass were unchanged. Thus, 3,5-T2 acts as a direct stimulator of energy expenditure and reduces body mass gain; however, TSH suppression may develop secondary to 3,5-T2 administration. Several years ago, it was shown that 3,5-diiodothyronine  is responsible for certain non-genomic effects of thyroid hormones on the resting metabolic rate (RMR) and oxygen consumption  levels as well as TSH secretion from rat pituitary fragments, and it was also able to decrease the TSH levels of hypothyroid rats (d2 (dio2) mRNA levels in the liver  with daily exposure to a 12 h light:12 h darkness cycle and were provided free access to water and standard rat chow. This investigation conforms to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and was approved (number: IBCCF 070) by the Institutional Animal Care and Use Committee . All animals were individually housed for a 1-week acclimation period.3, and T4 could be carried out. Hearts, retroperitoneal fat pads , and epididymal fat pads were removed and weighed. Liver, kidneys, thyroid glands, hypothalami, and pituitaries were dissected out and stored at −70 °C until processing for enzymatic measurements or for RNA extraction procedure.Three-month-old male Wistar rats were divided into the following groups: control (daily s.c. injections of vehicle were administered), 25 μg 3,5-T2 ), 50 μg 3,5-T2 , and 75 μg 3,5-T2 . The experiments were conducted for 3 months. Compound 3,5-T2 (Sigma) was dissolved in 0.04 M NaOH and subsequently diluted with saline solution  at the time of administration , which maintained equal alkalinity in all of the injected solutions. Animals were weighed at the beginning of the treatment and just before killing; food ingestion was determined for 24 h before killing. After the experimental period, animals were killed by decapitation, and blood was collected. The serum was obtained and stored at −20 °C until specific RIA to determine serum TSH, TThe fasting serum insulin concentrations were measured using a commercial RIA kit for rat insulin , and inter- and intra-assay coefficient of variation (CV) values were 5.0 and 8.9% respectively.The serum TSH levels were evaluated using a specific RIA obtained from the National Institute of Diabetes, Digestive and Kidney Diseases  and expressed in terms of the reference preparation 2 (RP2). The intra- and inter-assay CV values were 7.7 and 6.5% respectively and the sensitivity was 0.63 ng/ml.3 and T4 concentrations were measured using commercial RIA kits based on the presence of specific antibodies adhered to the internal surface of propylene tubes. T3: DLS – 3100 Active, sensitivity of 4.3 ng/dl, inter- and intra-assay CV values varied from 4.2 to 6.0% and from 5 to 6.5% respectively; T4: DLS – 3200 Active, sensitivity of 0.4 μg/dl, inter- and intra-assay CV values varied from 7.1 to 7.4% and from 2.9 to 5.1% respectively .Total serum TAll procedures were carried out following the manufacturer's recommendations.A glucose tolerance test was carried out before the commencement of 3,5-T2 treatment and after 1, 2, and 3 months of treatment, as described previously . Spontaneous activity was measured in the same metabolic cage that contains four sensors in the housing base to detect the movement of the animal. Oxygen consumption and spontaneous activity were recorded at 5-min intervals, and the results were expressed in ml/min per kg0.75 of O2 and horizontal activity in counts/min respectively. To avoid disruption caused by adaptation to the chamber, the first 3 h (from 1800 to 2100) were excluded from the analyses.We evaluated the RMR of rats that received 50 μg 3,5-T2  and control rats, as described previously , with a low pass signal filtered at 500 Hz, and digitalized at 1 kHz by a 16-bit A/D converter  using the Lab chart 7.0 software (AD Instruments).3 (Sigma), equal volumes of [125I] rT3  previously purified using Sephadex LH-20, and 10 mM DTT (USB) in 100 mM potassium phosphate buffer containing 1 mM EDTA (pH 6.9). The reaction volume was 300 μl, in the presence or absence of propylthiouracil (PTU) (1 mM) in order to measure specific D1 activity. For the type 2 deiodinase activity assay, duplicate homogenates  were incubated in a water bath for 3 h at 37 °C with 1 nM T4 (Sigma), equal volumes of [125I] T4 (PerkinElmer Life Sciences) previously purified using Sephadex LH-20, 1 mM PTU, and 20 mM DTT (USB) in 100 mM potassium phosphate buffer containing 1 mM EDTA (pH 6.9). The reaction volume was 300 μl, in the presence or absence of excess of T4 (100 nM) in order to inhibit D2 in the blank assay tube. For D1, blank incubations were carried out in the absence of protein and the presence of 1 mM PTU. For D2, blank incubations were carried out in the presence of 100 nM T4.The 5′-deiodinase activities in the liver, kidney, thyroid, pituitary, and hypothalamus were evaluated as described previously  and 200 μl trichloroacetic acid . Samples were centrifuged at 8000 d method  after in125I-NaI administration (short-term iodide uptake) reflects iodide transport through the sodium–iodide symporter (NIS) without the influence of in vivo thyroid iodine organification activity  15 min before decapitation. The thyroid glands were removed and weighed. The radioactivity of the thyroid glands was measured using a gamma counter (LKB) and expressed as percentage of total 125I injected per milligram of thyroid.We had previously demonstrated that the measurement of radioiodide uptake 15 min after g at 4 °C for 1 h. The pellet was suspended in 0.5 ml Triton  and incubated at 4 °C for 24 h to solubilize TPO. The triton-treated suspension was centrifuged at 100 000 g at 4 °C for 1 h, and the supernatant containing solubilized TPO was used for the assays. The protein content was determined by the Bradford method (Thyroperoxidase (TPO) extraction and activity measurement were carried out as described previously , 24 mM KI, 11 mM glucose, and increasing amounts of solubilized TPO. The final volume was adjusted to 2.0 ml with 50 mM sodium phosphate buffer (pH 7.4), and the reaction was initiated by the addition of 10 μl of 0.1% glucose oxidase (Boehringer Grade I). The increase in absorbance at 353 nm (triiodide production) was recorded for 4 min on a Hitachi spectrophotometer (U-3300). The ΔA353 nm/min was determined from the linear portion of the reaction curve and related to protein concentration. One unit of activity corresponds to ΔA353 nm/min=1.0.2O2 generation was quantified in thyroid particulate fractions by the Amplex red/HRP assay , which detects the accumulation of a fluorescent oxidized product. For particulate preparation, the excised thyroid glands remained at 4 °C for 24 h in 50 mM sodium phosphate buffer (pH 7.2) containing 0.25 M sucrose, 0.5 mM DTT, 1 mM EGTA, 5 mg/ml aprotinin, and 34.8 mg/ml phenylmethylsulfonyl fluoride (PMSF) before homogenization. Then, the homogenate was centrifuged at 100 000 g for 35 min at 4 °C and suspended in 0.25 ml of 50 mM sodium phosphate buffer (pH 7.2) containing 0.25 M sucrose, 2 mM MgCl2, 5 mg/ml aprotinin, and 34.8 mg/ml PMSF. This particulate fraction was incubated in 150 mM sodium phosphate buffer (pH 7.4) containing 100 U/ml superoxide dismutase (Sigma), 0.5 U/ml HRP (Roche), 50 μM Amplex red , 1 mM EGTA, with or without 1.5 mM CaCl2, and the fluorescence was immediately measured in a microplate reader  at 30 °C, using wavelength excitation at 530 nm and emission at 595 nm. The H2O2 production was quantified using standard calibration curves.H2, 2.7 mM KCl, 20 mM Tris (pH 8.0), 1% Triton, 10% glycerol, and protease and phosphatase inhibitors  and subsequently homogenized using an Ultra-Turrax Homogenizer (IKA). The samples were then centrifuged at 570 g for 10 min at 4 °C, and the supernatant was