We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca2+ signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca2+]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5′-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca2+ were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca2+ mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro‐inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (EThe online version of this article (doi:10.1007/s00424-016-1840-7) contains supplementary material, which is available to authorized users. Clinical observations suggested that reduced E2 levels were associated with greater risks of lung pathologies in menopausal women i using Felix software . In Ca2+ imaging experiments, the perfusion chamber was mounted on an inverted microscope  equipped with a scientific CMOS camera . Images were digitized and analyzed using MetaFluor Imaging Software . The data were also shown quantitatively as a change in Fura-2 ratios.Calcium signals in cells grown on glass coverslips were measured as previously described , 48. Fur2+ has a similar permeability as Ca2+ through most plasma membrane Ca2+ channels and quenches Fura-2 fluorescence at all excitation wavelengths, Ca2+ influx can be estimated by the Mn2+ quench of Fura-2 fluorescence at the Ca2+-insensitive 360-nm excitation wavelength. During the measurement, cells were treated with 10-μM UTP in the absence  or presence of E2 (100 nM) or G1 (10 nM) for 10 min. Then 1-mM MnCl2 was added in perfusion solution to observe the extent of Mn2+ entry. The rate of Mn2+ quenching was assessed by measuring the change of slope of Fura-2 fluorescence decrease before and after the addition of Mn2+ application , as well as the percentage decrease of Fura-2 fluorescence 120 s after Mn2+ application i i . Because pathway , we exam4o- Fig.  and prim4o- Fig.  cells we2 and G1 were used to examine their inhibitory effects on P2Y receptor-mediated Ca2+ signaling in 16HBE14o- cells. The 16HBE14o- cells were pretreated with E2 (1–300 nM) or G1 (0.01–100 nM) for 10 min and then stimulated by 10-μM UTP in the presence of E2 or G1. Both E2 and G1 inhibited the UTP-induced increases in Ca2+ in a concentration-dependent manner  values of E2 and G1 were 12.42 and 0.58 nM, respectively.Various concentrations of Ener Fig. . The hal2+ was reversed in both 16HBE14o-  on the increase in Ca2+ was also reversed in the presence of G15  and influx (second phase), epithelia were exposed to Ca2+-free solution in the presence or absence of G1 or E2 for 10 min and then stimulated with UDP or UTP together with G1 or E2. Subsequently, Ca2+ (2.5 mM) was added back to the perfusate to induce Ca2+ influx. Typical recordings of fluorescence ratios in control -induced Ca2+ release in 16HBE14o- cells -induced Ca2+ influx  was reversed by 1-μM G15  channels regulated by stromal interaction molecule 1 (STIM1) . To examrol Fig.  and E2-tted Fig.  cells shlls Fig. . Howeverlux Fig. . MoreoveG15 Fig. . Taken t2+ was measured at the Ca2+-independent excitation wavelength of Fura-2 (360 nm). When preincubating the cells with 100-nM E2 for 10 min, the rate of change on Mn2+ quenching was significantly decreased compared to untreated or G1 pretreated 16HBE14o- cells  in human airway epithelial cells, including 16HBE14o- cells i levels, but it did significantly inhibit P2Y receptor-mediated increases in Ca2+. This inhibitory effect was not P2Y receptor subtype-specific, because G1 inhibited the Ca2+ responses elicited by different P2Y receptor subtype ligands . The inhibitory effect could be reversed when pretreating with GPER-specific antagonist, G15. Besides, siRNA knockdown of GPER was also applied to further confirm the role of GPER. Various transfect reagents  have been applied in our experiment for GPER silencing; however, the knockdown efficiency was low and inconsistent (data not shown). Therefore, lentiviral-mediated silencing approach was adopted and a stable silenced cell line was generated with better knockdown efficiency on GPER. To reveal whether G1 inhibited intracellular increases in Ca2+ by blocking Ca2+ release and/or Ca2+ influx, we characterized the two phases of Ca2+ increase by perfusing cells with a Ca2+-free Krebs-Henseleit Buffer, followed by a Ca2+-containing