We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles.We generated a mouse with a CaV1.1 required for Ca2+ binding and/or permeation , we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca2+ stores during sustained activity. Decreases in these Ca2+-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca2+ binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers.Using mice with a pore mutation in Ca2+ binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance.While not essential for excitation-contraction coupling, CaThe online version of this article (doi:10.1186/s13395-014-0027-1) contains supplementary material, which is available to authorized users. Although Ca2+ entry through CaV1.1 is not required for skeletal muscle ECC [V1.1 opens after the initial voltage-gated release event to allow Ca2+ to both bind and permeate the channel pore [2+ channels, CaV1.1 undergoes multiple conformational changes driven by membrane depolarization and/or Ca2+ movement (and/or binding) through the channel [V1.1 actually occurs after the opening of RyR1 [V1.1 has been suggested to facilitate RyR1 closing [2+ permeation via CaV1.1, this influx has been suggested to regulate clustering of nicotinic acetylcholine receptors at the neuromuscular junction [Excitation-contraction coupling (ECC) in skeletal muscle involves a mechanical interaction between the L-type voltage-dependent Caannel CaV.1 and th channel ,4. The o of RyR1 , and CaV closing . Althougjunction -9 and mujunction .2+ binding and permeation through CaV1.1 contribute to the pathological development of several muscle diseases [V1.1 are associated with malignant hyperthermia (MH) in humans. The CaV1.1 mutations either enhance Ca2+ influx, increase the Ca2+ sensitivity of RyR1, and/or destabilize a closed state of RyR1. At least one of these MH mutations (R174W) ablates the L-type current and increases the sensitivity of RyR1 to caffeine, but does not alter ECC, demonstrating that the disease mechanism involves a distinct role for CaV1.1 [2+ permeation via CaV1.1 in muscle function than what is presently understood.Alterations in Cadiseases -14. Mutar CaV1.1 . Togethe2+ regulates multiple signaling pathways depending on the amplitude, frequency, duration, and location of the Ca2+ signal  due to its ability to cluster nearby and, upon Ca2+ binding, to interact with CaV1.x channels [V1.x channels prevail over other Ca2+ influx channels with respect to transducing excitation-transcription coupling (ETC) [et al. [V1.2 and phosphorylates the carboxy-terminal tail of the α1 subunit to increase Ca2+ influx. The interaction of CaMKII with CaV1.2, in turn, facilitates CaMKII auto-phosphorylation.In skeletal muscle, as in most cells, Casterblad ). CaMKIIchannels . CaV1.x ng (ETC) ,19. Hudm [et al.  demonstr2+ permeation through CaV1.1 in skeletal muscle function, we created a mouse with a Ca2+ binding and permeation defect in CaV1.1. We demonstrate that this CaV1.1-mediated pathway utilizes the flexibility of Ca2+ signaling  to regulate CaMKII and calcineurin activation, thereby enhancing SR Ca2+ store refilling and protein synthesis to modulate fatigue susceptibility, muscle size, and fiber type distribution.To directly delineate the role of CaN-benzyl-p-toluene sulfonamide (BTS) was purchased from Tocris Biosciences , latrunculin A from AdipoGen , and 4-chloro-m-cresol (4CmC) from Pfaltz & Bauer . The calcium dyes fura-2 AM and mag-fluo-4 AM were purchased from Invitrogen . Molecular weight marker for western blotting was purchased from GenDEPOT . Every other reagent including Insulin, KN-92, KN-93, and the myristoylated autocamtide 2-related inhibitory peptide (AIP) were purchased from Sigma-Aldrich .ad libitum. All procedures were approved by the Animal Care Committees at Baylor College of Medicine  and the University of Rochester .In this study, male mice between six and 12 weeks old were used ), unless otherwise indicated. All mice were housed at room temperature with a 12:12 hour light-dark cycle and provided with food and water V1.1. This selection marker-containing fragment was used to isolate a larger CaV1.1 genomic clone from a mouse 129 phage library via homologous recombination in Escherichia coli [The EK mutation was engineered on a genomic fragment of about 500 bp containing the exon-encoding residue E1014. A tetracycline resistance gene cassette  and a neomycin cassette  were inserted into the middle of the engineered fragment, flanked by about 250 bp homologies to Cahia coli . Severalhia coli .To expedite the transfer of the E1014K mutation from the 129SvEv mouse sub-strain background to a congenic C57BL/6 J background, speed congenics were used, in addition to conventional backcrossing. We identified 92 microsatellites of maximal base-pair length disparity between the 129SvEv and C57BL/6 J strains. These microsatellites were chosen to representatively span the entire genome and show distinct electrophoretic separation. Primer pairs were selected to PCR amplify the chosen microsatellites to be resolved by electrophoresis in Spreadex EL300 Wide Mini Gels . Finally, we compared 129SvEv and C57BL/6 J DNA standards to DNA from our backcrossed mice, and selected mice with the most sequence homology to C57BL/6 J DNA to be used in the next backcross. This method both ensured congenicity between the wild-type (WT) and EK mutant mice and provided a means to more quickly begin experimentation.Skeletal muscle fibers were isolated from the flexor digitorum brevis (FDB) muscle obtained from WT and EK mice as described .V1.1 currents (ICa) in FDB fibers isolated from WT and EK mice. FDB fibers were bathed in an external recording solution containing (in mM): 157 TEA-methanesulfonate, 2 CaCl2, 10 HEPES, 0.5 anthracene-9-carboxylic acid (9-AC), and 0.1 BTS, at pH 7.4, adjusted with TEA-OH. The patch pipette internal solution contained (in mM): 140 Cs-methanesulfonate, 10 HEPES, 20 Na-EGTA, and 4 MgCl2, at pH 7.4, adjusted with CsOH. All reagents here were purchased from Sigma Aldrich . The patch pipette resistance when placed in the external solution was between 0.6 and 1.0 Mohm. Fibers were voltage-clamped at a holding potential of −80 mV. Series resistance was compensated up to 80%. Data were sampled every 120 μs and filtered using a low pass Bessel filter  with a 2 kHz cut-off frequency. ICa was activated by 200 ms depolarizing pulses ranging from −40 mV to +60 mV in 10 mV increments delivered every 10 seconds. CaV1.1 current-voltage relationships (ICa-V) were obtained from peak currents measured during each depolarization normalized to cell capacitance and plotted against the corresponding test potential. ICa-V data were then fitted by the following modified Boltzman equation:max is the maximal L-channel conductance, V is test potential, Vrev is the L-channel reversal potential, V0.5 is the potential for half-maximal activation of Gmax, and kg is a slope factor.The whole-cell patch clamp technique was used to assess CaV1.1 currents were analyzed using Igor Pro 6  and Clampfit 9  software.Ca2, 1 MgCl2, and 10 HEPES, at pH 7.4. Fibers were then washed and incubated for 20 minutes in dye-free Ringer’s solution supplemented with 20 μM BTS, a skeletal muscle myosin inhibitor, to block contraction. Mag-fluo-4 AM-loaded FDB fibers were excited at 480 ± 15 nm and fluorescence emission detected at 535 ± 20 nm was collected at 10 kHz using a photomultiplier system. Electrical field stimulation  was elicited using a glass electrode placed adjacent to the cell of interest. Peak changes in mag-fluo-4 fluorescence for all 10 stimuli were measured as (Fmax-F0)/F0 and then averaged to generate a single peak value for each fiber. The rate of mag-fluo-4 fluorescence decay for the second, third, and fourth twitches for each fiber was fitted to a first order exponential function and the resulting amplitude and tau values were averaged.Acutely isolated FDB fibers were loaded for 20 minutes at room temperature with 4 μM mag-fluo-4 AM in a Kreb’s Ringer solution containing (in mM): 146 NaCl, 5 KCl, 2 CaCl2+ quench of fura-2 emission was measured in myotubes loaded with 5 μM fura-2 AM for 1 h at 37°C in Kreb’s Ringer solution. Briefly, prior to Mn2+ quench measurements, myotubes (primary cultured cells from muscle of mice) were treated with 100 μM ryanodine to block RyR1-mediated Ca2+ release during subsequent KCl application. Fura-2-loaded myotubes were excited at the experimentally determined fura-2 isosbestic point (362 nm) and emission monitored at 510 nm during perfusion of 50 mM KCl in the presence of 0.5 mM Mn2+. Maximum rates of fura-2 quench during KCl application were determined and evaluated for statistical significance.Mn2+ release during electrical stimulation, FDBs fiber were loaded with 5 μM of mag-fluo-4 AM for 30 minutes at room temperature in the presence of 20 μM of BTS. Loaded FDBs were placed on the stage of a confocal microscope with an adapted perfusion system (tyrode with 20 μM BTS at 0.5 mL/min) and imaged in line scan mode using the 20x objective (EC Plan-Neofluar) mounted in the confocal microscope , one line was acquired every 1.15 milliseconds (3.66 μsec/pixel time) using the 488 nm excitation laser and the LP 505 emission filter . FDBs were stimulated with 50 square electrical pulses (200 μsec duration) at 50 Hz and the produced florescence transients was normalized (F/F0) and plotted.To monitor Ca2+ store content, 1 mM of 4CmC was perfused at 3.25 ml/min, applied after 60 trains of electrical stimulation. Mag-fluo-4 fluorescence was collected at 20 Hz. Data were collected and analyzed using Metafluor version 6.2 software .Isolated FDB fibers were loaded with 5 μM of mag-fluo-4-AM for 30 minutes at room temperature, followed by washout with fresh DMEM . Electrical stimulation was performed using two platinum wires placed at each side of the fiber oriented longitudinally and fatigue was induced with uninterrupted application of electrical trains  for 300 seconds. For evaluation of RyR1-releasable SR Ca3VO4, 10 NaPyroPO4, 10 β-glycerophosphate, 10 NaF, PMSF, protease inhibitor cocktail (Santa Cruz), 1% NP40, 1% sodium deoxycholate, and 0.1% SDS . Equal amounts of total protein from whole muscle lysates were resolved by electrophoresis, transferred to PVDF  membrane and western blot analyses were performed using antibodies shown in Additional file Muscles were homogenized and lysed in ice-cold RIPA buffer consisting of (mM): 25 Tris pH 7.6, 150 NaCl, 1 Na2HPO4 and 81.4 mM NaH2PO4, pH7.2) for one hour at room temperature, washed twice with phosphate buffered saline (PBS) , permeabilized, and blocked in PBS containing 0.5% Triton X-100 and 5% BSA overnight at 4°C   according to the instruction of the user manual using an anti-goat MINUS PLA and anti-rabbit PLUS PLA probes and the orange detection reagent (Cy3) . Fibers were imaged with confocal microscopy . For analysis, Z stacks were projected and saved as a single image. The positive spots were counted with Image J , σ = 1, and same threshold per set adjusted at 15-30). The number of spots counted was normalized to the area of the fiber estimated from the width and length of the fiber in the image. For each set of experiments, the average counts of WT fibers (control) were set to 100% and the number of spots in each experimental condition was calculated as percentage change.We used proximity ligation assays (PLAs) to identify proteins that are within 40 Å of each other. PLA was performed on single FDB fibers plated on glass disks. Fibers were kept at 37°C in a 95% Oet al. [Eight-week-old WT and EK mice were fasted for 12 hours and then given an intraperitoneal injection of insulin (1 U/kg) diluted in saline. Control mice were injected with saline. After 7.5 minutes mice were sacrificed and muscles (soleus and EDL (Extensor Digitorum Longus)) were isolated, frozen in liquid nitrogen, and stored at −80°C until use. Muscle levels of pAkt1/2 and pGSK3β in the presence and absence of insulin were measured as described in Butler et al. .in vivo SUnSET technique [For measurement of protein synthesis we used an echnique ,25. BrieRas activity was measured using a Ras activity assay kit . Briefly, muscle was lysed in buffer and protein concentration was measured. Raf-Ras binding domain (RBD) beads (50 μl) were added to the muscle lysates  and the mixture was incubated at 4°C on a rotator for one hour. After incubation, Raf-RBD