collected. An aliquot was used to determine the protein concentration via the BCA protein assay kit , following the manufacturer's instructions. The protein samples were then resolved by SDS–PAGE, transferred onto PVDF membranes, and probed with the indicated antibodies. The NIS antibody was kindly provided by Dr Nancy Carrasco, the dual oxidase (DUOX) antibody was kindly provided by Dr Corinne Dupuy, the anti-NADPH oxidase 4 (NOX4) primary antibody (NB 110-58851) was obtained from Novus Biologicals , and TSHR antibody was purchased from Santa Cruz Biotechnology. GAPDH was used as an internal control , and an anti-rabbit IgG HRP-linked antibody  was used as a secondary antibody. The immunoblots were developed using ECL.Thyroids were placed in lysis buffer containing 135 mM NaCl, 1 mM MgClSupplementary Table 1, see section on CT.Thyroid and hypothalamic total RNAs were extracted using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's instructions. After DNase treatment, RT was carried out followed by real-time PCR as described previously , Tpo, D1 (Dio1) and Tshr mRNA levels were analyzed by the unpaired t-test. Statistical analyses were carried out using the GraphPad Prism software . Differences were considered to be statistically significant when P<0.05.The results are expressed as the mean±After 3 months of 3,5-T2 administration, the final body mass was significantly reduced when compared with the control group independent of the dose . Body mass gain during the treatment was ≈30% lower in rats treated with 3,5-T2 when compared with those in the control group A. AbsoluControl male Wistar rats developed impaired glucose tolerance at 6 months of age B, and ba3 and T4 levels were reduced in rats treated with 3,5-T2, and the reduction was dose dependent  protein levels. The protein level of both α- and β-TSHR subunits B and the content C were siTrh mRNA content did not significantly differ between the groups .Hypothalamic 2O2 generation  protein expression and insulin-induced Akt phosphorylation  mRNA levels . The regulation of the D1 gene by T3 is mediated by the TRβ isoform because T3 induction of hepatic and renal D1 is severely weakened in Thrb1 (Thrb)-null mice but is normal in Thra1 (Thra)-null mice (in vivo. By contrast, in the thyroid gland, D1 activity is under the positive control of TSH through the cAMP pathway (Hepatic and kidney D1 activities were significantly increased in rats treated with 3,5-T2 despite significantly reduced serum Tull mice . Thus, 3 pathway . As a re4. As T4 levels are reduced in 3,5-T2-treated rats, this could explain the increase in D2 activity in these animals.On the other hand, D2 activity was increased in both the hypothalamus and pituitary of rats treated with 3,5-T2, despite the thyromimetic effects of 3,5-T2. It has been shown that D2 protein levels and activity are regulated by post-translational mechanisms, especially ubiquitination (Tshr mRNA levels were increased in the thyroids of 3,5-T2-treated rats. It has been shown that TSH exerts inhibitory effects on Tshr promoter activity (Taken together, our results show that 3,5-T2 significantly downregulates thyroid function. In addition to the decreased levels of thyroid hormones, we have also observed a reduction in the thyroid D1, NIS, and TPO activities as well as their respective mRNA levels. Iodide uptake is a fundamental step in thyroid hormone biosynthesis, and TPO is a key enzyme in thyroid hormone biosynthesis ; therefoIn conclusion, chronic 3,5-T2 administration reduced body mass gain and retroperitoneal fat mass and increased the RMR regardless of decreased thyroid hormone levels. The reduction in thyroid hormone levels might be secondary to the decreased serum TSH levels, leading to the reduced activity and expression of NIS, thyroid D1, and TPO. These new data support the idea that exogenous 3,5-T2 causes TSH suppression. Thus, future studies on the possible deleterious effects of hypothyroidism in tissues where 3,5-T2 might not act as a thyromimetic agent are of great importance before this thyroid metabolite can be used as a pharmacological agent.http://dx.doi.org/10.1530/JOE-13-0502.This is linked to the online version of the paper at


Tityus fasciolatus. Tf2 is identical to Tb2-II found in Tityus bahiensis. We found that Tf2 selectively activates human (h)Nav1.3, a neuronal voltage-gated sodium (Nav) subtype implicated in epilepsy and nociception. Tf2 shifts hNav1.3 activation voltage to more negative values, thereby opening the channel at resting membrane potentials. Seven other tested mammalian Nav channels  expressed in Xenopus oocytes are insensitive upon application of 1 μM Tf2. Therefore, the identification of Tf2 represents a unique addition to the repertoire of animal toxins that can be used to investigate Nav channel function.We identified Tf2, the first β-scorpion toxin from the venom of the Brazilian scorpion  Tityus fasciolatus scorpion is found in the Cerrado biome in Brazil and is phylogenetically related to Tityus serrulatus, a scorpion responsible for many envenomation cases [T. serrulatus however, getting stung by T. fasciolatus is less likely since this scorpion is predominantly found in termite hills and rarely moves into human habitats. The venom toxicity of T. fasciolatus is considered moderate with a median lethal dose (LD50) of 56.65μg/mouse [v) channels [v channels [The on cases ,2. In coμg/mouse . So far,channels . Since tl dose LD0 of 56.6i.e. fast inactivation) [+ current when the channel should no longer conduct. In contrast to α-scorpion toxins, β-scorpion toxins activate Nav channels at voltages where they should normally be closed without affecting fast inactivation [v channel activation [v channel function [v channel isoforms found in mammals. Such a tool would be beneficial to probe the physiological role of a particular isoform in an organism [α-Scorpion toxins primarily interfere with the voltage-drive activation process of the domain IV voltage sensor within the channel –8. Sinceivation) –17, α-sctivation ,19. In gtivation –23. Givefunction . Howeverorganism .T. fasciolatus on an array of Nav channel isoforms, we came across a fraction that selectively activated human (h)Nav1.3, a neuronal subtype believed to be involved in epilepsy as well as pain perception after channel upregulation due to spinal cord injury [T. fasciolatus. Tf2 can shift hNav1.3 activation voltage to much more negative values, effectively opening the channel at resting membrane potentials. Remarkably, seven other tested mammalian Nav channels  are insensitive to Tf2 upon application of an identical quantity. As such, the identification of Tf2 represents a distinct addition to the range of animal toxins that can be used to investigate hNav1.3 function.While testing the venom of d injury –27. FurtT. fasciolatus were collected in Brasilia, Federal District, Brazil (15°46’S 47°50’W), under license n° 19138–1 (IBAMA- Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais). They were kept at appropriate facilities at the University of Brasilia with food and water ad libitum. Scorpions were submitted to electrical stimulation close to the telson, respecting a 30 day interval. The venom was collected, solubilized in a watery trifluoroacetic acid (TFA) 0.12% solution, and centrifuged at 15,000 × g for 15 min. The supernatant was collected and quantified at 280 nm and dried. Hereafter, the material was kept at -20°C.Specimens of T. fasciolatus venom was dissolved in purified water and separated by RP-HPLC (reverse-phase high performance liquid chromatography) using a C18 analytical column , using a linear gradient from 0% solvent A (0.12% TFA in water) to 60% solvent B (0.10% TFA in acetonitrile) over 60 min at a flow rate of 1 