solution. The results showed that E2 inhibited both P2Y receptor-mediated Ca2+ release and Ca2+ influx, whereas G1 only inhibited Ca2+ release. However, the observed difference in Ca2+ signals could be due to regulation of Ca2+ pumps. The inhibitory effect of E2 on Ca2+ influx was further confirmed by the data obtained from Mn2+ quench experiments. Taken together, these data suggest that the activation of GPER only inhibits P2Y-activated IP3-mediated Ca2+ release, whereas classical E2 receptors activated by E2 played a role in regulating Ca2+ influx. FRET microscopy further confirmed that the inhibitory effect of E2, but not G1, on SOC influx was due to the inhibition of STIM1 oligomerization. Our findings were similar to those reported recently i after P2Y receptor activation. Treating 16HBE14o- cells with the intracellular Ca2+ chelator, BAPTA-AM, but not H89, inhibited P2Y receptor-mediated IL-6 and IL-8 secretion [2+]i in human airway epithelia. A recent study reported that Ca2+-dependent calmodulin can regulate GPER-dependent signaling at the receptor level [2+ could, in turn, regulate GPER function, but the details of the possible cross talk between the two receptors require further investigation. 16HBE14o- cells were cultured in MEM without phenol red in some experiments since phenol red may serve as a weak estrogen mimic. However, no significant differences have been observed in terms of GPER expression and the inhibitory effect of G1 on P2Y receptor-mediated cytokine secretion and Ca2+ increase (data not shown) in cells cultured in MEM with or without phenol red.GPER couples to different signaling pathway(s), including the cAMP/PKA pathway . Our datow cells . The cAMow cells . Calciumow cells  and can ow cells . A recenow cells . Althoug actions , 9. The  actions . Therefoecretion . Taken tor level . Therefo2 or its specific agonist, G1, rapidly attenuated a nucleotide-evoked increase in Ca2+, whereas the specific GPER antagonist, G15, reversed this GPER-mediated inhibition. Furthermore, E2 and G1 also inhibited nucleotide-induced cytokine release. The inhibitory effects on P2Y receptor-mediated Ca2+ mobilization and cytokine secretion are likely due to GPER-mediated activation of a cAMP-dependent PKA pathway in human bronchial epithelia Below is the link to the electronic supplementary material.


CmLOX10 and CmLOX13 which have been isolated from oriental melon (Cucumis melo var. makuwa Makino) cultivar “Yumeiren”, encode 902 and 906 amino acids, respectively. Bioinformatics analysis showed that CmLOX10 and CmLOX13 included all of the typical LOX domains and shared 58.11% identity at the amino acid level with each other. The phylogenetic analysis revealed that CmLOX10 and CmLOX13 were members of the type 2 13-LOX subgroup which are known to be involved in biotic and abiotic stress. Heterologous expression of the full-length CmLOX10 and truncated CmLOX13 in Escherichia coli revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the lipoxygenase activities. The purified CmLOX10 and CmLOX13 recombinant enzymes exhibited maximum activity at different temperature and pH and both had higher affinity for linoleic acid than linolenic acid. Chromatogram analysis of reaction products from the CmLOX10 and CmLOX13 enzyme reaction revealed that both enzymes produced 13S-hydroperoxides when linoleic acid was used as substrate. Furthermore, the subcellular localization analysis by transient expression of the two LOX fusion proteins in tobacco leaves showed that CmLOX10 and CmLOX13 proteins were located in plasma membrane and chloroplasts respectively. We propose that the two lipoxygenases may play different functions in oriental melon during plant growth and development.Lipoxygenases (LOXs) are a class of non-heme iron-containing dioxygenases that catalyse oxidation of polyunsaturated fatty acids to produce hydroperoxidation that are in turn converted to oxylipins. Although multiple isoforms of LOXs have been detected in several plants, LOXs in oriental melon have not attracted much attention. Two full-length LOX cDNA clones,  Lipoxygenases  are a class of non-heme iron-containing dioxygenases that catalyze oxidation of polyunsaturated fatty acids (PUFAs) with a -1,4-pentadiene structure to produce unsaturated fatty acid