beads were pelleted by centrifugation at 5,000 × g at 4°C for one minute and washed with wash buffer. The bound active Ras was eluted in the two × sample buffer by boiling for three minutes. Eluted protein was run on 12% gel, transferred to PVDF membrane, and immunoblotted with Ras-specific antibody. The westerns were normalized to the amount of GAPDH in the muscle lysates (60 μg).2+-ATPase (SERCA) activity in tissue homogenates was performed as described [Sarco/endoplasmic reticulum Caescribed ,27.et al. [2/CO2, as above. After a 30 minute resting equilibration period, muscles to be stimulated underwent a fatigue protocol  for five minutes per muscle. At the completion of the stimulation protocol, rested and stimulated muscles were immediately frozen in liquid N2 and stored at −80°C. For muscles treated with KN-93, the drug was added to the chamber at the start of the 30 minutes equilibration period, at a final concentration of 5 μM.To assess stimulation-induced changes in signaling pathways we used the method of Sakamoto et al. . Intact 2/CO2. Muscles were tied with sutures at the musculotendinous junction and suspended between a force transducer and stationary anchor within a test chamber filled with warmed (35°C), oxygenated incubation medium. After a 20 minutes rest to allow mounted muscles to equilibrate, muscle optimal length (lo) was determined via single-twitch force generation measurements. Next, force frequency measurements were obtained at lo using frequencies from 15 to 300 Hz at 200 ms/train followed by a fatigue protocol performed over five minutes per muscle. The specific fatigue protocol for each muscle used was for the soleus: 15 Hz, 200 ms duration, one second intervals) and for EDL: 60 Hz, 200 ms duration, one second intervals. Muscle stimulation occurred within the test chamber using platinum electrodes attached to a Grass S48 stimulator and recorded within Chart5 (version 5.2) software .Intact soleus and EDL muscles were removed and immediately immersed in incubation medium comprised of KRS (oxygenated with a 95/5% mixture of O2b), MHCIIa , and/or MHCIIb  antibodies diluted 1:50 in PBS. After washing with PBS, muscle sections were incubated at room temperature for 90 minutes with isotype-specific AlexaFluor-594-conjugated goat anti-mouse IgG2b, AlexaFluor-488-conjugated goat anti-mouse IgG1, and AlexaFluor-594-conjugated goat anti-mouse IgGM secondary antibodies diluted 1:200. After three consecutive washes with PBS, muscle slides were mounted with VECTASHIELD mounting media . Images were taken under a fluorescence microscope . The relative numbers of the different fiber types were quantified and normalized by the total number of muscle fibers per field.Skeletal muscles (soleus and EDL) were dissected, embedded in OCT compound , and frozen in 2-methylbutane  precooled in liquid nitrogen. The frozen muscles were sectioned with 10-μm thickness using a SHANDON cryostat microtome . Immunofluorescent staining was performed using specific antibodies against myosin heavy chain I (MHCI), IIa (MHCIIa), and IIb (MHCIIb) . Briefly, sections were rehydrated with PBS for 10 minutes, followed by incubation at 4°C overnight with MHCI  calculations, 10-μm-thick frozen sections were obtained from the mid-belly area of the soleus and EDL muscles. Sections were immunostained for fiber type as described in the previous section, imaged at 10x magnification through an Olympus DP70 camera , and saved in .tif format. Saved images were then imported into Photoshop CSE version 10.0  for analysis. First, measurement scale was established by tracing a within-image scale bar (μm). Next, myofiber CSA was measured by tracing the external border of individual myofibers using the Magnetic Lasso tool. Myofibers exhibiting evidence of tears or processing artifacts were excluded from the analysis. Recorded measurements were then exported into Excel for analysis, with resulting CSA values reported in μmt-test. P <0.05 was considered to be statistically significant. *P <0.05, **P <0.01, and ***P <0.001 were used to indicate statistical significance.We performed a statistical analyses of two groups using the Student’s 2+ influx via CaV1.1, we created mice with a knock-in mutation (E1014K or EK) in the pore region of CaV1.1. The mutation of a glutamate to lysine residue in the repeat III pore region of CaV1.x abolishes both Ca2+ binding to this site [To explore the role of Cahis site  and diva2+ influx through CaV1.1, we compared whole-cell Ca2+ currents . The magnitude and decay kinetics of voltage-gated Ca2+ transients  elicited by a low frequency train of electrical stimulation (1 Hz) were not significantly different between FDB fibers from WT and EK mice  is unliand Na+;  relative2+ quench of fura-2 fluorescence assay to determine the effect of the EK mutation on excitation-coupled Ca2+ entry (ECCE) [V1.1 channels, providing a pathway for Mn2+ entry to quench fura-2 fluorescence  is absent in myotubes from EK mice  . In WT mr et al. . The EK 2+ transients during repetitive stimulation. During a single train of high frequency stimulation, the amplitude of the Ca2+ transients was lower in EK compared to WT fibers  of 4-chloro-m-cresol (4CmC) were significantly lower in EK compared to WT fibers .While the data in Figure 2+ indicator, fura-2 AM, to measure changes in cytosolic Ca2+ levels. Resting cytosolic Ca2+ levels did not differ between EK and WT fibers. However, following stimulation  [2+ transients  , and fous Figure H, and res Figure I in FDB 1 Figure J.2+ is required for mTORC1 activation of p70 ribosomal S6 kinase 1 (S6K1) [2+ permeation through CaV1.1 altered protein synthesis, we used the SUnSET technique [2+ permeation through CaV1.1, we examined the phosphorylation status of several key regulators of protein synthesis. We found that pS2448 mTOR, p-T37/T46-4EBP1/4EBP1, and p-S235/S236-S6/S6 were reduced in the soleus and EDL of insulin-treated EK mice compared to insulin-treated WT mice  upstream of mTORC2, PDK1, and ERK signaling, especially in the steps that lead to Ras activation.Ca1 (S6K1) -39. To dechnique  to asses2+ permeation and binding to CaV1.1 and activation of CaMKII in the regulation of these pathways, we isolated soleus and EDL muscle from WT and EK mice, electrically stimulated in the presence and absence of KN-93 (an inhibitor of CaMKII) and again analyzed the pathways described in Figure The data with and without insulin suggest that the effects of the EK mutation on insulin-stimulated protein synthesis involve both Akt phosphorylation (PDK1 and mTORC2) and ERK signaling. To assess Ras activation in EK and WT muscle, we pulled down activated Ras (GTP bound) with Raf-RBD-beads from soleus and EDL muscle and western blotted with RAS specific antibody to evaluate the amount of Ras that pulled down. As shown in Figure 2+ permeation though or binding to CaV1.1 activates CaMKII which, in turn, phosphorylates Raf to activate Ras/Raf and ERK1/2. ERK1/2 then activates mTORC1  was not different between muscles of young (between eight and 12 weeks of age) WT and EK mice  decrease of approximately 25% . However, we found no significant differences in grip strength, voluntary monitored wheel running, or endurance running in young (eight to 12 weeks of age) EK compared to WT mice, suggesting that the effects of the EK mutation on fiber type distribution, muscle contractility, and fatigue are relatively mild in young mice but increase in severity with age.The observed changes in activity dependent Cae Figure A and B, e Figure C and D. e Figure E and fure Figure F. Changee Figure G-J, but e Figure I. A decre Figure K and L. 2+ and in the presence of dihydropyridines were previously reported [2+ permeation through CaV1.1 in skeletal muscle has largely been ignored because Ca2+ influx through this channel is not required for ECC [2+ signaling in skeletal muscle is significantly altered by a permeation defect in CaV1.1 . This mutation resulted in concurrent inhibition of multiple Ca2+-sensitive pathways. These signaling changes translated to significant effects on muscle contractile function, providing compelling evidence that Ca2+ binding and/or permeation via CaV1.1 is important for maintaining optimal muscle physiology.Although acute changes in depolarization-induced contraction in low Careported , the rol for ECC . We demo2+ entering the muscle fiber through CaV1.1 is adequate to directly drive these changes in intracellular Ca2+ handling during repetitive stimulation. If so, previous studies have either underestimated the magnitude and relative contribution of Ca2+ influx through CaV1.1 during repetitive stimulation of muscle, or CaV1.1 Ca2+ binding and permeation activates a pathway (such as CaMKII) that promotes Ca2+ entry through a second Ca2+ influx channel . Another possibility is that Ca2+ binding to the selectivity filter triggers a conformational change in CaV1.1 that drives autophosphorylation of CaMKII bound to the channel (such as the C-terminus). CaMKII, activated by this mechanism, would then modulate multiple Ca2+ handling and downstream signaling pathways . The activation of these pathways would then alter numerous aspects of muscle physiology including fatigue, muscle fiber CSA, type II fiber type specification, and protein synthesis. Indeed, we demonstrate that autophosphorylation of CaMKII is decreased in the muscle of EK mice that have undergone repetitive stimulation, a condition that significantly enhances p-CaMKII levels in WT muscle.The question that arises is whether the amount of Ca2+-sensitive signaling pathways depends on the location, frequency, amplitude, and duration of the Ca2+ signal , since Ca2+ store refilling is decreased by CaMKII inhibitors in WT, but not EK fibers. However, at this point it is unclear whether the observed reduction in activity-dependent SR Ca2+ store content in FDB fibers from EK mice is due to loss of CaV1.1 Ca2+ influx or a downstream effect of CaMKII activation on SR Ca2+ reuptake and/or store-operated Ca2+ entry.The activation of different Casterblad ). ImportV1.1/CaMKII-dependent event that modulates insulin-mediated protein synthesis is upstream of PDK1 and/or mTORC2 activation, since phosphorylation of both Akt T308 (a PDK1 target) and Akt-S473 (an mTORC2 target) [2+-dependent regulation of the PI3K/Akt/mTOR and Ras/Raf/ERK pathways [One of the major findings in this study was a substantial decrease in insulin-mediated protein synthesis in EK muscle, as assessed by puromycin labelling and immunoblot analysis of the mTOR signaling pathway. The mTORC1 pathway activation in response to electrical stimulation is also decreased in EK compared to WT muscle. Decreased protein synthesis in response to growth factors such as insulin and repetitive stimulation may underlie the observed decrease in muscle fiber size in EK muscle. Activation of downstream substrates of mTORC1 was significantly decreased in EK muscle, as was activation of the upstream regulators of mTOR, Akt, and ERK1/2. We suggest that the Ca target) ,45 are dpathways -39.et al. [2+ influx associated with a deficiency in the β-subunit of CaV1.1 altered the formation of the neuromuscular junction. This may reflect a recently identified transcriptional role for the β1a-subunit [V1.1 Ca2+ currents, since mice with the EK mutation in CaV1.1 lack Ca2+ currents but do not exhibit alterations in neuromuscular junction preformatting (Additional file The phenotypic changes associated with the EK mutation do not arise from any obvious changes in the neuromuscular junction. Chen et al.  found th-subunit , as oppo2+ permeation through CaV1.1 is small, there is a significant impact of this pathway on muscle function. Changes include increased fatigue, decreased muscle fiber diameter, increased type IIb fiber specification, and decreased protein synthesis. Thus, our results indicate that small changes in CaV1.1 Ca2+ permeation, and CaMKII activation are amplified by changes in the activity of multiple critical downstream signaling pathways that impact a broad range of skeletal muscle functions. We propose that Ca2+ permeation through CaV1.1, while not required for ECC, modulates multiple downstream Ca2+-sensitive signaling pathways to improve muscle function.While the magnitude of Ca