mL/min, with detection at a wavelength of 216 and 280nm. The optimized chromatography protocols used to purify Tf2 consisted of three extra steps with a gradient from 25% to 45% solvent B in 40 min with the two last steps performed at 45°C. The purity of the toxin was verified by MALDI-TOF mass spectrometry and by the area under curve on HPLC and was higher than 97%.Dried The molecular mass and purity of the sample was checked on an Ultraflex III MALDI TOF mass spectrometer . The sample was dissolved in an α-cyano-4-hydroxycinnamic acid matrix , spotted onto the MALDI target plate and dried at room temperature for 15 min and analyzed in positive reflected and linear modes. External calibration was preceded by a Peptide Calibration Standard for mass spectrometry  for reflector mode and Protein 1 mixture  for linear mode. Spectra were analyzed with FlexAnalysis 3.3 . In order to check the accurate monoisotopic molecular mass of Tf2, a micrOTOF-QII  equipped with an orthogonal electrospray ionization source (ESI) operated in the positive mode was used. Sample was diluted in variable concentrations of 1% formic acid in a water/acetonitrile mixture  and applied to the mass spectrometer source by direct infusion.T. fasciolatus venom gland . The signal peptide was identified using SignalP 4.1 server [http://www.ncbi.nlm.nih.gov/blast) with an e value cutoff set to <10–5 to identify putative functions. ClustalW version 2.0 [The complete amino acid sequence was obtained from a transcriptomic library performed with RNA isolated from the 1 server , and thesion 2.0  was usedsion 2.0 . The proModel construction of Tf2, Ts2 and CssIV was performed by the fold recognition approach for identifying potential templates using the Phyre2 server . This sev1.4, hNav1.5, and rat (r)Nav1.8 were a gift from Peter Ruben , Chris Ahern , and John Wood , respectively. hNav1.1–1.3, hNav1.6–1.7 were obtained from Origene Technologies, Inc. . Accession numbers are NM_001165963.1 (hNav1.1), NM_021007.2 (hNav1.2), NM_006922.3 (hNav1.3), NM_000334 (hNav1.4), NM_198056 (hNav1.5), NM_014191.2 (hNav1.6), NM_002977.2 (hNav1.7), and NM_017247.1 (rNav1.8). cRNA of hNav1.1–1.7 and rNav1.8 was synthesized using T7 polymerase  after linearizing the fully-sequenced DNA with appropriate restriction enzymes. Channels were expressed together with the hβ1 subunit  (1:5 molar ratio) in Xenopus laevis oocytes  that were incubated at 17°C in 96 mM NaCl, 2 mM KCl, 5 mM Hepes, 1 mM MgCl2, 1.8mMCaCl2, and 50 g/mL gentamycin (pH 7.6 with NaOH) for 1–4 days after cRNA injection, and then were studied using two-electrode voltage-clamp recording techniques  with a 150-μl recording chamber. Data were filtered at 4 kHz and digitized at 20 kHz using pClamp10 software . Microelectrode resistances were 0.5–1 MΩ when filled with 3 M KCl. The external recording solution contained 100 mM NaCl, 5 mM Hepes, 1 mM MgCl2, and 1.8 mM CaCl2 (pH 7.6 with NaOH). All experiments were performed at room temperature (22°C). Leak and background conductance, identified by blocking the channel with tetrodotoxin , were subtracted for all Nav channel currents. The G-V curves were plotted with OriginPro8 (3<n<6), normalized, and fitted with the Boltzmann equation. A t-test was carried out to calculate the statistical relevance of each experiment with p<0.05.Human (h)NaT. fasciolatus venom was divided into 60 fractions using reversed-phase HPLC with a linear acetonitrile gradient and Tf2 was collected at 38.5% of acetonitrile  obtained by using the micrOTOF QII  or an aspartic acid . Another feature observed in all analyzed structures is the presence of a positive region close to the negative core, i.e. a lysine residue situated at position 1 for the β-scorpion toxins, and position 2 for the α-scorpion toxin AaHII. Comparing Tf2 to other scorpion toxins suggests that positively charged groups at positions 1 and 12 , as well a negative charge at position 2 are likely determinants of β-scorpion toxin specificity [58 in AaH2, 12 and A58   (v channel isoforms.The primary sequences of Tf2 and Ts2 differ in three residues (Snd N51H) , among wosition) , face A.osition) , face B.50 found in Ts2   Figs  and 3B. ly) Figs  and at fly) Figs . As expechannels . Howeverv channel isoforms expressed in X. laevis oocytes before and after toxin addition. At 1 μM, Tf2 does not influence currents generated by hNav1.1–1.2, 1.4–1.7, and rNav1.8 . To a certain extent, this effect resembles that of Ts2, which promotes opening of rNav1.3 when applying 1 μM toxin [v channel isoforms whereas Tf2 does not appear to influence activation or fast inactivation of other Nav channel isoforms. Thus, Tf2 activity seems to be unique among β-scorpion toxins and may be valuable to further hNav1.3 research.To examine the biological activity of Tf2, we used the two-electrode voltage-clamp technique to measure sodium currents from 8 mammalian Na rNav1.8 ; howeverμM toxin . HoweverT. fasciolatus. In contrast to related β-scorpion toxins [v1.3 opening, resulting in a dramatic shift in activation voltage. As a result, delivery of 1 μM Tf2 to Nav1.3 expressed in neurons may cause the channel to open at resting membrane voltages, an effect that would be detrimental to the envenomed organism.In this work, we report the discovery and characterization of Tf2, the first β-scorpion toxin from the venom of n toxins ,44,45,4920A, S50D, and N51H). As a result of the substitution S50D, Ts2 has a lower overall charge that may affect toxin ability to interact with its binding site, which may be located within the lipid membrane. In Ts1, the residue W50, equivalent to the residues H51 in Ts2 or N51 in Tf2, is crucial for toxin activity [v channels are due to this substitution or due to the charge difference caused by S50D. Hence, in order to elucidate the different effects of Tf2 and Ts2, it will be interesting to perform site-directed mutagenesis and test both Tf2 and Ts2 under similar electrophysiological conditions.Given its exquisite selectivity pattern, it is interesting to consider the mechanism underlying Tf2 activity. When comparing the amino acid sequence of Tf2 and Ts2, only three substitutions can be found , a negative potential at position 2 (E2), a positively charged group at position 12 (K12) and a solvent-exposed aromatic core . In all presented structures, the central negative residue (E2 or D3) is surrounded by the aromatic core. At face B, Tf2 and Ts2 possess an Y28 instead of the E28. As discussed by Cohen et al., [12, the toxin AaHII has a lysine residue at position 58, as a proposed conserved feature in α-scorpion toxins.Overall, Tf2 possess all the important characteristics mentioned by Polikarpov et al.,  that are et al., , the chav channels [v1.1-r1.8  from hNav1.3. In contrast to α-scorpion toxins for which an interaction with the paddle motif is the domain IV voltage sensor is sufficient to exert their effect [v channel isoform selectivity of Tf2 may be determined by amino acids in regions outside of the anchoring point in the domain II paddle motif. Further mutagenesis studies will therefore help delineate the complete binding site of Tf2. Finally, it is also worth considering that the extent of the effect on hNav1.3 as observed in the X. laevis system, may differ in mammalian neurons due to a different membrane lipid composition or the presence of β-subunits [v1.3 in epilepsy or nociception after spinal cord injury [e.g. FLIPR) in which voltage-control is impractical to open the channel in the presence of therapeutics [In general, β-scorpion toxins interact primarily with the S3b-S4 paddle motif within the domain II voltage sensor in Nachannels ,18–23. H1.1-r1.8 . Even thr effect ,56–59, ir effect ,51,60–62subunits ,64. Altod injury –27. Moreapeutics ,65.