hydroperoxides . Plant LEscherichia coli, positional specificity of LOXs in diverse plants was identified. Pepper CaLOX1 encodes a 9-specific lipoxygenase [Based on the positional specificity of the oxygen insertion, plant LOXs are grouped into 9-LOXs and 13-LOXs which produce 9- and 13-hydroperoxides respectively. Both hydroperoxides can be enzymatically cleaved to aldehydes and ω-oxo acids . Accordixygenase , while txygenase . In addixygenase .PvLOX6 in common bean showed that wounding or non-host pathogen infections, as well as signaling molecules like H2O2, SA and MeJA regulated PvLOX6 expression [In plants, LOXs are related to many biological processes and physiological functions, such as tuber development , generatpression .Cucumis melo var. makuwa Makino) is an important agricultural commodity and widely grown in China and eastern Asian countries. In comparison with other plants, there is relatively little known about oriental melon lipoxygenases. With the completeness of melon genomic sequencing project which provides an ideal way for melon gene cloning and functional analysis [CmLOX10 and CmLOX13 cDNAs which they get together closely in phylogenetic tree from the tender stem of 30-day-old oriental melon (Cucumis melo var. makuwa Makino) cultivar “Yumeiren” and expressed CmLOX10 and CmLOX13 in Escherichia coli to characterize the two proteins. The results show that the recombinant CmLOX10 and CmLOX13 preferentially consume linoleic acid and introduce oxygen onto the 13th carbon of the fatty acid. The subcellular localization analysis by transient expression of CmLOX10 and CmLOX13 in tobacco leaves shows that CmLOX10 and CmLOX13 proteins are located in plasma membrane and chloroplasts respectively. This study provides knowledge on the physiological function of CmLOX10 and CmLOX13 genes in oriental melon.The oriental melon  cultivar “Yumeiren” were individually grown in a greenhouse at 25 ± 2°C, under a 14-h light/10-h dark photoperiod at Shenyang Agricultural University in China. The tender stems of 30-day-old oriental melon were immediately frozen in liquid nitrogen and stored at -80°C until used for the cloning of CmLOX10 and CmLOX13. Tobacco (Nicotiana benthamiana) plants for agro-infiltration were raised in a growth chamber at 25°C under a 16/8 h day/night cycle for six weeks.Oriental melon  in a 50 μl reaction mix containing 2 μl first-strand cDNA and 0.2 μM of each primer. The sense primer (CmLOX10-F and CmLOX13-F) and the antisense primer (CmLOX10-R and CmLOX13-R) were designed based upon the CmLOX10 and CmLOX13 sequences from the melon genome database and used for the cloning of the two full-length LOX genes. The PCR amplification conditions were as follow: 30 cycles of 10 s at 98°C, 15 s at 54°C for CmLOX10 and 52°C for CmLOX13 respectively, and 3 min at 72°C; 10 min at 72°C. The amplified about 3.0 kb LOX fragments were purified using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit  according to the manufacturer’s protocols and inserted by TA-cloning into the pMD18-T vector  following adding A-tailing. The plasmids showing inserts of the expected size following plasmid PCR were sequenced by invitrogen  and positive clones were designated as p18T-LOX10 and p18T-LOX13, respectively.Total RNA was extracted from the tender stem of melon using Ultrapure RNA Kit  and treated to eliminate genomic DNA using RNase-Free DNaseI . First-strand cDNAs were synthesized using M-MLV RTase cDNA Synthesis Kit  following the manufacturer’s protocol and used for CmLOX10 and CmLOX13 from the melon genome database acquired by RT-PCR were truly full-length, 5’ rapid amplification of cDNA ends (RACE) was performed using a commercial kit . Following the manufacturer’s recommendations, 5’ RACE of the CmLOX10 was performed with the three gene-specific primers: LOX10-GSP1, LOX10-GSP2 and LOX10-GSP3, while 5’ RACE of the CmLOX13 used LOX13-GSP1, LOX13-GSP2 and LOX13-GSP3.To identify whether the coding sequences of CmLOX10 and CmLOX13 cDNAs acquired by a combination of RT-PCR and 5’ RACE-PCR were analyzed using several bioinformatics software. The calculated molecular mass and conserved domains of the deduced amino acid sequences of CmLOX10 and CmLOX13 were determined by ProtParam (http://web.expasy.org/protparam/) and the NCBI