In this study, we investigated if increasing CO2 levels stimulate the intestinal acid–base regulatory machinery of Atlantic cod (Gadus morhua) and whether temperatures at the upper limit of thermal tolerance stimulate or counteract ion regulatory capacities. Juvenile G. morhua were acclimated for 4 weeks to three CO2 levels  covering present and near-future natural variability, at optimum (10°C) and summer maximum temperature (18°C), respectively. Immunohistochemical analyses revealed the subcellular localization of ion transporters, including Na+/K+-ATPase (NKA), Na+/H+-exchanger 3 (NHE3), Na+/−/+-ATPase subunit a (VHA), and Cl− channel 3 (CLC3) in epithelial cells of the anterior intestine. At 10°C, proteins and mRNA were generally up-regulated for most transporters in the intestinal epithelium after acclimation to higher CO2 levels. This supports recent findings demonstrating increased intestinal 2 induced seawater acidification. At 18°C, mRNA expression and protein concentrations of most ion transporters remained unchanged or were even decreased, suggesting thermal compensation. This response may be energetically favorable to retain blood e, but may negatively affect intestinal salt and water resorption of marine teleosts in future oceans.CO Therefon et al.  suggeste+ and Cl− to create an osmotic driving force to absorb water from the luminal space  and Na+/K+/Cl− co-transporter 2 (NKCC2) located in apical membranes, which import Na+ and Cl− ions. Ion transport against adverse gradients is fueled by the electrochemical gradient created by the Na+/K+-ATPase (NKA) located in basolateral membranes of the intestinal epithelium , anion exchangers  and basolateral Na+/+ secretion via VHA and Na+/H+ exchangers (NHE), respectively were hypothesized to contribute to H+ export and mask parts of the secreted 2O and CO2. This molecular CO2 can diffuse back into the cell for re-hydration and repeated apical anion exchange   550 μatm as the average pCO2 at the study site at 10°C; (ii) 1200 μatm as the present extreme pCO2 at the study site at 18°C and model projection for the average pCO2 at 10°C within the next 100 years and, (iii) 2200 μatm at both temperatures in accordance with model predictions for the next 100 years  and the Ensembl genome browser system (http://www.ensembl.org/), we selected expression sequence information of 29 acid-base transporters and their mRNA expression levels were assessed using quantitative Real-Time PCR (qRT-PCR). Finally, functional capacities of NKA were determined, as this enzyme is believed to provide the major driving force for most energy-dependent ion transport in the fish intestine. We hypothesized that in response to elevated pCO2 levels (hypercapnia) at 10°C, selected transporters of the intestinal acid-base regulatory machinery will increase expression levels to support an increased secretion of This species, found in temperate to polar marine habitats, is of high ecological and commercial importance. Immunohistological analyses demonstrated that various acid-base relevant transporters including NKA, NHE3, NBC1, SLC26a6, VHA, and CLC3 are localized in apical  and basolateral membranes of epithelial cells of the cod anterior intestine Figure . These cpCO2, whereas temperature had large effects  compared to those acclimated to 10°C, when measured at a common temperature of 25°C. No interaction between temperature and CO2 has been found (p = 0.957). The patterns of NKA capacities related to fresh weight were similar (data not shown).Determination of NKA enzyme activities demonstrated no significant effects of pCO2 treatments were found for NKA, NBC1, and VHA using two-way ANOVA  treatment compared to the low pCO2 (pH 8.1) and intermediate pCO2 (pH 7.8) groups, with about 1.5-fold higher expression in high pCO2 treated animals compared to animals kept at low pCO2 conditions. NKA protein concentrations decreased with decreasing pH resulting in a 2.5-fold reduction in the high pCO2 treatment compared to the low pCO2 group at 18°C. This observation is also reflected in a strong correlation of NKA protein levels at different pCO2 levels. Correlation analyses (ANCOVA) also demonstrate a significant difference between the linear regressions at different acclimation temperatures  whereas no significant differences were observed in the warm acclimated fish. This resulted in significantly (p < 0.01) decreased NBC1 protein levels in the 18°C treatment compared to animals kept at 10°C, at high pCO2 levels. Furthermore the significant correlation between NBC protein levels and pCO2 at 10°C disappeared in the warm-acclimated animals , NHE1a, NBCa, NBCb, SLC26a3.2, SLC 26a6 and the carbonic anhydrase isoforms, CA4b and CA15a, in the anterior intestine of Atlantic cod. For NHE3 and the carbonic anhydrase isoform CA2b, 0.5 and 2-fold higher transcript abundances were determined, whereas the carbonic anhydrase isoform CA2a was highly dominating , NHE3, NBCa, SLC26A3.2, SLC26a6, CA2a, and CA15a  were detected for VHAa, NHE1a, NBCb, CA2b, and CA4b. At 10°C, transcript abundance of NKA increased at high pCO2 conditions compared to animals kept at low pCO2 (pH 8.1) conditions. No change in transcript levels was observed in response to elevated pCO2 at 18°C leading to significantly lower transcript levels under elevated seawater pCO2 conditions in animals acclimated at 18°C. A similar pattern was observed for NHE3, where transcript levels increased about 2.5-fold in response to high pCO2 at 10°C. No significant CO2 effect on NHE3 transcript levels was observed at 18°C, which led to about 4-fold decreased NHE3 mRNA expression levels at 18°C compared to the high pCO2 treatment at 10°C. NBCa expression responded in a similar fashion as NKA and NHE3 with a pronounced pCO2 effect on expression levels at 10°C, whereas no pCO2-dependent change was observed in warm acclimated fish. Transcript levels of the two SLC26 isoforms A3.2 and A6 increased in response to elevated pCO2 at 10°C, with a significant increase of SLC26A6 expression at high pCO2. At 18°C, no change was observed for SLC26A6, whereas SLC 26A3.2 transcript levels significantly decreased at high pCO2 compared to expression levels in the intermediate pCO2 treatment. A pCO2 effect on the expression at 10°C was also evident for CA2a and CA15a, where transcript levels significantly increased in response to seawater hypercapnia of 0.2 kPa pCO2. At 18°C, the carbonic anhydrases showed unchanged CA15a expression but a drastic decrease in expression of the dominating CA2a in response to elevated seawater pCO2.Elevated seawater pCO2 and temperature conditions. Strong correlations (p < 0.0001) of mRNA and protein expression were found for NKA and NBCa at 10°C as well as for SLC26A6 and NHE3 at 18°C. Weak correlations were found for VHA at both, 10 and 18°C as well as for NHE3 at 10°C.Correlation analyses using mRNA levels and protein concentrations of different ion regulatory proteins were used to detect co-regulations on mRNA and protein level during acclimation to different O. mykiss) , suggesting that these transporters operate in concert with each other during environmental hypercapnia. Strong correlation of mRNA and protein levels was found for NKA, NBCa/NBC1 and SLC26A6 at optimum temperature  in response to acute exposure (48 h) to a seawater pCO2 of 0.7 kPa  in seawater acclimated medaka  under prospected future seawater pCO2  increased cytosolic − absorption  did not affect the drinking rate in toadfish  in the same animals as used for the present work  activity in branchial epithelia to compensate blood pH  higher fractions of energy were spent on acid-base regulatory processes in response to acute high CO2 treatment   leading to an unchanged energy budget of the whole animal  at 18°C. Additionally, the counter-productive base loss in response to elevated serosal + export as demonstrated in Kreiss et al., + export and This work demonstrated that the intestinal ion/acid-base regulatory machinery responds to moderately increased seawater 2 effect became visible at the mRNA and protein level corroborating previous findings  mixed in gender and size  were separately incubated to CO2 concentrations of 552.97 ± 78.1 μatm, 1469.93 ± 459.93 μatm, and 2227.95 ± 312.33 μatm at water temperatures of 10.2 ± 0.2°C and 18.1 ± 0.2°C, respectively , and length and weight were determined. Fish were killed by a cut through their spine as close to the cranium as possible. Animals were dissected and tissue samples from the anterior intestine were cleaned and immediately shock frozen in liquid nitrogen and stored at −80°C for RNA and protein extraction. Samples for immunohistochemical analyses were fixed in Bouin's solution over-night, and stored in 75% ethanol.The animal experiments were conducted according to ethical commission Dnr.: 23-2012, approved by Sweden's Ethical Committee on Animal Experiments. Atlantic cod, l et al.  and Kreil et al. . For eac+/K+-ATPase , a polyclonal antibody raised against NBC1 of Atlantic cod  , a polyclonal antibody raised against the V-type H+-ATPase of squid (Sepioteuthis lessoniana)  in PBS and incubated for 1 h with small droplets (100 μl) of secondary antibody, anti-mouse alexafluor 488 or anti- rabbit alexafluor 568 . After rinses in PBS (3 × 5 min), sections were examined with a fluorescence microscope (Zeiss imager A1) with an appropriate filter set and a phase-contrast device.For immunohistochemistry, tissues were fixed by direct immersion in Bouin's fixative for 24 h followed by rinses in 70% ethanol. Samples were fully dehydrated in a graded ethanol series and embedded in Paraplast . Sections of 4 μm were cut on a Leitz Wetzlar microtome, collected on poly-L-lysine-coated slides, and stored at 37°C for 48 h. The slides were deparaffinized in Histoclear II® for 10 min and passed through a descending alcohol series . Slides were washed in phosphate- buffered saline (PBS), pH 7.3. Subsequently, samples were transferred to a PBS solution containing 5% bovine serum albumin (BSA) for 30 min to block non-specific binding. The primary antibodies (Table +/K+-ATPase activity was measured in crude extracts in a coupled enzyme assay with pyruvate kinase (PK) and lactate dehydrogenase (LDH). Crude extracts were obtained by quickly homogenizing the tissue samples in a conical tissue grinder in 10 volumes of ice-cold buffer containing 50 mM imidazole, pH 7.8, 250 mM sucrose, 1 mM EDTA, 5 mM β-mercaptoethanol, 0.1% (w/v) deoxycholate, proteinase inhibitor cocktail from Sigma-Aldrich . Extraction was performed on ice to avoid protein degradation. Cell debris was removed by centrifugation for 10 min at 1000 g, 4°C. The supernatant was used as a crude extract. The reaction was started by adding 1.5 μl of the sample homogenate to the reaction buffer containing 100 mM imidazole, pH 7.8, 80 mM NaCl, 20 mM KCl, 5 mM MgCl2, 5 mM ATP, 0.24 mM Na-NADH2, 2 mM phosphoenolpyruvate, and about 12 U/ml PK and 17 U/ml LDH in a PK/LDH enzyme mix (Sigma-Aldrich). The oxidation of NADH coupled to the hydrolysis of ATP was followed photometrically at 25°C in a temperature controlled plate reader  over a period of 15 min with the decrease of extinction being measured at λ = 339 nm. The fraction of Na+/K+-ATPase activity in total ATPase (TA) activity was determined by the addition of ouabain to a final concentration of 5 mM to the assay. Each sample was measured in three replicates with and without the addition of ouabain. Enzyme activity was calculated by using the extinction coefficient for NADH of ε = 6.31 mM−1·cm−1 and given as micromoles of ATP consumed per mg protein per hour.Na10 μL of crude tissue extract were fractionated by SDS-PAGE on 10% polyacrylamide gels, according to Lämmli , and traAnterior intestine tissue (without pyloric caecae) was homogenized in Trizol reagent  using the Tissue Lyser (Quiagen). Total RNA was purified using the RNeasy Mini Kit (Quiagen) following the manufacturer's protocol and DNA contamination was removed with DNase I . The amount of mRNA was determined by spectrophotometry , and the RNA integrity was checked by electrophoresis in RNA denatured gels , we selected sequence information of 29 acid-base transporters. Primers for all genes were designed  acclimated to the respective temperature and pCO2 conditions in two experimental replicates. Enzyme activities, protein levels and transcript levels were compared using two-way analysis of variance (ANOVA) followed by Tukey's Post-Hoc tests (Tukey's Post-Hoc with variable n = 5–8). Statistical analysis of