Studies have shown that a major metabolite of the red ginseng ginsenoside Rb1, called 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (GPD), exhibits anticancer properties. However, the chemotherapeutic effects and molecular mechanisms behind GPD action in human melanoma have not been previously investigated. Here we report the anticancer activity of GPD and its mechanism of action in melanoma cells. GPD, but not its parent compound Rb1, inhibited melanoma cell proliferation in a dose-dependent manner. Further investigation revealed that GPD treatment achieved this inhibition through the induction of autophagy and apoptosis, while Rb1 failed to show significant effect at the same concentrations. The inhibitory effect of GPD appears to be mediated through the induction of AMPK and the subsequent attenuation of mTOR phosphorylation. In addition, GPD activated c-Jun by inducing JNK phosphorylation. Our findings suggest that GPD suppresses melanoma growth by inducing autophagic cell death and apoptosis via AMPK/JNK pathway activation. GPD therefore has the potential to be developed as a chemotherapeutic agent for the treatment of human melanoma. Malignant melanoma is one of the most aggressive forms of cancer, and advanced melanoma has a very poor prognosis. Every year in the US, more than 75,000 people are diagnosed with melanoma, of which 10,000 patients will die of the disease Autophagy is a complex process that enables cells to recycle cellular components during times of stress. In certain situations, its activation can function as a tumor suppressor by blocking pathways that lead to cancer cell proliferation in vivoIn East Asia, ginseng has been employed as a traditional medicine for a variety of ailments for more than 5,000 years Our study is the first to demonstrate the therapeutic potential of GPD and associated signaling mechanisms involved in the induction of autophagy and apoptosis in human melanoma. We report that GPD induces AMPK/JNK activation, leading to the inhibition of malignant cell growth by increasing autophagic and apoptotic processes.GPD was obtained from the Ambo Institute . The antibody against LC3 was purchased from ABGENT Inc. , while antibodies against phosphorylated AMPKα (Thr172), total AMPKα, phosphorylated c-Jun NH2-terminal kinases , total JNK, Beclin-1, phosphorylated Bcl-2, cytochrome C, PARP, caspase-3, 7, 9, phosphorylated ATF2, ATF, phosphorylated c-Jun (Ser73), total c-Jun, phosphorylated mTOR (Ser2448) and total mTOR were purchased from Cell Signaling Technology . The antibodies against p62/SQSTM1, Bcl-2, α-tubulin and β-actin were purchased from Santa Cruz Biotechnology, Inc. . The BCA protein assay kit and mitochondrial isolation kit for cultured cells were obtained from Thermo Scientific . Puromycin was purchased from InvivoGen . 5-bromo-2′-deoxyuridine and anti-bromodeoxyuridine were purchased from Roche Applied Science .2 incubator at 37°C. For transfection experiments, jetPEI  transfection reagent was used, following the manufacturer's instructions.SK-MEL-28 cells were obtained from ATCC . The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, antibiotics and 1× MEM non-essential amino acids in a 5% CO5) were seeded in 60 mm dishes and treated with 20 µM, 40 µM and 60 µM of GPD or 60 µM of ginsenoside Rb1 for 48 h. Cells were labeled by incubation with 10 µM BrdU for 30 min before harvesting. The cells were harvested and fixed with 70% ethanol at 4°C for at least 30 min. After denaturing cellular DNA, cells were suspended in 100 µL solution containing 2 µg/mL anti-BrdU antibody diluted in PBS containing 0.1% BSA and incubated for 30 min at room temperature. Cells were washed with PBS twice and re-suspended in 100 µL of diluted goat anti-mouse IgG-FITC. Cells were analyzed on a FACS Calibur flow cytometer (BD Biosciences) and the ratio against untreated control of BrdU labeled cells was measured.The BrdU assay was used to evaluate the effects of GPD on SK-MEL-28 cell proliferation. Cells (1.2×103 per well) were suspended in BME (1 mL with 10% FBS and 0.33% agar) and plated over a layer of solidified BME/10% FBS/0.5% agar (3 ml) with various concentrations of GPD or 40 µM of Rb1. The cultures were maintained at 37°C in a 5% CO2 incubator for up to 7 days, and the cell colonies were counted under a microscope with the aid of Image-Pro Plus software .Cells (8×103) were seeded in 4-chamber polystyrene vessel tissue culture glass slides , and treated with 20, 40 µM GPD or 40 µM Rb1 for 72 h in a 37°C incubator. Cells were fixed with 4% formalin for 15 min at room temperature in the dark. After washing twice for 10 min each with PBS containing Ca+ and Mg+, cells were blocked with PBS/0.02% Tween20/1% BSA in a 37°C incubator for 1 h. Cells were then incubated with a 1∶100 dilution of p62/SQSTM1 mouse antibody at 37°C for 1 hr. After washing three times for 5 min each with PBS/0.02% Tween20/1% BSA, cells were incubated with a 1∶1000 dilution of Alexa Fluor 568 goat anti-mouse antibody for 1 h in the dark. Co-localization of proteins was observed by laser scanning confocal microscopy  using a CFI Plan Fluor 40× oil objective and then analyzed using the EZ-C1 (v3.90) software program.SK-MEL-28 cells were stably transfected with GFP-LC plasmids and selected using 2 µg/ml of puromycin. Cells (5×106) were seeded and incubated for 24 h. Cells were then treated with GPD  or Rb1 (40 µM) for 72 hr. The cells were disrupted by adding lysis buffer  with 5 µl of proteinase K and incubated at 50°C for 4 h, and centrifuged at 12,000× g for 30 min at 4°C. DNA was extracted twice with phenol∶chloroform∶isopropyl alcohol (25∶24∶1). The supernatant fraction containing fragmented DNA was mixed with 0.5 ml of isopropanol at room temperature for 10 min and centrifuged at 12,000 g for 10 min at 4°C. The DNA pellet was washed with 75% ethanol and resuspended in Tris-HCl (pH 8.0) with 100 µg/ml RNAse for 2 h. DNA fragments were separated using 1.8% agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV light.SK-MEL-28 cells . The ratio of total apoptosis was measured in SK-MEL-28 cells that were treated with various concentrations of GPD or 40 µM of Rb1 for 48 h. The cells were harvested and washed with PBS and incubated for 5 min at room temperature with annexin V-FITC, plus propidium iodide following the protocol instructions. Cells were analyzed on a FACS Calibur flow cytometer (BD Biosciences).Proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore), which were blocked with 5% skim milk and incubated with specific primary antibodies overnight at 4°C. The protein bands were visualized using ECL solution (Thermo Scientific) after hybridization with a horseradish peroxidase–conjugated secondary antibody.Bcl-2 antibody was used for immunoprecipitation of GPD treated SK-MEL-28 cell lysates (1 mg of each). The cell