Conserved Domain Search program (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), respectively. The deduced protein sequences of CmLOX10 and CmLOX13 were aligned using ClustalW2 software and displayed with GeneDoc. ClustalW2 software was used to align the deduced protein sequences of CmLOX10 and CmLOX13 with the protein sequences of other plant LOXs identified. The phylogenetic tree was then constructed using MEGA 5.0 based on the neighbor-joining method and a bootstrap value was calculated from 1000 replicates. The corresponding protein accession numbers of the identified LOX genes were acquired from NCBI GenBank (http://www.ncbi.nlm.nih.gov/Genbank). Subcellular localization was predicted by three different programs: WoLFPSORT (http://wolfpsort.org/), TargetP (http://www.cbs.dtu.dk/services/TargetP/), and ChloroP (http://www.cbs.dtu.dk/services/ChloroP/).The full-length CmLOX10 and CmLOX13 containing EcoRI and XhoI restriction site were acquired using four PCR primers and two PCR reactions respectively according to the method described in the book Purifying Proteins for Proteomics: A Laboratory Manual [CmLOX13 was amplified by the first pair of primers containing LOX13t-F-1 and LOX13t-R-1 and the second pair of primers containing LOX13t-F-2 and LOX13t-R-2 respectively to eventually generate two different PCR fragments of truncated CmLOX13. Similarly, the two different PCR fragments of the truncated CmLOX10 were acquired by the primers including LOX10t-F-1, LOX10t-R-1, LOX10t-F-2 and LOX10t-R-2 and the full-length CmLOX10 were acquired by the primers containing LOX10f-F-1, LOX10f-R-1, LOX10f-F-2 and LOX10f-R-2 using the p18T-LOX10 plasmid as template DNA. Typical PCR reactions were performed with high-fidelity PrimeSTAR™ HS DNA Polymerase  according to the manufacturer’s instructions and the PCR condition was as similar as that described above. The PCR products were purified using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit  according to the manufacturer’s protocols.The DNA fragments of y Manual . Based oCmLOX13 acquired above were mixed in a 50 μl reaction mixture containing 1 μl T4 polynucleotide kinase  and 1 μl 100 mM ATP  and the reaction conditions were as follow: 90 min at 37°C, 5 min at 95°C and 10 min at 25°C. The mixture containing the DNA fragments of truncated CmLOX13 was directly ligated into the expression vector pET-30a (+)  digested by EcoRI and XhoI  using T4 DNA ligase  to generate pETt-CmLOX13 which encodes CmLOX13 with a His tag fused to the N terminus. The truncated and full-length CmLOX10 expression vectors were constructed using the same method above designated as pETt-CmLOX10 and pET-CmLOX10, respectively. The truncated CmLOX13 and the truncated and full-length CmLOX10 ORF of these constructs were checked by sequencing prior to transform into Escherichia coli BL21 (DE3).The same amount of two different purified PCR fragments of the truncated −1 kanamycin and grown overnight at 37°C. The overnight culture of the E. coli BL21 (DE3) was transferred into one liter of LB medium containing the same amount of kanamycin and grown at 37°C until its optical density at 600 nm (OD600) reached approximately 0.6 followed by adding the isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM to induce the expression of the target proteins. The culture was subsequently incubated at 20°C for 18 h. Bacteria were harvested by centrifugation at 7000 rpm for 10 min at 4°C and the pellet was then resuspended in 40 ml of ice-cold lysis buffer  followed by lysing cell using sonication (5 s bursts and 7 s breaks for 30min on ice at 30% power) SONICATOR JY99-IIDN . After sonication on ice, the resulting slurry was centrifuged at 10000 g for 30 min at 4°C to pellet cell debris. The supernatant (soluble protein fraction) obtained as described above was filtered through a 45-μm filter and mixed with the same volume binding buffer . Then the mixture above and 2 ml of nickel-nitrilotriacetate agarose resin  pre-equilibrated with binding buffer was mixed and vibrated at 4°C for 1 h followed by adding to a 12-ml purification column .The transformants harboring pETt-CmLOX13, pETt-CmLOX10 and pET-CmLOX10 plasmids were inoculated in 2 ml of Luria-Bertani medium containing 50 μg mlThe column was washed with the