qRT-PCR results was performed on mRNA quantities normalized to the geometric mean of the housekeeping genes RPL4 and UCE2a  and p < 0.01(**).Values are presented as the mean ± standard error (SE). Statistical analyses were performed on biological samples n = –8 acclimMH, KM, and CK designed and conducted the experiments of the present work and wrote the manuscript. ML, MS, and SD helped with the experimental design, data analysis and performed statistical analyses. YT conducted gene expression analyses and helped with the immunocytochemical staining. All authors equally contributed to the writing and completion of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The removal of acetylene from ethylene/acetylene mixtures containing 1% acetylene is a technologically very important, but highly challenging task. Current removal approaches include the partial hydrogenation over a noble metal catalyst and the solvent extraction of cracked olefins, both of which are cost and energy consumptive. Here we report a microporous metal–organic framework in which the suitable pore/cage spaces preferentially take up much more acetylene than ethylene while the functional amine groups on the pore/cage surfaces further enforce their interactions with acetylene molecules, leading to its superior performance for this separation. The single X-ray diffraction studies, temperature dependent gas sorption isotherms, simulated and experimental column breakthrough curves and molecular simulation studies collaboratively support the claim, underlying the potential of this material for the industrial usage of the removal of acetylene from ethylene/acetylene mixtures containing 1% acetylene at room temperature through the cost- and energy-efficient adsorption separation process. The removal of acetylene from ethylene/acetylene mixtures is an industrially important but challenging task. Here the authors report a microporous metal–organic framework with optimized pore/cage space design and chemical functionalization, capable of removing acetylene from low concentration mixtures. Ethylene is one of the most essential raw chemicals and widely used to produce polymers and other useful chemicalsExtensive efforts have been pursued to remove acetylene from ethylene/acetylene mixtures32H2/C2H4 have not been fully explored. We realized the first microporous MOF for this challenging separation in 20112H2/C2H4 are quite high because of their extraordinarily high sieving effects; their extremely narrow pores have also limited their acetylene uptake, which eventually affects their overall performance for separation of C2H2/C2H4, as clearly demonstrated in the simulated breakthrough curves2H2/C2H4 separation in 201238402H2/C2H4 separation. The ideal MOFs for C2H2/C2H4 separation are those with high C2H2/C2H4 sieving effects but without sacrificing acetylene uptake. From the MOF structural point of view, these MOFs should still have comparatively narrow pores to enforce their high sieving effects, but these narrow pores should still take up moderate amount of acetylene molecules. Furthermore, some additional intercrossed cage spaces will be required to exclusively bind acetylene molecules and thus to maximize acetylene uptake. It should be theoretically feasible for us to design and realize MOFs to meet these criteria; however, in the reality, it is still a daunting challenge. Recently, there has been some progress on microporous MOFs for C2H2/C2H4 separation; however, their pore structures still cannot meet the above mentioned criteria and their separation performances are comparable to established ones422ATBDC=5-(5-Amino-1H-tetrazol-1-yl)-1,3-benzenedicarboxylic acid; G=guest molecules), based on our extensive research endeavours on microporous MOFs for C2H2/C2H4 separation, which can indeed meet those mentioned criteria. UTSA-100 is thus superior to other MOFs, exhibiting highly efficient removal of acetylene from ethylene/acetylene mixtures containing 1% acetylene.Among diverse porous materials, the emerging microporous metal–organic frameworks (MOFs) are of particular interest and are important for gas separation and thus removal of acetylene from ethylene/acetylene mixtures. This is because the pores within microporous MOFs can be straightforwardly and rationally tuned to enforce their size selective sieving effects while their pore surfaces can be readily functionalized to induce preferential interactions with specific gas molecules5678910111213141516171819202122232425262728293031323334H-tetrazol-1-yl)-1,3-benzenedicarboxylic acid, H2ATBDC) was prepared based on alkaline decomposition of the tetrazole ring and heterocyclization of the resulting N-arylcyanamides on interaction with ammonium azide generated in situ. Reaction of CuCl2·2H2O with H2ATBDC in the solvothermal condition at 353 K formed UTSA-100 as green block single crystals. It was formulated as [Cu(ATBDC)]·G (UTSA-100) by single-crystal X-ray diffraction (SXRD) studies, and the phase purity of the bulk material was independently confirmed by powder X-ray diffraction (PXRD)  . The desn (PXRD) .c-direction (2(COO)4 units, which are bridged by 3-connected ATBDC2− anions to form a -connected apo-type network with Schläfli symbol {4.62}2{42.69.84} (3OH), and there are small cages with the diameter of about 4.0 Å between the 1D channels with window openings of 3.3 Å  surface area based on the N2 adsorption isotherm at 77 K is 1,098 (970) m2 g–1 for UTSA-100a, within the pressure range of 0.05<P/P0<0.3 (2 g–1 matches well with the simulated one (913 m2 g–1) from its crystal structure data.To assess the permanent porosity, the acetone-exchanged UTSA-100 was further activated under high vacuum to obtain the desolvated UTSA-100a. The porosity of UTSA-100a was evaluated by Ns nature . The nitP/P0<0.3 . The exp2H2/C2H4. The low-pressure sorption isotherms of acetylene and ethylene were collected at 273 and 296 K, respectively. At 296 K and 1 atm, the acetylene and ethylene uptake amounts of UTSA-100a are 95.6 and 37.2 cm3 g−1, respectively The highest adsorption selectivity is with M'MOF-3a, and this is followed by UTSA-100a. MOFs with coordinately unsaturated metal atoms MgMOF-74, CoMOF-74 and FeMOF-74 have selectivities that are in the range of 1.6 to 2.2. NOTT-300 has selectivities in the range 1.8–2.1.et al.et al., we carried out similar comparison, also including UTSA-100a; the results are shown in et al., We note that UTSA-100a has significantly higher selectivity than both NOTT-300 and FeMOF-74.In the paper by Yang 2H2/C2H4 mixtures containing 1% C2H2 is compared with other five MOFs . At a total gas phase pressure of 100 kPa, the hierarchy of uptake capacities for acetylene is UTSA-100a>MgMOF-74>FeMOF-74>CoMOF-74>M'MOF-3a≈NOTT-300.As shown in 2H2/C2H4  mixture, whose composition is typical of industrial mixtures, in a fixed-bed to demonstrate the feasibility of purification of ethylene in a Pressure Swing Adsorption operation. The transient breakthrough simulations show the concentrations of C2H2/C2H4 exiting the adsorber packed with UTSA-100a as a function of the dimensionless time, τ , that corresponds to the purity requirement of the feed to the polymerization reactor. The adsorption cycle needs to be terminated at that time τbreak and the regeneration process needs to be initiated. From a material balance on the adsorber, the amount of acetylene captured during the time interval 0–τbreak can be determined. The amount of acetylene captured in UTSA-100a during the time 0–τbreak is 137.6 mmol l−1, which is the best one among the compared six MOFs and approximately twice that of NOTT-300  mixture was flowed over a packed bed of UTSA-100a solid with a total flow of 2 ml min−1 at 296 K. As shown in 2H2/C2H4  mixture through the column packed bed of UTSA-100a solid can be efficiently achieved. To the best of our knowledge, this is the first example of porous materials whose separation for C2H2/C2H4  has been clearly established by experimental breakthrough, enabling UTSA-100a to be a potential high-performance material for real industrial ethylene purification application.To evaluate the performance of UTSA-100a in the actual adsorption-based separation and purification processes, breakthrough experiments were performed in which an C2H2/C2H4 separation performance of UTSA-100a to its structure, we carried out more detailed pore structure analysis. As mentioned earlier, the overall MOF pore size is centred at 4.3 Å, with rather narrow distribution (c axis direction) is shown in 2H2/C2H4 are ∼3.3/4.2 Å (3.32 × 3.34 × 5.70 Å3 for C2H2 versus 3.28 × 4.18 × 4.84 Å3 for C2H4), respectively464748To correlate the excellent Cribution . Gas adsr−6 terms2H2' structures using DFT-D. Interestingly, the guest acetylene molecules all get relaxed to a particular adsorption sites. In 2 groups, 2H2 can be assigned as hydrogen bonding interactions. Given the fact that aromatic –NH2 has weak basicity, while C2H2 has weak acidity (pKa=25)2 groups and C2H2 molecules. Because C2H2 is more acidic than C2H4 (pKa=44)2 groups have stronger interactions with C2H2 than C2H4, which enforces the selective binding of UTSA-100a for C2H2 than C2H4 as well. It is suggested that the relatively narrower pore size allows and reinforces one acetylene molecule to interact mutually with –NH2 group and one metal center O atom. We note that the adsorbed acetylene is slightly distorted, with an induced dipole moment. The H–C–C bond angle of acetylene is 178.8°, comparable to that of acetylene adsorbed on the open-Cu site in HKUST-1 (∼178°)−1. This value  is somewhat larger than the experimental Qst value but still reasonable, considering the accuracy limitation of the DFT-D approach.To further understand the acetylene adsorption in UTSA-100a, we performed detailed computational investigations . We firs2 further maximize the acetylene uptakes. It is speculated that weak acid–base interactions between –NH2 and C2H2 molecules also play the important roles for the preferential binding of UTSA-100a with C2H2 over C2H4. Incorporation of stronger basic sites such as alkaneamines into porous MOFs might significantly differentiate their interactions with C2H2 over C2H4, leading to even more efficient MOF materials for C2H2/C2H4 separations in the future.Removal of acetylene from ethylene/acetylene mixtures containing 1% acetylene is a very important but challenging industrial separation task. It has been speculated that adsorption-based porous materials could be the good alternative for the highly efficient removal of acetylene from ethylene steam, but have not been fully fulfilled. To evaluate a porous material for acetylene removal from ethylene, adsorption selectivity and saturation uptake capacity have been deemed as two most important criteria, and high values for both of them are needed to achieve high effectiveness and high efficiency for acetylene removal. However, which factor plays the dominant role depends on the composition of gas mixtures. For the ethylene/acetylene mixtures containing 1% acetylene, adsorption selectivity will be enough more important than acetylene adsorption capacity. Comparing with other five well-known MOFs , the significantly superior performance of UTSA-100a in removing acetylene from ethylene/acetylene mixtures containing 1% acetylene is attributable to the collaboration of high adsorption selectivity and high uptake capacity at ambient conditions. From the structure point of view, UTSA-100a has suitable pores and opening windows to enforce its high sieving effects and thus high adsorption selectivities, while the suitable cages and immobilized functional sites such –NHIt is to be noted that our comparisons of different MOFs are for ethylene/acetylene mixtures containing 1% acetylene that is representative of compositions encountered in industry. If we were to compare the performance of UTSA-100a, NOTT-300 and FeMOF-74 for 50/50 ethylene/acetylene mixtures, the conclusions are different because in this case, capacity considerations would be very important. In conclusion, we have prepared a microporous MOF (UTSA-100) for highly efficient removal of acetylene from ethylene/acetylene mixtures containing 1% acetylene. Experimental and computational simulation results demonstrate the high efficiency of UTSA-100a in the removal of acetylene from ethylene/acetylene mixtures containing 1% acetylene, which is a very important but challenging industrial separation task. The results of this research have important significance on the practical design and preparation of porous materials for light hydrocarbon separations. It will also provide some guidance on the design and synthesis of microporous MOFs for other gas separations.H-tetrazol-1-yl)-1,3-benzenedicarboxylic acid (H2ATBDC) was synthesized according to the literature method1H NMR and 13C NMR spectra were obtained using a Varian INOVA 500 MHz spectrometer at room temperature. FTIR spectra were performed on a Bruker Vector 22 spectrometer at room temperature. Thermal gravimetric analysis (TGA) was performed under a nitrogen atmosphere with a heating rate of 3 °C min−1 using a Shimadzu TGA-50 thermogravimetric analyzer. PXRD patterns were measured by a Rigaku Ultima IV diffractometer operated at 40 kV and 44 mA with a scan rate of 1.0 deg min−1.Commercially available reagents were purchased in high purity and used without further purification. 