lysates were precleared by incubating with 30 µl agarose A/G beads (50% slurry) for 2 h at 4°C. The beads were removed and 30 µl of fresh agarose A/G beads (50% slurry) and appropriate antibodies were added to the precleared lysates, followed by overnight incubation at 4°C. The beads were washed, mixed with 6× SDS sample buffer, boiled, and then resolved by 15% SDS–PAGE. The protein was analyzed by Western blot as described above.All quantitative results are expressed as mean values ±SE. Statistically significant differences were obtained by Student's t test. A value of p<0.05 was considered to be statistically significant.To determine whether GPD  can affeGPD treatment was found to induce significant alterations in cell morphology, including enlargement of the cytoplasm and generation of membrane vacuoles, phenotypes which have been reported to be representative characteristics of autophagy Since the release of cytochrome c from the mitochondria is known to trigger apoptosis, we further investigated apoptotic cell death in SK-MEL-28 cells with GPD treatment. Annexin V and propidium iodide (PI) staining revealed that GPD significantly induced apoptosis in SK-MEL-28 cells . MoreoveTo investigate the possibility that AMPK was playing a key role in upregulating autophagic cell death Melanoma remains one of the leading causes of cancer-related deaths Numerous studies have reported that various natural chemicals can be effective at inducing both autophagy and apoptosis Autophagy is an essential mechanism for maintaining cell homeostasis and involves lysosomal degradation of cytoplasmic components The AMPK pathway plays an important regulatory role in the metabolism of both normal and malignant cells, and AMPK activators have been shown to suppress mTOR activity, negatively regulating malignant transformation and cell proliferation in various cancer cell types Autophagy is also negatively regulated by mTOR signaling, particularly in relation to mTOR Complex 1, which senses nutrient conditions through the actions of GTP-bound Rheb Bcl-2, an anti-apoptoic protein, is a major regulator of the crosstalk between apoptosis and autophagy. Bcl-2 binds with Beclin-1 and inhibits autophagy. Phosphorylation of Bcl-2 by JNK dissociates Bcl-2 from beclin-1, and free beclin-1 plays a crucial role in inducing autophagy In the current study we have shown that GPD exhibits potent cancer inhibitory effects against human melanoma cells by inducing autophagic cell death and apoptosis. The inhibitory effect of GPD is mediated through AMPK/JNK signaling . These rFigure S1GPD inhibits anchorage-independent cell growth in PANC-1 human pancreatic carcinomas. Anchorage-independent cell growth in pancreatic carcinoma, PANC-1 cells was inhibited by GPD in a dose-dependent manner. A soft agar assay was performed with or without 10, 20, 40 µM of GPD or 40 µM of Rb1 and cell colonies were counted under a microscope with the aid of Image-Pro Plus software (Version 6). Results are shown as means ±SE (n = 3). The asterisks (*), (**) and (***) indicate statistical significance  compared with untreated control groups.(TIF)Click here for additional data file.


Terminal deletion of 6q27 produces a rare syndrome associated with unexplained mental retardation, hypotonia, epilepsy, and multiple malformations. Structural brain malformations are consistently observed, including agenesis of the corpus callosum, hydrocephalus, periventricular nodular heterotopia, polymicrogyria, and cerebellar malformations. Here we report a fetal risk assessment of a 27-year-old woman with mental retardation, hypotonia and dysmorphic features at 17 weeks of pregnancy.Cytogenetic analyses revealed an addition at chromosome 6qter in the mother. Haploinsufficiency of 6q27 to 6qter (1.3 Mb) and trisomy of the entire short arm of chromosome 18 (15.2 Mb) were found using a single nucleotide polymorphism based array. Results were confirmed by molecular cytogenetics and multiplex ligation-dependent probe amplification. The karyotype of the mother was 46,XX dic(6pter → 6q27::18p10 → 18pter).arr [hg19]6q27 × 1,18p11.3p10 × 3.ish dic. Deletion of 6q27 was associated with the structural brain malformations, whereas trisomy of 18p had minor clinical effects. The unbalanced rearrangement of chromosome 6 and chromosome 18 was de novo and was not inherited by the developing fetus.A rare rearrangement between 6q27 and 18p was identified, which led to a de novo 1.3 Mb deletion of 6q27 and a 15.2 Mb duplication of 18p in an adult with mental retardation, hypotonia, epilepsy, and multiple malformations. THBS2, PHF10, DLL1, and C6orf70 [Terminal deletion of 6q27, ranging from 0.4 Mb to 10.8 Mb, produces a rare syndrome with variable clinical features, including mental retardation, hypotonia, epilepsy, and multiple malformations . With thHere, we report a smallest 6q27 deletion with trisomy18p in a 27-year old patient at 17 weeks gestation. The patient presented with severe mental retardation (MR), hypotonia, and other malformations  and hypotonia. The patient was delivered at 41 weeks gestation. During the entire pregnancy, the mother of the patient felt sick and experienced emesis. Antiabortifacients were taken from 18 weeks of gestation until birth. The patient presented intra-uterine growth retardation; birth weight was approximately 1,500 g (>3SD), and birth length was 40 cm (>2SD); she also exhibited a weak cry and suckling. No apparent congenital abnormalities were noticed, although global developmental delays were observed.At age two, the patient had few single words and walked with an unstable gait. At age 27, evaluation revealed an IQ score of 46, hypotonia, prominent forehead, hypertelorism, long philtrum and, slow nystagmus, ataxic gait, joint laxity, bilateral clinodactyly, talipes cavus, scoliosis, pelvic obliquity, genu valgum on right, and femur length discrepancy of 1 cm  showed the presence of ACC, colpocephaly, asymmetric lateral ventricles, and choroid fissure cysts on the left Figure B. X-ray SNP-Array analysis of the present patient revealed a 1.3 Mb de novo deletion from 6q27 to 6qter and a 15.2 Mb de novo duplication including the entire short arm of chromosome 18. The proximal breakpoint in 6q27 mapped to  (q27) Figure A. Her paC6orf70 gene, related to the clinical phenotype of 6q27 deletion syndrome, was confirmed by qPCR  gene in 6q27 seemed the most likely causal mutation [TBP deletion and trisomy18p.Mental retardation and seizures are associated with brain malformation in 6q27 deletion patients. The TATA-binding protein mutation . TATA-bimutation . AlthougHypotonia is a clinical phenotype that affects approximately half of 6q27 deletion patients. Brain malformation may cause this phenotype, and long-lasting repercussions of hypotonia without corrective procedures, include scoliosis, pelvic