binding buffer and the nonspecific bound proteins were eluted. His-tagged proteins were then eluted with the elution buffer . The purified CmLOX10 and CmLOX13 were subjected to 7.5% SDS-PAGE and stained with Coomassie Brilliant Blue to confirm purity. Fractions of 1.5 ml showing LOX activity and containing pure protein on SDS-PAGE were concentrated and exchanged into 30 mM sodium phosphate, 50 mM NaCl (pH 7.5) by using an Amicon Ultra-15 membrane with a molecular weight cutoff of 30,000  as described by Palmieri-Thiers et al . All purSDS-PAGE was performed using 7.5% polyacrylamide gels. The proteins were stained with Coomassie brilliant blue R-250 or transferred onto nitrocellulose using a Trans-Blot Turbo blotting System . Blots were blocked overnight using 5% nonfat milk powder and the membrane was incubated for 12 h at 4°C with the anti His-tag mouse monoclonal antibody  at 1:3000 in western antibody dilution buffer . Then the membrane was washed three times for 10 min in TBST  followed by incubating 30 min with HRP conjugated goat anti-mouse antibody  at 1:10000 in western antibody dilution buffer and washing three times for 10 min in TBST. Finally the membrane pretreated using Pro-light HRP Chemiluminescent Kit  according to the manufacturer’s instructions was exposed using ChemiDoc imaging system  for 1 min to detect the proteins containing histidine-tag.LOX activity was determined by continuously monitoring the formation of conjugated dienes at 234 nm. Linoleic acid and linolenic acid were purchased from Sigma-Aldrich . The substrates were prepared according to the method described by Gata et al .To estimate the optimum pH of the two recombinant LOX proteins, the reaction was run across a pH range of 4.0–9.0 at 25°C. Reaction buffers were prepared with 0.5 pH unit increments as follows: 100 mM sodium acetate (pH 4.0–5.5), 100 mM sodium phosphate (pH 6.0–7.5) and 100 mM sodium borate (pH 8.0–9.0). The effect of temperature on the activity of the two recombinant LOX proteins was determined at the temperatures ranging from 20 to 50°C with 5°C unit increments at pH 5.0 for CmLOX10 and 5.5 for CmLOX13.Enzymatic assays were performed at saturating oxygen concentration at 25°C. The standard reaction mixture (3 ml) consisted of 0.33 mM linoleic acid or linolenic acid in 100 mM reaction buffer and 5 μl purified enzyme. The control reaction was carried out using the same pH reaction buffer but using water instead of the enzyme. Enzymatic activity was measured according to the method described by Liu et al  with a fThe identity of the reaction products of recombinant CmLOX10 and CmLOX13 proteins was determined according to the method described by Long et al . The 9-HCmLOX10 and CmLOX13 without stop codon were re-amplified by the primers containing LOX10G-F/R and LOX13G-F/R, respectively. Typical PCR reactions were performed with high-fidelity PrimeSTAR™ HS DNA Polymerase  according to the manufacturer’s instructions and the PCR conditions were as follow: 30 cycles . The PCR product was purified using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit  according to the manufacturer’s protocols and cloned into a pENTR™/D-TOPO® vector  followed by transforming into DH5α competent E. coli cells . Plasmids from five randomly selected clones for each gene were extracted, checked by restriction digest and sequenced by invitrogen . The resulting entry vectors were used to deliver the CmLOX10-GFP and CmLOX13-GFP constructs into the plant expression vector pB7FWG2 [® LR Clonase™ II Enzyme Mix  according to the manufacturer’s instructions. After transformation and sequencing, the binary vectors which were designated as LOX10-GFP and LOX13-GFP were transferred into the Agrobacterium tumefaciens strain GV3101 by freeze-thaw transformation.Two GFP fusion constructs were produced to investigate the subcellular localization of CmLOX10 and CmLOX13. Using the p18T-LOX10 and p18T-LOX13 plasmids as templates, the full-length  pB7FWG2  using GaAgrobacterium harboring LOX10-GFP and LOX13-GFP binary vectors were inoculated in 5 ml of YEB medium containing 50 μg ml−1 rifampicin, 50 μg ml−1 spectinomycin, 10 mM ethanesulfonic acid (pH 5.7 MES) and 20 μM acetosyringone (AS) and grown overnight at 28°C, 200 rpm. Cells were harvested by centrifugation