5-: δ 13.78 , 10.33 , 8.65 , 8.55 .Glacial acetic acid (10.0 ml) was added with stirring to a suspension of 5-aminoisophthalic acid , and sodium azide  in triethyl orthoformate , and the mixture was stirred at 80–90 °C for 6 h. The reaction mixture was cooled, and concentrated hydrochloric acid  and water (12.5 ml) were added. The precipitated solid was separated by filtration, washed with water and dried. The obtained raw product was recrystallized from DMF. Yield: 82% (4.79 g). H-tetrazol-1-yl)-1,3-benzenedicarboxylic acid  in 22% aqueous KOH solution (12.0 ml). Gas evolution was observed, accompanied by self-heating of the reaction mixture. Stirring of the reaction mixture was continued for 2 days. The mixture was then diluted to 160 ml with water, acidified with concentrated hydrochloric acid to pH 3–4 and stored at 5–10 °C until precipitation of solid. The obtained product was filtered off and dried in vacuum. Another portion of product was reprecipitated from the filtrate through salting out method using sodium salt. Yield: 93% (3.83 g). 1H NMR : δ=7.67 , 7.40 .DMSO (20.0 ml) was added dropwise with constant stirring to a suspension of 5-: δ=8.47 , 8.14  : δ=173.00, 138.49, 132.35, 130.43, 126.94 (A suspension of 5-(cyanoamino)-1,3-benzenedicarboxylic acid , sodium azide  and ammonium chloride  in DMF (25 ml) was stirred at 70–80 °C for 6 h, after which water (100 ml) was added to the reaction mixture. The white solid was precipitated through salting out method using sodium salt. The obtained product was filtered off, and dried in vacuum. Yield: 82% (2.04 g). , –C6H3) . 13C NMR, 126.94 .2·2H2O  and the organic linker H2ATBDC  was dispersed into an 8 ml mixed solvent  in a screw-capped vial (20 ml). And five drops of HBF4 (48% w/w aqueous solution) were added. The suspension was sonicated until homogenous. The vial was capped and heated in an oven at 80 °C for 24 h. Green block crystals were obtained by filtration and washed with DMF several times to afford UTSA-100. IR : 1,739w; 1,630s; 1,596m; 1,490w; 1,457m; 1,377s; 1,255m; 1,128w; 1,097m; 1,062w; 908m; 780m; 725s; 677m; 661m.A mixture of CuCl−1 before measurements. A sample of activated UTSA-100a (100–150 mg) was used for the sorption measurement and was maintained at 77 K with liquid nitrogen, at 273 K with an ice-water bath. As the center-controlled air conditioner was set up at 23 °C, a water bath was used for adsorption isotherms at 296 K.A Micromeritics ASAP 2020 surface area analyzer was used to measure gas adsorption isotherms. To remove all the guest solvents in the framework, the fresh sample of UTSA-100 was guest exchanged with dry acetone at least 10 times, filtered and degassed at room temperature (296 K) for one day, and then at 358 K for another 3 days until the outgas rate was 5 μmHg minExperimental data on pure component isotherms for acetylene and ethylene in UTSA-100a were measured at temperatures of 273 and 296 K. The pure component isotherm data for acetylene and ethylene were fitted with the dual-Langmuir–Freundlich isotherm modelT-dependent parameters bA and bBwith The fitted parameter values are presented in et al.et al.For FeMOF-74, the dual-site Langmuir–Freundlich parameters are from Bloch ρ ; length of packed bed, L (0.12 m); voidage of packed bed, ɛ (0.75); superficial gas velocity at inlet, u . For breakthrough simulations with NOTT-300, we use calculated the framework density from the crystal structure information provided in the paper of Yang et al.ρ . The framework densities for all other MOFs are available in the papers by Bloch et al.et al.τ, defined by dividing the actual time, t, by the characteristic time, The performance of industrial fixed-bed adsorbers is dictated by a combination of adsorption selectivity and uptake capacity. For a proper comparison of various MOFs, we perform transient breakthrough simulations using the simulation methodology described in the literature5354in situ activated under vacuum (6.5 × 10−4 Pa) at 353 K to remove adsorbed molecules and make the active sites accessible. The sample was then purged with He flow (2.0 ml min−1) for 1 h while the temperature of the column was decreased to 296 K. The mix gas (C2H2:C2H4=1: 99 by volume) flow was then introduced at 2.0 ml min−1. Effluent from the column was monitored using a mass spectrometer.The breakthrough separation experiments were conducted on a home-made apparatus  with a sCaution: Because of its wide flammability limits and a potential for explosive decompositionAccession codes: The X-ray crystallographic coordinates for structures reported in this Article have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition number CCDC 1044083. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.How to cite this article: Hu, T.-L. et al. Microporous metal–organic framework with dual functionalities for highly efficient removal of acetylene from ethylene/acetylene mixtures. Nat. Commun. 6:7328 doi: 10.1038/ncomms8328 (2015).Supplementary Figures 1-13, Supplementary Tables 1-5, Supplementary Methods and Supplementary ReferencesCrystal data.


Clinical studies with montelukast show variability in effect and polymorphic OATP2B1-dependent absorption has previously been implicated as a possible cause. This claim has been challenged with conflicting data and here we used OATP2B1-transfected HEK293 cells to clarify the mechanisms involved. For montelukast, no significant difference in cell uptake between HEK-OATP2B1 and empty vector cell lines was observed at pH 6.5 or pH 7.4, and no concentration-dependent uptake was detected. Montelukast is a carboxylic acid, a relatively potent inhibitor of OATP1B1, OATP1B3, and OATP2B1, and has previously been postulated to be actively transported into human hepatocytes. Using OATP1B1-transfected HEK293 cells and primary human hepatocytes in the presence of OATP inhibitors we demonstrate for the first time that active OATP-dependent transport is unlikely to play a significant role in the human disposition of montelukast. Montelukast, a leukotriene receptor cysLT1) antagonist, is used for the treatment of asthma. In mono-therapy it is used as an alternative to low-dose inhaled corticosteroids (ICS) or in addition to ICS for improving clinical management of the disease in various phenotypes of asthma patients  antagoni. In cont3H]-Montelukast was supplied by The American Radiolabeled Chemical Company  and non-radiolabeled montelukast was supplied by Chemtronica . Montelukast is light sensitive and was stored in darkness. [3H]-Estrone 3-sulfate (E13S) and [3H]-estradiol 17-beta-glucuronide (E217βG) were obtained from PerkinElmer , whilst non-radiolabeled E13S, E217βG, rifampicin, dimethyl sulfoxide (DMSO), 2-(N-morpholino)ethanesulfonic acid (MES), and fetal calf serum (FCS) were obtained from Sigma–Aldrich . Erlotinib was obtained from Chemtronica ; all cell culture reagents, Leibovitz medium supplemented with l-glutamine, Hank’s balanced salt solution (HBSS), phosphate buffered saline (PBS), and HEPES from Invitrogen Life Technologies ; sodium hydroxide (NaOH) and hydrochloric acid (HCl) from Merck KgaA , and the scintillation cocktail Optiphase “Hisafe” 2 was provided by PerkinElmer . Pierce® BCA Protein Assay kit was used for protein determination . compound (1 µCi/mL) and non-radiolabeled compound. Uptake experiments were performed in a thermostated shaker, set to 37 °C and 250 rpm . At the start of the experiment, the cell culture medium was removed and the cells were washed twice with pre-heated transport media (700 µL per well). Thereafter, the cells were incubated in pre-warmed transport media (200 µL) for 10 min and experiments initiated by the addition of an equal volume of pre-warmed dose solution (200 μL) to each well. All uptake experiments were carried out in transport medium consisting of HBSS buffered with 25 mM MES adjusted to pH 6.5 with NaOH or buffered with 25 mM HEPES adjusted to pH 7.4 with NaOH. All experiments were performed in triplicate and the total concentration of DMSO and ethanol never exceeded 1% and 0.2% (v/v), respectively, in the incubations.Montelukast, E13S (0.5 µM) and montelukast (1 µM) were incubated at pH 6.5 and 7.4 from 0.5 to 20 min. Concentration-dependent and transport inhibition experiments were performed for 3 min at pH 6.5, with E13S (0.02 to 50 μM) and montelukast (0.02 to 6.6 μM). E217βG (1 µM) and montelukast (1 µM) were incubated at pH 7.4 from 0.5 to 20 min with HEK-OATP1B1 and corresponding mock transfected cells. Concentration-dependent experiments were performed for 1 min with E217βG (0.02 to 50 µM).For the OATP2B1 time-dependent experiments, EThe uptake experiments were terminated by removing the incubation media and washing the cells twice with PBS. Ice-cold NaOH  was added to each well and the plates were placed in a refrigerator for at least one hour to lyse the cells. The cell homogenate was neutralized with an equal volume of HCl (0.2 M) prior to sampling for protein determination and scintillation counting for radioactivity analysis. The absorbance was read at 562 nm using a SpectraMax 190 plate reader . The samples were assayed for radioactivity using a Wallac WinSpectral™ 1414 liquid scintillation counter and a Tri-Carb 3100TR Liquid Scintillation analyzer, both supplied by PerkinElmer . Scintillation cocktail Optiphase “Hisafe” 2 was added to each sample before analysis.Km and Vmax for OATP2B1 and OATP1B1 substrates were obtained by fitting to a Michaelis–Menten equation . OATP-mediated uptake, calculated by subtracting the uptake in HEK-mock from that in HEK-OATP cells was used for the Michaelis–Menten fit.The enterocyte blood flow method  was used13S (1 µM) were performed in serum free Leibovitz L-15 medium in absence and presence of OATP transport inhibitors rifampicin (10 µM) or erlotinib (4 µM) at a final hepatocyte concentration of 1,000,000 cells/mL. Briefly, cells were thawed and re-suspended in Leibovitz medium according to the manufacturer’s protocol, counted using a CASY cell counter  and further diluted to reach a concentration three times that of the final incubation. Incubations were initiated as follows: substrate and inhibitor solutions were prepared in Leibovitz medium to a concentration three times that of the final incubation. A blank (no inhibitor) solution was also prepared with the same DMSO concentration as the inhibitor solutions. Equal volumes (300 µL) of blank/inhibitor solution and cell suspension were mixed in a glass vial and allowed to pre-incubate for 15 min in water bath set to 100 rpm (linear shaking) at 37 °C. Experiments were started by addition of substrate solution (300 µL). The final DMSO content in all incubations was 0.15% v/v. At designated time points  a 100 µL aliquot of incubate was removed and dispensed into a microtube containing an upper layer of oil and a lower layer of cesium chloride. Upon rapid centrifugation  hepatocytes are passed through the oil layer separating them from the incubation medium. The microtubes were directly placed on dry ice before cutting (just below the oil/cesium chloride interface) of the frozen tube tips containing the cell pellets into a 96-well plate for sample processing as previously described [Pooled cryopreserved primary human hepatocytes were obtained from Bioreclamation IVT . Hepatocyte uptake was assessed using an “oil-spin” procedure, as previously described . Incubatescribed . IncubatSamples were