obliquity, genu valgum, and leg discrepancy. In this case study, chromosome 6 is dicentric, one centromere is from chromosome 6 and the other is from chromosome 18. When human chromosome are dicentrics or multicentrics, faulty alignments may result . Three dAssessment of risk for the offspring was a major goal of this study. Results indicated no genetic abnormalities were present in the amniotic fluid, and the fetus was developmentally normal. The baby was born with no complications.de novo 1.3 Mb deletion of 6q27 and 15.2 Mb duplication of 18p in an adult with mental retardation, hypotonia, epilepsy, and multiple malformations. Analyses revealed that the 6q27 deletion contributed to the majority of the patient’s clinical features.In conclusion, this study identified a rare rearrangement between 6q27 and 18p, which led to DNA was analyzed with the Illumina Human CytoSNP-12 array to 100 kb resolution following the instructions provided in the Illumina Infinium® HD Assay Ultra manual. The iScan scanner was used to translate electronic signals into digital signals. Initial analyses and quality control were performed using Illumina GenomeStudio software. Copy number variation was determined using the Illumina KaryoStudio software.Chromosome preparations were obtained from cultured peripheral lymphocytes and amniotic fluid cells using standard cytogenetic protocols at 320-450 bands resolution. For FISH, probes RP11-614P3 (=6q27-Red), RP11-196G15 (=6p22.3-Orange), D18Z1 (=cep18-Green), RP11-1035E2 (=18p11.32-Green),  were used according to standard cytogenetic protocols.C6orf70 gene were designed for use in real-time qPCR analyses on an ABI 7900 HT fluorescence quantitative PCR instrument . Primers sequences and PCR conditions are available by request.P036-E2 probe mix contains a probe designed to detect deletions and duplications for every subtelomeric region. MLPA reactions were performed following the manufacturer’s instructions . Fragment separation was performed using the ABI-3130 sequencer , and data was directly analyzed using Coffalyser software. Primers for the Written informed consent was obtained from the patient for publication of this case report and accompanying images.


Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na Rab GTPases are essential regulators of intracellular membrane trafficking Rab27a regulates the transport and secretion of lysosome-related organelles and secretory granules from a variety of cell types including melanocytes, haematopoietic cells , endothelial cells, platelets and neuroendocrine cells SF2) maintained interactions with known Rab27a effectors yet failed to target to melanosomes SF1/F4) failed to bind to any known Rab27a effector yet maintained its melanosomal localisation showing that effector binding is not necessary for targeting SF2 mutant was found to be efficiently GTP-loaded by R3G in vitro yet mis-targeted in melanocytes, therefore indicating that the GEF activity alone is not sufficient for Rab27a targeting to melanosomes in melanocytes +,K+-ATPase isoform 1 (ATP1a1) as a key Rab27a targeting factor in melanocytes.Previous work in our laboratory has investigated the involvement of the aforementioned mechanisms in the targeting of Rab27a to melanosomes in melanocytes. We demonstrated that exchange of the C-terminal hypervariable domain of Rab27a with Rab1a or Rab5a did not re-target the hybrid protein and similarly, the Rab27a C-terminal domain was insufficient to retarget Rab5a or Rab1a For Western blotting the following antibodies were used at the concentrations indicated: mouse anti-Rab27a, 1∶10000 For immunofluorescence the following antibodies were used at the concentrations indicated: rabbit anti-Rab27a, 1∶200 ; mouse anti-ATP1a1, 1∶200 (Thermo MA1-16731); rabbit anti-Rab5a, 1∶50 (Abcam ab18211); rabbit anti-Rab38, 1∶100  and anti-FLAG (Sigma F3165) were used.5′-GUAUGAGCCUGCCGCUGUA-3′ (J-052829-06).All siRNA oligonucleotides were purchased from Dharmacon (Thermo Scientific). Non-targeting (NT) ON-TARGETplus (D-001810-02) was used as a negative control. siRNA oligos for Mlph and R3G were detailed previously WT and Rab27aSF1/F4 from pEGFP-C2 vectors described previously HTF-Rab27a constructs were generated by PCR amplification of rat Rab27aashen (melan-ash2) 2. Cells and melanosome distribution was assessed using a light microscope. For immunofluorescence experiments, cells were grown on glass coverslips in 24-well plates. Transfection of plasmid DNA was performed using FuGENE6 (Roche Diagnostics) in a 1∶3 ratio of µg of DNA to µl of FuGENE6, according to the manufacturer's recommendations. For each well of a 24-well plate, 0.5 µg of DNA was mixed with 1.5 µl of FuGENE6 in 25 µl of serum-free medium OptiMEM. Following a 5 h incubation, the medium was replaced with RPMI (+L-Glutamine) supplemented with 10% (v/v) FBS. After overnight culture the medium was replaced with complete RPMI medium. For siRNA analyses, cells were transfected with siRNA to a final concentration of 100 nM using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Following 5 h incubation the medium was removed and replaced with complete RPMI medium. For optimal siRNA depletion the siRNA treatment was sometimes repeated after 72 h. For membrane/cytosol fractionation experiments soluble (S100) and insoluble (P100) fractions were separated by centrifugation at 100'000×g of the PNS. The P100 was then resuspended in the same volume of loading buffer as the total volume of S100. Equal volumes of S100 and P100, relative to the protein concentration of the PNS for each siRNA treatment, were loaded on the gel. Quantification of ECL signals was carried out using ImageJ software.All tissue culture material was purchased from Invitrogen unless otherwise specified. Mouse WT melanocytes (melan-INK4a) and melan-ink4a-ash2 was carried out as described above. Two days post transfection, the medium was replaced with complete RPMI medium supplemented with 100 µg/ml Hygromycin B (Invitrogen). Cells were grown under selection for three weeks before Hygromycin-resistant cells were sub-cultured to one cell per well in a 96-well plate. The isolated clones were expanded and sub-cultured to increasing culture volumes.Transfection of melan-2 flasks of stable HTF-Rab27a expressing melan-ash2 cells were harvested by trypsinisation, washed in PBS and the pellets flash frozen in liquid nitrogen. Cell pellets were resuspended in 300 µl Buffer A  (Roche)) and lysed by passage through a 25G needle. CHAPS was added to a final concentration of 1% (Buffer A+CHAPS) and the sample incubated for 1 h at 4°C, rotating. The soluble fraction (S100) was isolated by centrifugation at 100,000×g for 1 h at 4°C. The S100 was pre-cleared by incubation with 100 µl pre-equilibrated Protein A/G Ultralink Resin  for 1 h at 4°C, rotating. 