at 5000 rpm for 10 min at room temperature. After supernatant was removed, added 2 ml of infiltration media  and resuspend. Repeat the step above and diluted using the infiltration media to OD600 = 0.8. Agrobacterium suspensions harboring LOX10-GFP and LOX13-GFP binary vectors were injected into the abaxial surfaces of the leaves of the tobacco (Nicotiana benthamiana) grown for about 6 weeks using a 1ml needleless syringe. Then the tobacco plants were maintained in a growth cabinet under a 16/8 h day/night cycle at 25°C. The leaves of tobacco plants at 4 days after infiltration were collected to analyze the subcellular localization of CmLOX10 and CmLOX13 fusion proteins.Transformant Both green fluorescent signals and spontaneous red fluorescent signals of chloroplasts were detected by confocal laser scanning microscopy . GFP fluorescence was excited with the 488-nm Ar laser line and the emission signals were collected between 505 and 525 nm for GFP and between 640 and 720 nm for chlorophyll autofluorescence. All fluorescence experiments were repeated three times.We isolated two full-length cDNAs by a combined strategy using RT-PCR and RACE-PCR on total RNA extracted from the tender stem of oriental melon. The nucleotide and amino acid sequences of oriental melon CmLOX10 and CmLOX13 were analyzed by bioinformatics software .CmLOX10 (GenBank: KT613843) harbours a 2709-bp open reading frame (ORF) flanked by a 47 bp 5’-untranslated region (UTR) and 118 bp 3’-UTR. A TAA stop codon in the 5’- UTR of CmLOX10 is found in 9 bp upstream from the first ATG which is the start codon of CmLOX10. The deduced CmLOX10 protein sequence consists of 902 amino acid residues with a calculated molecular mass of 102.301 kDa and a theoretical isoelectric point (pI) of 6.0. The CmLOX13 (GenBank: KT613844) harbours a 2721-bp ORF encoding a predicted protein of 906 amino acid residues with a calculated molecular mass of 102.312 kDa and a theoretical pI of 6.34. The ORF of CmLOX13 is flanked by the 5’-UTR of 48 bp and 3’-UTR of 21 bp.The Cucumis melo var. makuwa Makino) and from the melon (Cucumis melon L.) genome database (https://melonomics.net) were done and seven different nucleotides and three different amino acids are found  and Thr/Phe motif (T621 and F622) respectively and are predicted to be 13-LOXs. The conserved Ala residue which determines the S-stereospecificity of most LOXs is also observed [By analyzing the deduced amino acid sequences of CmLOX10 and CmLOX13, the PLAT_LH2 and LOX domains of the deduced CmLOX10 protein which are the two conserved domains of plant LOXs and located in 73–205 and 215–885 amino acids respectively were identified, while the PLAT and LOX domains of the deduced CmLOX13 protein are located in 108–209 and 218–890 amino acids respectively . Sequencobserved .To predict the regiospecificity and potential subcellular location of the CmLOX10 and CmLOX13, multiple sequence alignments of the putative amino acid sequences of the two oriental melon LOXs and other plant LOXs of which the functions were characterized were calculated with ClustalW2 software and a phylogenetic tree was constructed using MEGA 5.0 software . AccordiE. coli, without their chloroplast transit peptide, but with a poly-His tag. Total proteins were extracted by sonication and the soluble fusion proteins were obtained in the supernatant and purified by affinity chromatography. The purity of the purified recombinant CmLOX10 and CmLOX13 proteins were checked by SDS-PAGE and Western blotting with an anti-His-tag antibody. SDS–PAGE and Western blotting analysis  was detected in both soluble supernatant fractions of the cells and purified recombinant CmLOX10 , using linoleic and linolenic acids as substrates which are the two major PUFAs in plants. As shown in Vmax values indicats that CmLOX10 and CmLOX13 oxidize linoleic acid about 2.3 and 1.3-fold more quickly than linolenic acid, respectively. The recombinant CmLOX10 and CmLOX13 show a higher kcat/Km values for linoleic acid than for linolenic acid, suggesting that CmLOX10 and CmLOX13 have the highest catalytic efficiency with linoleic acid. These results above indicate that linoleic acid is the preferred substrate for the recombinant CmLOX10 and CmLOX13.To determine the kinetic characteristics