analyzed by LC–MS–MS: HTS PAL injector , HP 1100 LC binary pump  and a Micromass Quattro Ultima mass spectrometer . A mobile phase  with a flow rate of 0.7 mL/min was used with a Sunniest C18 RP-aqua column  for chromatography.13S profiles the optimal statistical confidence in slope determination and was, therefore, included in analysis. Significance of inhibition of uptake by rifampicin and erlotinib was calculated from a two-tailed linear regression t-test . Obtained p-values were corrected for multiple comparisons using the Holm–Bonferroni method [p-values < 0.05 were considered as significant.Relative rates of uptake were determined by linear regression. Data generated up to 60 s after substrate addition gave for montelukast as well as for Ei method . Adjuste13S (0.02–50 μM), also incubated for 3 min at pH 6.5 in HEK-OATP2B1 and HEK-mock  of montelukast failed to show a difference in cell uptake rate between the HEK-mock and the HEK-OATP2B1 cells A. The soHEK-mock B, were u13S (0.5 µM) and montelukast (1 µM) were used. At pH 7.4, the uptake of montelukast observed in both HEK-OATP2B1 and HEK-mock cells was similar  was used as a positive control for validation of the OATP1B1-mediated uptake and the uptake rate was notably higher in HEK-OATP1B1 than in HEK-Mock cells  with HEK-OATP1B1 and HEK-mock cells performed at pH 7.4, no difference in uptake rate was detected between the two cell lines A. E217βGck cells B. Km andkinetics . The OAT13S were taken up into human hepatocytes linearly with respect to time between 15 and 60 s, when incubated at 1 μM. Rifampicin-mediated (10 μM) statistically significant 51% inhibition of E13S uptake (adjusted p < 0.001) but no significant impact on montelukast uptake (adjusted p = 0.16)  . Erlotinin vitro Caco-2 data in which the relationship between permeation rate and montelukast concentration was interpreted as being indicative of transporter mediated transport that could be described by Michaelis–Menten kinetics. Only the single concentration of 170 µM montelukast deviated from the linear relationship between rate and concentration, this data point being associated with the greatest variance, possibly due to limitations in the aqueous solubility of montelukast being in the 10–50 µM range [13S (only 2.8-fold higher cell uptake in OATP2B1 cells compared to control). Additionally, the cell uptake was shown to be inhibited 27% by 125 µM sulfobromophthalein. In this context it is noteworthy that when E13S has been previously used as a substrate of OATP2B1, an IC50 of 2 µM was reported for sulfobromophthalein [et al. provided MDCKII (control and OATP2B1-expressing) cellular uptake data that showed no significant difference in the amount of montelukast taken into control and OATP2B1 cells when incubated at 1 µM, pH 6.0. The same finding was observed at incubation pH 7.4, but in both cell lines the amount of drug taken up was approximately 1.5-fold lower compared to incubations performed at pH 6.0. A similar experiment was performed using 1 µM E13S and the rate of uptake was approximately 10-fold higher in the OATP2B1 cells compared to control cells. Thus, an assay with a relatively large window for the positive control compound contradicted the findings of the earlier publication. Furthermore, cellular accumulation data for 1 µM E13S and 3 µM montelukast presented by Chu et al., concluded that E13S accumulated to a greater extent in MDCKII OATP2B1 cells when added to the basolateral side than the apical side, but that this was not true for montelukast. However, higher montelukast accumulation in the control cells after apical administration, compared to basolateral administration, together with only a two-fold difference between basolateral and apical administration for E13S, reduces the clarity of these results.Previously, it has been reported that OATP2B1 may be important in the human intestinal absorption of montelukast . These cµM range . Vectorihthalein . Other i13S, shown to have a Km value of 7.5 µM, in excellent agreement with previously observed values [13S uptake were observed when comparing the OATP2B1- and mock-transfected HEK cell lines at both pH 6.5 and 7.4. This gave an advantageous window for defining test compounds as substrates of the drug transporter. In contrast to E13S, montelukast uptake at pH 6.5 was very similar in the OATP2B1 and mock cell lines over a range of concentrations. Altering the pH in the incubation from 6.5 to 7.4 did not affect the OATP2B1/mock ratio substantially for E13S. We, therefore, conclude that montelukast is not pronouncedly transported by OATP2B1 due to high inherent passive permeability and given the low variability in observed human exposure there is no reason to consider that the transporter is important in driving human intestinal absorption [Here we performed experiments in an attempt to describe montelukast time- and concentration-dependence for HEK293 OATP2B1 cellular uptake. A novel human OATP2B1 expressing HEK293 cell line was used, created at Uppsala University . This ced values ,16. Drugd values . Since hsorption . This dasorption .in vitro, although the mechanism of the uptake has, to our knowledge, never been investigated [P values in the range 3.5 to 5.5 [P of over 7. Whilst acidic drugs that are highly bound to plasma proteins typically have low steady state volume of distribution (Vss) values in the order of 0.1 to 0.3 L/kg, for those whose hepatic disposition is influenced by OATP1B1/3 transport, the Vss is likely to be larger [Vss of approximately 0.15 L/kg [217βG as a standard OATP1B1 substrate since Km and Vmax values of 4.1 µM and 697 pmol/mg/min were in line with previously published data [13S uptake rate was reduced by 51%. The back-extrapolated Y-intercept of the plots represents instantaneous non-specific drug binding which was understandably higher for montelukast than E13S due to the higher lipophilicity. Taking into account this intercept, the uptake rate between 15 and 60 s appears a good measure of the true initial rate (13S intracellular concentration was less than 70% of the concentration achieved by 3 min, whereas montelukast uptake was complete after 1 min. This more rapid cellular uptake may be more suggestive of a drug concentration-driven passive process. Nevertheless, the total amount of montelukast entering the cell was reduced in the presence of rifampicin and it cannot be ruled out that the active process plays a minor part in the overall hepatic permeability of the drug. Since 10 µM rifampicin should cause only minimal inhibition of OATP2B1 activity, 4 µM erlotinib was used to selectively inhibit this drug transporter [13S is an OATP2B1 substrate.It is interesting to note that montelukast has been shown to be a relatively potent inhibitor of OATP1B1, 1B3, and 2B1 ,21. It istigated . Drugs t5 to 5.5 . Montelue larger ,25. Mont.15 L/kg . In the hed data  but no sial rate . It is insporter . ErlotinIn summary, it appears unlikely that OATPs are involved in the human absorption of montelukast or distribution into hepatocytes.


Four groups were established and referred to as the control, atorvastatin, resveratrol and R+A groups. Atorvastatin  and/or resveratrol  treatments were administered for 14 days. On the 15th day, the thoracic aortas and hearts of the rats were dissected and placed into isolated organ baths. Vascular responses to cumulative doses of H2O2 (1×10−8–1×10−4 M H2O2) with and without N (G)-nitro-L-arginine methyl ester (L-NAME) incubation were measured. In addition, myocardial electrical stimulation (ES) responses to various H2O2 concentrations (1×10−7–1×10−5 M H2O2) were evaluated. In the control and atorvastatin groups, H2O2 application caused a significant dose-dependent decrease in the ES-induced contractions in the myocardial tissue of rats. In the resveratrol and R+A groups, H2O2 application did not significantly affect myocardial contraction at any dose. In all groups, incubation with L-NAME caused a significant augmentation in the H2O2 response, revealing that this effect was mediated via the vascular endothelium. In conclusion, pretreatment with R+A for CVD appears to be superior to pretreatment with either agent alone.Oxidative stress has been implicated in the pathophysiology of several types of cardiovascular disease (CVD). Statins are widely used to inhibit the progression of atherosclerosis and reduce the incidence of CVD. Certain over-the-counter products, including resveratrol, show similar effects to statins and may thus be used in conjunction with statins for the treatment of the majority of patients with CVD. The aim of the present study was to evaluate the effects of atorvastatin, resveratrol and resveratrol + atorvastatin (R+A) pretreatment on myocardial contractions and vascular endothelial functions in the presence of H It is also considered to play a role in the progression of atherosclerosis –4. PreviIn vitro experiments have revealed that the cardiovascular protective effects of resveratrol may occur through a number of mechanisms. Resveratrol inhibits the proliferation of smooth muscle cells, platelet aggregation and the oxidation of low-density lipoprotein cholesterol, and reduces the synthesis of lipids and eicosanoids, which promote inflammation and atherosclerosis (The β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used to inhibit the progression of atherosclerosis and reduce the incidence of CVD. As well as their cholesterol-lowering effects, statins improve endothelial function in normocholesterolemia ,9. Certaclerosis . These m2O2 as an experimental model of oxidative stress in rats.A number of studies have demonstrated the aggravating effects of statins on oxidative stress in organisms ,12. Suchad libitum. The experimental procedures were approved by the Animal Ethics Committee of the Meram School of Medicine . All chemicals were purchased from Sigma-Aldrich  and stored according to the manufacturers’ instructions unless otherwise specified. For 14 days, the control group (n=8) received 1.5 ml drinking water by oral gavage and 1 ml 10% v/v dimethyl sulfoxide , the vehicle for resveratrol. The atorvastatin group (n=6) received 40 mg/kg atorvastatin (Lipitor®), which was prepared daily and dissolved in drinking water, by oral gavage and 1 ml 10% v/v DMSO i.p. for the same period of time. The resveratrol group (n=6) was treated with 30 mg/kg i.p. resveratrol and 1.5 ml drinking water by oral gavage for the 14 days, and the R+A group (n=8) was treated with 40 mg/kg atorvastatin by oral gavage and 30 mg/kg i.p. resveratrol. Rats were weighed every five days for adjustments to the dosing schedule and observed every day or as necessary. On day 15, the rats were anesthetized with an i.p. injection of 50 mg/kg body weight sodium pentobarbital. The heart and thoracic aorta were removed from each rat and placed immediately into fresh, oxygenated, ice-cold Krebs-Henseleit solution (KHS) composed of 119 mmol/l NaCl, 4.7 mmol/l KCl, 1.5 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 2.5 mmol/l CaCl2, 25 mmol/l NaHCO3 and 11 mmol/l glucose.A total of 28 male Wistar albino rats, aged 8 weeks and weighing 260–280 g, obtained from the Animal Care Facility of Meram Medical Faculty  were used in the present study. Animals were housed identically in cages in an air-conditioned room under a 12-h light/dark cycle. Temperature and relative humidity were controlled within the limits of 21±2°C and 55±15%, respectively. All animals were acclimated for ≥7 days prior to the onset of the study. A standard diet and tap water were provided 2O2 on myocardial contractions were then evaluated at concentrations of 1×10−7, 1×10−6 and 1×10−5 M. Following each dose, the organ bath was washed with KHS and the next dose was applied after 20 min resting time. The same procedure was conducted for all four groups. Contractions are given as a percentage of the initial contractions.Myocardial strips (10–13 mm long and 2–3 mm wide) were prepared from the left ventricle using a previously described method . The str−8, 1×10−7, 1×10−6, 1×10−5 and 1×10−4 M H2O2 were cumulatively added to the organ bath. Once the contractions reached a plateau, the tissues were washed twice every 15 min and incubated with 1×10−4 M N (G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) formation, for 30 min to evaluate the effect of the vascular endothelium on the H2O2 results. Following incubation, 