50 µg of mouse anti-HA antibody was added to the pre-cleared S100 and incubated for 2 h at 4°C, rotating. This was added to 250 µl pre-equilibrated beads and incubated for 2 h at 4°C, rotating. The beads were washed 3 times in Buffer A+CHAPS and resuspended in 200 µl Buffer A+CHAPS including 10 µl (10 U) AcTEV Protease (Invitrogen) and incubated overnight at 4°C, rotating. The supernatant was isolated and incubated with 13.6 µg of mouse anti-FLAG antibody for 2 h at 4°C, rotating. This was added to 50 µl pre-equilibrated beads and incubated for 2 h at 4°C, rotating. The beads were washed 3 times in Buffer A+CHAPS and resuspended in 100 µl Buffer A+CHAPS including 45 µg of FLAG peptide (Sigma) and incubated for 2 h at 4°C, rotating. The eluted sample was then isolated. Multiple TAPs were performed with lysates of HTF-Rab27aWT and HTF-Rab27aSF1/F4 expressing melan-ash2 cell lines and untransfected melan-ash2 cells; the samples were pooled and sent for Mass Spectrometry analysis at Dundee Cell Products.Three 150 cm2S2O3 for 2 min, then washing 3 times for 5 min in H2O. The gel was stained in Silver Nitrate  and washed twice for 1 min in H2O before adding to Developer . Developing was stopped by transferring to Stop Solution  for 5 min.The SDS polyacrylamide gel was fixed in Fixer  for 2 h at room temperature. The gel was then washed 3 times for 20 min in 35% EtOH before sensitising in 0.02% NaEscherichia coli BL21-codon plus (DE3) RILP (Stratagene) and purified using a method adapted from 600 = 0.1 and grown for 3 h until A600 = 0.6. Isopropyl β-D-1-thiogalactopyranoside (VWR) was added to a final concentration of 0.1 mM and the culture grown for an additional 4 h. The bacterial pellet was washed in PBS then stored at −20°C before resuspension in lysis buffer . This was incubated on ice for 30 min before sonication. The soluble proteins were separated from cell debris by centrifugation 12,000×g for 10 min. The bacterial lysate was incubated with 1.5 ml Glutathione Sepharose  for 2 h at 4°C, rotating. The Sepharose was washed with Buffer A before eluting GST-ATP1a1-M4M5 with elution buffer . Eluted fractions containing protein as assessed using Bradford Reagent (Pierce), were dialysed  in 1000× volume of wash buffer overnight and the buffer replaced for an additional 4 h dialysis. GST and GST-Slp1 6-tagged Rab27a, -Rab5a and -Rab27a mutant variants Recombinant GST-tagged ATP1a1-M4M5 was expressed in 6 melan-INK4a cells per condition were resuspended in 500 µl Buffer A and lysed by passing 20 times through a 25G needle. The lysate was cleared by centrifugation at 700×g for 5 min at 4°C. CHAPS was added to a final concentration of 1% and incubated for 30 min at 4°C, rotating. The soluble fraction was isolated by centrifugation at 100,000×g for 40 min at 4°C. 400 pmol of GST or GST-ATP1a1-M4M5 was added to the soluble fraction with 50 µl equilibrated glutathione-Sepharose and incubated overnight at 4°C, rotating. The Sepharose was washed 5 times with 1 ml Buffer A+CHAPS. Bound proteins were eluted by boiling the Sepharose in SDS loading buffer, and the proteins were separated by SDS-PAGE and analysed by immunoblotting using antibodies specific for Rab27a and GST.5×10in vitro GST pull-down method was adapted from 6-Rab was mixed with either 100 pmol GST or GST-ATP1a1-M4M5 in Buffer A+CHAPS and incubated for 30 min at RT, rotating. Equilibrated glutathione-Sepharose (20 µl) was added to each reaction and incubated for 30 min at room temperature, rotating. The Sepharose was pelleted by centrifugation and washed five times with 1 ml Buffer A+CHAPS. Bound proteins were eluted by boiling the Sepharose in SDS loading buffer, and the proteins were separated by SDS-PAGE and analysed by immunoblotting using antibodies specific for Rab27a, GST or His. Nucleotide loading of Rab27a was as described previously 2+-agarose beads were incubated with 2 nmol of his6-Rab27a in Buffer A for 20 min at room temperature. The beads were then washed twice with Buffer A before the bound nucleotide was eluted by washing with 1 M Guanidine-HCl, and followed by two washes with ice-cold Buffer A. The immobilized his6-Rab was nucleotide loaded by incubation for 10 min on ice in 200 µl of Buffer A supplemented with 200 µM of either GTPγS or GDP. The beads were then washed rapidly, three times with Buffer A, before the his6-Rab was eluted from the Ni2+-agarose beads by incubating for 15 min at room temperature with 2 bed volumes of Elution Buffer . The eluted Rab was then diluted 4-fold in Buffer A+CHAPS and 0.5 mM GTPγS or GDP, and incubated with GST fusion proteins in the presence of 20 µl of glutathione-Sepharose beads for 20 min at room temperature, rotating. The beads were then washed 4 times in Buffer A+CHAPS containing the appropriate nucleotide. Bound proteins were eluted by boiling the Sepharose in SDS loading buffer, and the proteins were separated by SDS-PAGE and analysed by immunoblotting using antibodies specific for Rab27a and GST.The g for 10 min at 4°C. 25 µg total protein PNS, 20 µl pre-equilibrated glutathione-Sepharose and 20 µM GST or GST-Slp1 were combined. Samples were incubated for 45 min at room temperature, rotating. Sepharose was washed three times with Buffer A. Bound proteins were eluted by boiling the Sepharose in SDS loading buffer, and the proteins were separated by SDS-PAGE and analysed by immunoblotting using antibodies specific for Rab27a and GST. Band intensities were measured using Image J. Rab27a band intensities were normalised to GST-Slp band intensities and then measured relative to NT siRNA control. The experiment was repeated five times and the mean values relative to NT controls presented with standard errors of the mean.Melan-INK4a cells were plated in a 6-well plate and treated with NT, ATP1a1 and/or R3G siRNA. Following siRNA depletion, the cells were washed in PBS then 150 µl Buffer A was added. Cells were harvested by scraping and lysed by passage through a 25 G needle. PNS was isolated by centrifugation at 800×4Cl in PBS, and then permeabilised in PBS containing 2% FBS and 0.05% Saponin (PFS). Permeabilised cells were incubated with primary antibody diluted in PFS, washed three times in PFS and incubated with the appropriate fluorescently-conjugated secondary antibody (Molecular Probes) diluted 1∶400 in PFS. The coverslips were washed three times in PFS, twice in PBS and once in H2O, then mounted on microscope slides with either ImmunO-fluore mounting medium  or Prolong Gold antifade reagent (Invitrogen). Microscopy was performed in the Facility for Imaging Light Microscopy (FILM) at Imperial College London. Immunofluorescence images were acquired at room temperature on a Zeiss AxioVert 200M laser scanning inverted confocal microscope ((LSM)-510) using Plan-Apochromat 63× 1.40 Oil DiC M27 objective, Zeiss AxioCam HRm camera and LSM 510 acquisition software. Images