of the recombinant CmLOX10 and CmLOX13 proteins, the enzyme activity was assayed by monitoring the formation of hydroperoxides over a range of concentrations of the main substrates (linoleic and linolenic acid) between 16.5 and 330 μM using standard conditions. Furthermore, the kinetic parameters using linoleic and linolenic acid as substrates were calculated by a Michaelis-Menten plot analysis program . The kinTo determine the positional specificity of purified CmLOX10 and CmLOX13, we further identified the reaction products from the CmLOX10 and CmLOX13 enzyme reaction following the SP-HPLC analysis. As shown in In order to determine the in vivo localization of CmLOX10 and CmLOX13, we transiently expressed CmLOX10-GFP and CmLOX13-GFP fusion constructs in tobacco leaves under the control of the cauliflower mosaic virus 35S promoter. We examined the CmLOX10-GFP and CmLOX13-GFP fusion protein localization using confocal laser scanning microscopy. As shown in CmLOX10 and CmLOX13 which were isolated from the tender stem of oriental melon cultivar “Yumeiren” grown for about 30 days were cloned into T vector and sequenced. Comparing the sequencing results with the sequences of CmLOX10 and CmLOX13 from melon genome database, a few different nucleotides and deduced amino acids were found. The differences above should due to varieties difference. The oriental melon cultivar “Yumeiren” is used in our experiment, while the melon doubled-haploid line DHL92 is used in genome sequence [Using RT-PCR and RACE-PCR combined approach, the full length of sequence .Pisum sativum) LOXN2, olive (Olea europaea L.) LOX and tea plant (Camellia sinensis) CsLOX1 [In plants, which type LOXs are classified into is based on the positions where the oxygenation of linoleic acid occurs. Generally, there are some conservative sites in plant LOXs that are related with the positional specificity and stereo-specificity of LOXs. A space-filling His or Phe residue at the bottom of the substrate-binding pocket was identified in nearly all plant 13-LOXs, while a Val residue was identified in plant 9-LOXs ,38. For ) CsLOX1 ,12,16. BThe intracellular localization of LOXs may provide some predictions about the physiological functions of the different LOXs . BesidesIt might be helpful in predicting biochemical features and physiological functions of the LOXs by phylogenetic tree analysis , so the E. coli. Chromatogram analysis of reaction products revealed that the full-length CmLOX10 and truncated CmLOX13 possessed the predicted 13-LOX activity and produced 13(S)-HPODE using linoleic acid as substrate. Although producing the same products, CmLOX10 and CmLOX13 exhibited different kinetic properties and optimal reaction conditions (kcat/Km) was higher with linoleic acid than with linolenic acid, so both CmLOX10 and CmLOX13 recombinant enzymes exhibited a preference for linoleic acid.Expression of the truncated CmLOX10 protein with putative transit peptide sequences removed failed to detect LOX activity, so we produced full-length His-tagged CmLOX10 expression vectors and expressed in nditions . CmLOX10Nicotiana attenuate and rice OsHI-LOX supply substrates for the AOS branch but not the HPL branch [The metabolism of PUFAs via the LOX-catalyzed step and the subsequent reactions are collectively named the LOX pathway . The LOXL branch ,44. In mL branch . FurtherL branch ,47.CmLOX10 expressed during fruit ripening, while the expression of CmLOX13 mRNA appeared at earlier stage of fruit ripening Click here for additional data file.S2 FigCmLOX13 is framed. The locations of the three gene-specific primers for 5’ RACE of the CmLOX10 which are GSP1, GSP2 and GSP3 are single, double and triple underlined, respectively. The five different nucleotides between the CmLOX13 and GeLOX13 are indicated by asterisks.The ATG of the ORF of (DOCX)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigThe samples identities are as follows: 1, root; 2, stem; 3, young leaf; 4, female flower; 5, male flower; 6, seeds without soaking; 7, seeds were soaked in tap water for 24 h; 8, seeds were soaked in tap water for 72 h; 9, 5d after pollination (DAP); 10, 10 DAP; 11, 15 DAP; 12, 20 DAP; 13, 25 DAP; 14, 30 DAP; 15, 35 DAP; 16, 40 DAP.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S1 TableEcoRI and XhoI restriction site.Nucleotides underlined were used for producing (DOCX)Click here for additional data file.