1×10−8, 1×10−7, 1×10−6, 1×10−5 and 1×10−4 M H2O2 were cumulatively added to the organ bath once more. All results are expressed as a percentage of the previous contraction induced by 80 mM KCl.Thoracic aorta rings (2–3 mm wide) were placed into a 10-ml chamber containing oxygen-enriched (0.5 l/min) KHS at 37°C. One end of the strip was attached to a force transducer  by a thin silk thread, while the other end was pinned to a hook in the tissue bath. The strips were left for 15 min to spontaneously recover their isometric tension, following which they were gradually stretched to a resting force of 1 × g. The tissues were allowed to equilibrate for 30 min with repeated washing every 10 min with KHS. Following the equilibration period, the thoracic rings were contracted with 80 mM KCl. After the 30-min wash-out period in which the tissues were repeatedly washed every 10 min with KHS, 1×10Data are expressed as the mean ± standard error of the mean. The statistical significance of differences between the groups was analyzed by one-way analysis of variance or the Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.2O2 was applied to the organ bath at doses of 1×10−7, 1×10−6 and 1×10−5 M. To observe the effects of increasing doses of H2O2, an intragroup comparison of the myocardial results was carried out for each group. In the control group, H2O2 significantly reduced the contractions induced by ES at all doses . The results were 94.16±5.94, 80.35±5.66 and 62.61±8.28% for doses of 1×10−7, 1×10−6 and 1×10−5 M, respectively. In the rats treated with atorvastatin, H2O2 caused a significant dose-dependent decrease in myocardial contractions . In the resveratrol group, no significant changes in contraction were observed following H2O2 application at all doses . In the R+A group, the contraction results following H2O2 application were 76.57±1.40, 66.34±5.91 and 66.55±11.10% for 1×10−7, 1×10−6 and 1×10−5 M H2O2, respectively; H2O2 application did not significantly decrease the contractions when its concentration was increased.H2O2. At 1×10−7 M H2O2 the atorvastatin group showed a significantly lower contraction percentage when compared with the control and resveratrol groups (P<0.01). The R+A group also demonstrated a significant decrease in contraction percentage when compared with the control group (P<0.01). However, no significant difference was observed between the contraction percentages of the resveratrol and control groups . The resveratrol group tissues showed a significantly higher percentage contraction than those of the atorvastatin and R+A groups (P<0.01). No significant difference was observed between the myocardial contractions in the resveratrol and control groups (2O2 (1×10−5 M), the atorvastatin group exhibited a significant decrease in contraction compared with all the other groups . The results of the present study demonstrated that resveratrol treatment alone attenuated the decrease in contraction percentage and that this effect was significant compared with all groups at 1×10−5 M H2O2 (P<0.01 vs. atorvastatin and control and P<0.05 vs. R+A). In the R+A group, the contraction percentages were higher than those in the atorvastatin group but significantly lower than those in the resveratrol group (P<0.05)  .2O2 (between 1×10−8 and 1×10−4 M) in aortic segments for the control, resveratrol, atorvastatin and R+A groups, with and without 1×10−4 M L-NAME incubation. In all groups, H2O2 caused vasoconstriction and the contraction responses increased in a concentration-dependent manner. The aortic rings reached their maximum contraction at 1×10−4 M H2O2, and the maximum contraction responses were 16.14±1.09, 7.50±0.75, 6.82±1.33 and 5.58±1.37% for the control, atorvastatin, resveratrol and R+A groups, respectively. The H2O2 responses were significantly lower in the treatment groups than those in the control group at 1×10−4 and 1×10−5 M H2O2 . When the maximum vasoconstriction responses of the treatment groups were examined, no statistical differences were identified among the resveratrol, atorvastatin and R+A groups  in body fluids –29. Althet al  demonstrependent . The proependent .2O2, but that its co-treatment with resveratrol recovered this effect. R+A treatment also exhibited a protective effect on H2O2-induced vascular responses. From these results, resveratrol appears to be a promising treatment for the improvement of myocardial function in diseases associated with the development of oxidative stress. Resveratrol has been a popular choice in OTC products. Resveratrol is frequently used for the prevention of atherosclerosis; thus, the indications for its administration appear to be similar to those for the administration of statins. The combined treatment of R+A provides a superior treatment for CVD compared with treatment with either agent alone.Increased levels of pro-oxidants have been associated with vascular diseases and they are considered to be an important initial step in the development of vascular diseases, including atherosclerosis and hypertension . The pre


We investigated associations of sixteen SNPs across seven genes with prostate-specific antigen-detected prostate cancer.Vitamin D pathway single nucleotide polymorphisms (SNPs) are potentially useful proxies for investigating whether circulating vitamin D metabolites . We quantified associations of SNPs with stage (T1–T2/T3–T4) and grade (<7/≥7). Multiple variant scores included SNPs encoding proteins involved in 25(OH)D synthesis and metabolism.p = 0.04; rs7041-T: OR 1.19, CI 1.02, 1.38, p = 0.03). Low 25(OH)D metabolism score was associated with high (vs low) grade ; there was a similar association of its component variants: rs6013897-A in CYP24A1  and rs10877012-T in CYP27B1 . There was no evidence that associations differed by level of sun exposure.We included 1,275 prostate cancer cases  and 2,062 healthy controls. Vitamin D-binding protein SNPs were associated with prostate cancer .The online version of this article (doi:10.1007/s10552-014-0500-5) contains supplementary material, which is available to authorized users. Thus, if the intermediate phenotype is causally associated with the outcome (prostate cancer) then we would expect the genetic variants to be associated with the outcome, to the extent that the genetic variants affect the intermediate phenotype. There are a number of single nucleotide polymorphisms (SNPs) involved in the vitamin D pathway which are potentially useful proxies for investigating whether circulating vitamin D is causally related to prostate cancer: vitamin D 25-hydroxylase enzyme (CYP2R1) converts provitamin D (from sun exposure or dietary intake) into circulating 25(OH)D . A case-only analysis was used as all cases have undergone biopsy, therefore removing potential detection bias which could otherwise occur through misclassification of control status because of imperfect sensitivity of the PSA test . SNPs wet tests and Chi-squared tests, respectively.The association of SNPs with prostate cancer was repeated, stratified by level of sun exposure. Since serum vitamin D level is an effect common to vitamin D pathway SNPs and confounders, attempting to estimate this association using serum vitamin D levels would have resulted in biased estimates which may have led to spurious associations between SNPs and prostate cancer risk p value for interaction. A dichotomized indicator of sun exposure was created based on levels above and below the median level of sun exposure.A likelihood ratio test, comparing the main effects model with the model including an interaction term between the SNP and level of sun exposure, was used to calculate a The top 10 principal components (PCs) that reflect the population’s genetic structure were estimated according to Price et al.  from genAll analyses were carried out in Stata 12 . We used ice for multiple imputation with chained equations  for impu2D measurement  localized, 141 (11.1 %) locally advanced, 3 (0.24 %) missing stage; 887 (69.6 %) low grade, 385 (30.2 %) high grade, 3 (0.24 %) missing grade], and 2,062 controls that have at least one available SNP. 39.3 % of participants had data available on all SNPs, and 43 % were missing data on three or fewer SNPs. No man was missing more than 10 SNPs. Of these, 926 cases and 872 controls had an available 25(OH)D measurement and 779 cases and 737 controls had an available 1,25(OH)p for difference = 0.62). The mean (SD) 1,25(OH)2D concentration in cases was 40.6 pg.mL (18.4) and in controls was 40.9 pg/mL (18.1) (p for difference = 0.74). There were no differences between mean storage times of blood samples between cases and controls. Only VDR SNP rs7975232 was out of Hardy–Weinberg equilibrium in controls (p = 0.01).The mean (SD) 25(OH)D concentration in cases was 23.7 ng mL (8.7) and in controls was 23.5 ng/mL (8.7) (VDBP rs4588-A with BMI (p = 0.01), CYP27B1 rs703842-G with smoking status (p = 0.02), diabetes (p = 0.01) and social class (p = 0.01) and CY27B1 rs10877012-T with smoking status (p = 0.01), and diabetes (p = 0.01). These associations are possibly due to chance as we carried out multiple tests, i.e., we tested 16 SNPs against eight covariates.For the most part, there was no evidence of associations between vitamin D pathway SNPS and the covariates (data not shown). There was evidence of an association of 2D measurements. Of the 16 SNPs investigated, two SNPs in CYP2R1  and two SNPs in VDR  were associated with 25(OH)D concentrations (with ss ranging from ≤0.001 to 0.04) (Table DHCR7 (rs12785878-G) and four SNPs in VDR  were associated with 1,25(OH)2D concentrations   were associated with both 25(OH)D and 1,25(OH)2D concentrations  (Table 2D concentrations did not differ by case–control status (results not shown). Based on the proportion of trait variability explained and on the F-statistic, which is related to the strength of the instrument, the best instruments for 25(OH)D concentrations in this population were VDBP SNPs (F = 12.2–22.3). Strong associations were detected between all VDBP SNPs and 1,25(OH)2D (F = 5.88–9.62). However, each polymorphism explained only ~1 % of the trait variance, and the F-statistics were below 10, the conventional lower limit of a strong instrument .Our study includes a large sample, with more men with prostate cancer and matched controls than previous studies, about which we have extensive information recorded. All of our men were resident in the UK and 99 % of our subjects self-reported their ethnicity as white. Since the decision to biopsy was based on PSA level, some of the controls with PSA <3 ng/mL will have unidentified prostate cancer  . Circulating vitamin D levels were measured in plasma collected at the prostate check clinic prior to diagnosis, with measurement of vitamin D concentrations blind to diagnosis. The study is population-based and thus subject to little selection bias. Circulating levels of 25(OH)D may vary by season of tissue collection . Circulating 25(OH)D and 1,25(OH)2D may not reflect the amount of 25(OH)D and 1,25(OH)2D available for use within the target tissues [Circulating 25(OH)D and 1,25(OH)llection , which iOur Mendelian randomization (MR) approach is more reliable than results from observational studies and can be used to strengthen the evidence of causality since genotypes are unlikely to be affected by confounding or reverse causation and are not subject to high levels of measurement error. Our SNPs and genetic scores were mostly not associated with the confounders, thus satisfying one of the main assumptions of MR. However, we did find evidence of an association of rs4588 with BMI and rs10877012 with smoking. These associations may be chance findings, but, if real, may pose a problem to our inference of causality, as BMI has been associated with prostate cancer . All SNPCYP24A1 and rs10877012T in CYP27B1), were associated with Gleason grade (high grade vs low grade). There was no association of other vitamin D pathway polymorphisms being associated with overall prostate cancer risk, stage, or grade. There was no evidence of an association in men with deficient vitamin D (measured by having low sun exposure).Our study found evidence that two SNPs in vitamin D-binding protein were associated with prostate cancer risk (rs4588-A and rs7041-T). A score measuring metabolism, and its component variants Below is the link to the electronic supplementary material.