were processed with LSM Image Browser and Adobe Photoshop 8.0 software before compiling in Adobe Illustrator.Cells plated on coverslips were washed in PBS and fixed with 4% (w/v) paraformaldehyde (PFA) in PBS for 20 min. They were washed twice in PBS and once with 50 mM NH2 flask and then harvested by trypsinisation. Washed cells were then resuspended in 5 ml of chilled Homogenisation Buffer (HB)  and, lysed on ice using a cell cracker. PNS was then collected by centrifugation at 600× g for 5 min at 4°C. The PNS was then centrifuged 3×10 min at 2500× g at 4°C and the pellets collected and pooled (P2500). The P2500 was resuspended in 2 ml of chilled HB and 500 µl aliquots layered on top of 500 µl 50% Percoll in microcentrifuge tubes. The sample was then centrifuged 20 min at 8000× g at 4°C. The pellets were collected, pooled and diluted up to 1.5 ml HB and centrifuged 10 min at 5000× g at 4°C. The final pellet was resuspended in 200 µl HB.Melanosome purification was adapted from g for 5 min at 4°C. Coverslips were fixed in 4% PFA and immunostained as described above.For immunofluorescence analysis, the purified melanosomes were diluted 1∶500 in HB then 500 µl aliquoted onto coverslips in a 24-well plate. The plate was centrifuged 1500× Immunofluorescence images were taken on a fluorescent confocal microscope as described above. The images were then analysed using Simple PCI Software (Hamamatsu) to measure the average fluorescence intensity of each melanosome. For each experiment five images of melanosomes was taken. The mean fluorescence intensity of the melanosomes in a field of view (60×) was measured and then the average from five images was normalised to experimental NT siRNA controls. The experiment was repeated five times and the mean of the five experiments presented with standard error of the mean.ash2) were transfected with vectors allowing stable expression of tagged Rab27a , either wild-type or the Rab27aSF1/F4 mutant. The latter was used as it targets to melanosomes, but does not interact with known Rab27a/b effectors e.g. Slp/Slac and Munc13-4 proteins WT, rescued melanosome transport defects in Rab27a null melanocytes. These observations indicate that it is highly likely that HTF-Rab27a proteins expressed in Rab27a null melanocytes interact with a group of proteins similar to that encountered by the endogenous Rab27a in wild-type melanocytes.To identify protein(s) involved in targeting of Rab27a to melanosomal membranes, a tandem affinity purification (TAP) of epitope tagged Rab27a from melanocytes was performed. To this end, Rab27a null melanocytes  presence in previous proteomic studies of melanosomes from melanoma and retinal pigment epithelial cells +,K+-ATPase isoform 1 (ATP1a1) and Annexin A2 (Anxa2). Of note, based on the candidate size, ATP1a1 (113 kDa) and Anxa2 (36 kDa) could be assigned to two of the bands observed on the silver stained gel  indicated that overall Rab27a expression, relative to tubulin, was not strongly affected by ATP1a1 knockdown  interacted with ATP1a1 with similar efficiency, the interaction was reduced approximately ten-fold for the non-targeted mutant (Rab27aSF2)  . This suAs current models indicate that Rabs initially associate with target membranes in a GDP-bound state These data indicate that ATP1a1 is not a Rab27a effector and provide further support for the hypothesis that ATP1a1 regulates Rab27a targeting.In vitro we then showed that the large intracellular loop of ATP1a1 (ATP1a1-M4M5) directly interacts with Rab27a. Furthermore, we showed that this interaction is Rab27a specific, independent of the Rab27a nucleotide-bound state and that the ability of Rab27a mutants to interact with ATP1a1 correlates with their ability to target to melanosomes. Crucially, we showed that siRNA depletion of ATP1a1 resulted in mis-localisation of Rab27a to non-melanosomal membrane structures, which coincided with a reduction in Rab27a GTP-loading and Mlph association with melanosomes. Based on these findings we propose a working model for Rab27a targeting in which a direct interaction between ATP1a1 and Rab27a, involving the RabSF2 motif and ATP1a1-M4M5, targets Rab27a to the melanosome, whereupon it becomes GTP-loaded by R3G. Rab27a-GTP may then interact with effectors e.g. Mlph/MyoVa, and function in melanosome transport.In this study we investigated the mechanism of Rab targeting to intracellular compartments using Rab27a targeting to melanosomes in melanocytes as a model. We present findings that strongly support an essential role for ATP1a1 in the targeting of Rab27a to melanosomes. Firstly, using tandem affinity purification we identified ATP1a1 as a Rab27a interacting protein in melanocytes. +,K+-ATPase is present at the plasma membrane and may be enriched in caveolae While our proposed model advances our understanding of the mechanism targeting Rab27a to melanosomes, it also raises interesting questions about how ATP1a1 contributes to this process. Foremost among these is whether a melanosome associated pool of ATP1a1 functions like a receptor mediating the first contact between the melanosome membrane and Rab27a. In support of this, two previous studies reported ATP1a1 to be present in melanosomes purified from melanoma and RPE cells 2+ from ER stores independent of IP3 in vitro. These observations support the idea that ATP1a1 at the plasma membrane may regulate the function of intracellular proteins and organelles via direct protein∶protein interactions.A direct protein-protein interaction-type functional link between plasma membrane ATP1a1 and intracellular organelles has been shown previously. Work in rat renal proximal tubules showed that ATP1a1 and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) interact in response to ouabain, resulting in release of Ca+, K+-ATPase ion pump function might regulate melanosomal Rab27a recruitment via regulation of melanosomal ion homeostasis. It is likely that the luminal ion environment would influence the conformational state of ATP1a1. This conformational state could relay across the melanosomal membrane the maturation stage of the melanosome and subsequent ability to recruit Rab27a. A precedent for ion transporters recruiting small GTPases comes from work showing recruitment of Arf6 and its GEF ARNO on to maturing endosomes is dependent on luminal pH and an interaction with the V-ATPase +,K+-ATPase may additionally contribute to melanosomal ion homeostasis and subsequent melanosome maturation.Another interesting question is the extent to which NaThis is the first reported incidence of a Rab protein interaction required for correct localisation involving a protein with no known Rab regulatory or effector function. Further work should unravel the precise mechanism by which ATP1a1 regulates Rab27a targeting and how this is coordinated with R3G activity.