The porous cylindrical β-TCP scaffolds doped with 0.5 wt % SiO2, 1.0 wt % MgO, 0.5 wt % SiO2 + 1.0 wt % MgO were fabricated via selective laser sintering respectively and undoped β-TCP scaffold was also prepared as control. The phase composition and mechanical strength of the scaffolds were evaluated. X-ray diffraction analysis indicated that the phase transformation from β-TCP to α-TCP was inhibited after the addition of MgO. The compressive strength of scaffold was improved from 3.12 ± 0.36 MPa (β-TCP) to 5.74 ± 0.62 MPa (β-TCP/SiO2), 9.02 ± 0.55 MPa (β-TCP/MgO) and 10.43 ± 0.28 MPa (β-TCP/SiO2/MgO), respectively. The weight loss and apatite-forming ability of the scaffolds were evaluated by soaking them in simulated body fluid. The results demonstrated that both SiO2 and MgO dopings slowed down the degradation rate and improved the bioactivity of β-TCP scaffolds. In vitro cell culture studies indicated that SiO2 and MgO dopings facilitated cell attachment and proliferation. Combined addition of SiO2 and MgO were found optimal in enhancing both the mechanical and biological properties of β-TCP scaffold.Nano SiO They proved that SiO2 and ZnO dopings increased the mechanical strength and facilitated cellular proliferation [et al. studied the effect of ZnO, SiO2 and SrO dopings on the mechanical and biological properties of β-TCP ceramics. And it was concluded that the dopings could tailor the strength and degradation behavior [et al. introduced MgO into dense hydroxyapatite (HA)/β-TCP ceramics to improve the sintering ability. The results showed a transgranular fracture mode and enhanced mechanical properties of HA/β-TCP ceramics doped with 1.0 wt % MgO [Carlisle verified that Si (up to 0.5 wt %) often localized in active growth areas and played an important role in bone formation and calcification . SiO2/Znferation . Bandyopbehavior . Ryu et wt % MgO .Besides, an interconnected porous structure in bone scaffold is essential to facilitate cell infiltration, bone growth and vascularization . So far,2 (0.5 wt %) and nano MgO (1.0 wt %) were used as dopants to improve the mechanical and biological properties of β-TCP scaffolds. The scaffolds were fabricated by SLS and characterized in terms of phase composition, morphology and compressive strength. The biodegradation behavior was evaluated by immersing the scaffolds in simulated body fluid (SBF) for 2 and 4 weeks. In addition, the human osteosarcoma MG-63 cells were cultured on the scaffolds to assess cell proliferation in vitro.In light of the present scenario, small quantities of nano SiO2. The designed porosity for all scaffolds was 48.3%. A representative cylindrical scaffold was fabricated via SLS  patterns from sintered scaffolds were presented in o et al. . A quante et al. . However3) to obtain the relative density of sintered scaffolds (2 increased the density slightly (91.35% ± 1.46%). The relative density of β-TCP/MgO and β-TCP/SiO2/MgO increased to 93.28% ± 1.08% and 95.53% ± 0.94%, respectively. The phase transformation from β-TCP to α-TCP prevented further densification due to the volume expansion. It was concluded that MgO could inhibit the phase transformation and improve the densification of β-TCP scaffolds.The apparent densities of the scaffolds were determined in water using Archimedes principle and then normalized with the theoretical density of β-TCP  [2. Higher compressive strength for β-TCP/MgO and β-TCP/SiO2/MgO scaffolds might be ascribed to the reduced formation of α-TCP phase and increased density after MgO doping, thereby significantly improving the mechanical properties of the scaffold. Impens et al. prepared β-TCP scaffolds utilizing gel-casting method. The scaffolds had a porosity of 80% and exhibited a compressive strength of 2.5 MPa [The typical stress-strain curve from compression testing was shown in –16 MPa) . The res 2.5 MPa , which wThe bone-like layer on scaffold surfaces was revealed in 2 scaffold was covered with petal-like apatite layer after soaked for 2 weeks  displayed lower degradation rates. The weight losses were 5.70 ± 0.72 wt %, 6.62 ± 0.68 wt %, and 4.43 ± 0.76 wt % respectively after 2 weeks. And β-TCP/SiO2/MgO showed the lowest weight loss of 5.44 ± 0.52 wt % (4 weeks). The weight loss results demonstrated that the degradation dominated over apatite formation for all scaffolds. Nevertheless, the addition of SiO2 and MgO slowed down the degradation of β-TCP scaffolds. This was because Si and Mg increased the mineralization rate in SBF. Si and Mg acted as nucleation or created a favorable condition for apatite formation and at the same time slowed down the dissolution process in vitro [2/MgO scaffold was significantly lower than that of β-TCP scaffold. As Ca ions were released to the solution, hydrogen ions formed a hydrated silica leaching layer containing Si–OH groups on the scaffold surface, which acted as a catalyzing agent for the nucleation and growth of bone-like apatite layer. Li et al. found that SiO2 can form a Si–OH functional layer easily in vitro and thereby enhance the mineralization of apatite-like phase. It was believed that the fast formation of bone-like layer on the surfaces of these scaffolds was closely related to the increasingly provided silanol groups [The scaffolds underwent both apatite layer formation and degradation processes in SBF. The weight change percent of scaffolds in SBF after 2 and 4 weeks were shown in in vitro . Weight l groups .2 and MgO could be used as additives to adjust the biodegradation characteristics of β-TCP scaffold.The high degradation rate of β-TCP made it difficult to match the growth rate of new bone tissues. Results of this study revealed that SiOin vitro biological evaluation, MG-63 cells were cultured and seeded on each scaffold. Morphologies of MG-63 cells on the scaffolds after 7 days were shown in 2 compared with β-TCP  and nano MgO  were purchased from Nanjing Emperor Nano Material Co. . Pure β-TCP powder (β-TCP), β-TCP powder doped with 0.5 wt % SiO2 (β-TCP/SiO2), β-TCP powder doped with 1.0 wt % MgO (β-TCP/MgO), β-TCP powder doped with 0.5 wt % SiO2 and 1.0 wt % MgO (β-TCP/SiO2/MgO) were prepared by mechanical mixing, respectively. The amounts were chosen according to previous research.β-TCP powder with an average particle size of 0.2 μm was obtained from Kunshan Chinese Technology New Materials Co. . Nano SiO2, β-TCP/MgO and β-TCP/SiO2/MgO) were used to fabricate scaffolds. All scaffolds were fabricated on a home-developed SLS system using a 100 W CO2 laser  with wavelength of 10.6 μm [The powders of different compositions  to investigate the surface morphologies of the scaffolds. The compressive strengths of the scaffolds were determined on a mechanical tester  with a constant cross-head speed of 0.4 mm/min. Compressive strength was calculated from the stress-strain curve based on the maximum load recorded and scaffold dimension. For each group, five scaffolds with radius 7 mm and height 8 mm were tested to ensure reproducibility. The data were analyzed statistically.2 (JCPDS No. 76-1390). The relative amounts of β-TCP after sintering were determined by integrated intensities of their peaks in XRD patterns. Reference intensity ratio (RIR) method was employed to identify and quantify the weight percent of β-TCP in the sintered samples according to Equation (1):iX is the weight percent of crystalline phase i, iRIR and jRIR are the reference intensity ratios of crystalline phase i and j, iI and jI are the integrated intensities of the diffraction peaks of phase i and j, and n is the number of crystalline phases.The phases of the scaffolds were characterized with XRD diffractometer  using Cu-Kα radiation at 40 mA and 40 kV. Samples were grinded into powders and tested with 2θ values from 20° to 60° at a step size of 0.02°. Phases were identified by comparing the diffraction patterns of samples with the standard PDF cards: α-TCP (JCPDS No. 9-348), β-TCP (JCPDS No. 9-169), MgO (JCPDS No. 45-946), SiO+, 5.0 K+, 1.5 Mg2+, 2.5 Ca2+, 148 Cl−, 4.2 HCO3−, 1.0 HPO42− and 0.5 SO42−) was almost equal to that of human plasma and buffered at similar pH. The scaffolds were immersed into the SBF solution at 37 °C for 2 and 4 weeks. The solution was changed every 3 days during the immersion process. At each time point, four samples of each composition were removed, washed with distilled water, and then dried at 65 °C in an oven for 48 h. After the drying process, the weight of each sample was carefully recorded and compared with their net weight before immersion. The morphology of apatite layers on scaffold surfaces was observed under SEM. The mineral layer was also analyzed by energy dispersive spectrometer (EDS) .All scaffolds were immersed in SBF solution to investigate their degradation behavior and bioactivity ability. SBF solution with ionic composition  were then directly seeded on the scaffolds after removing PBS solution completely. To avoid cell leakage during cell seeding, the concentrated MG-63 suspension was dropped onto the scaffolds and cultured for 1 h to allow the attachment of cells onto the surfaces. Then the medium was supplemented to 1 mL well. The medium was changed every 3 days. The medium was removed after 7 days, and the scaffolds with attached cells were washed with PBS and dehydrated in graded ethanol (30%–100%) and Freon. The fully dried samples were coated with a thin gold film and the morphology of the cells was analyzed with SEM . For each group, five scaffolds with radius 7 mm and height 8 mm were cell-cultured and tested to ensure reproducibility.Human osteosarcoma MG-63 cells were used to explore the cell-materials interactions. Before seeding, the scaffolds were sterilized with 70% ethanol for 30 min and then further sterilized under UV light for 30 min, followed by drying under sterile conditions at room temperature for 2 h. Then the scaffolds were washed and immersed in phosphate-buffered saline (PBS) for 24 h. Cells were cultured in α-MEM medium  supplemented with 10% FBS  and 100 U/mL penicillin/streptomycin (Hyclone) at 37 °C under 5% CO2 and MgO were used to improve the mechanical and biological properties of β-TCP scaffolds. Porous cylindrical scaffolds were successfully fabricated via SLS technique. MgO improved the thermal stability and reduced the phase transformation from β to α-TCP. The compressive strength of β-TCP scaffold doped with SiO2/MgO showed the maximum increase from 3.12 ± 0.36 to 10.43 ± 0.28 MPa. In vitro mineralization study in SBF indicated that SiO2 and MgO could decrease the biodegradation rate of β-TCP scaffold and facilitate apatite growth. In vitro cell culture study demonstrated that SiO2 and MgO dopings promoted the cell attachment and proliferation of β-TCP scaffold. Thus, β-TCP scaffold doped with SiO2 and MgO was a potential candidate for bone substitutes applied in bone tissue engineering.Nano SiO