AII triggers Ca2+ release from internal stores that is followed by store-operated and voltage-dependent Ca2+ entry, whereas K+-evoked depolarization activates voltage-dependent Ca2+ channels. ACTH acts primarily through the formation of cAMP and subsequent protein phosphorylation by protein kinase A. Both Ca2+ and cAMP facilitate the transfer of cholesterol to mitochondrial inner membrane. The cytosolic Ca2+ signal is transferred into the mitochondrial matrix and enhances pyridine nucleotide reduction. Increased formation of NADH results in increased ATP production, whereas that of NADPH supports steroid production. In reality, the control of adrenocortical function is a lot more sophisticated with second messengers crosstalking and mutually modifying each other’s pathways. Cytosolic Ca2+ and cGMP are both capable of modifying cAMP metabolism, while cAMP may enhance Ca2+ release and voltage-activated Ca2+ channel activity. Besides, mitochondrial Ca2+ signal brings about cAMP formation within the organelle and this further enhances aldosterone production. Maintained aldosterone and cortisol secretion are optimized by the concurrent actions of Ca2+ and cAMP, as exemplified by the apparent synergism of Ca2+ influx (inducing cAMP formation) and Ca2+ release during response to AII. Thus, cross-actions of parallel signal transducing pathways are not mere intracellular curiosities but rather substantial phenomena, which fine-tune the biological response. Our review focuses on these functionally relevant interactions between the Ca2+ and the cyclic nucleotide signal transducing pathways hitherto described in the adrenal cortex.The major physiological stimuli of aldosterone secretion are angiotensin II (AII) and extracellular K Coricularis  are beyo+, and AII, termed “classical” here have been described in several reviews  activates T-type voltage-dependent Ca2+ channels the current of which was detected in rat  acts on hormone-sensitive lipase    –40. Actite (IP3) –43 whichte (IP3) –46] genesa cells , the dom+ influx , 49 thro+ influx , 51 and channels , 33 [butchannels ]. In isochannels  brought /K+ pump  and by tnel TASK , 54. In tentials , 33.2+ signals evoked by high [K+] , lipoxygenase, and MAPK pathways .via MC2R receptors and cAMP. The mode of cAMP action is identical to that described above for glomerulosa cell m and the ensuing NAD(P)H formation play an essential role in the stimulation of aldosterone production. Targeted mitochondrial expression of a Ca2+ binding protein reduces both [Ca2+]m and NAD(P)H and ensuing aldosterone production in response to AII H formation and enhanced aldosterone production  was prepared from testis (2+ (2+ signal in HeLa and CHO cells and in rat cardiomyocytes (A recently recognized and biologically significant action of Cam testis . Its actm testis  and Ca2+stis (2+ . The expstis (2+ , 160. Thmyocytes . Importamyocytes .2+ signal increased the formation of mt-cAMP, and this response was enhanced by the PDE2A inhibitor EHNA. Mitochondrial cAMP signaling was attenuated with the sAC inhibitor 2-OH-estradiol, after silencing of the sAC gene and by the buffering of mitochondrial Ca2+ by S100G protein. All these maneuvers also attenuated aldosterone production, showing the cell-type-specific significance of mt-cAMP for the first time (The sAC is also expressed in H295R adrenocortical cells, and it is found in the particulate fraction predominantly. In these cells, AII-induced mitochondrial Carst time .2+ signaling and cyclic nucleotide metabolism. Importantly, these intracellular pathways are rarely, or probably never, independent. As postulated by Berridge in 1975 (2+ signaling may be antagonistic or synergistic in nature and, as hopefully accentuated by this review, the adrenal cortex is no exception to this rule. As shown in Figures Adrenocortical steroid production is under the control of both Ca in 1975 , cAMP an2+ release/influx is accompanied by the sufficient formation of cAMP. Albeit the secretagogues AII and K+ invoke predominantly Ca2+ signaling and the effects of ACTH are mediated chiefly by cAMP, the increase of both factors at the same time may potentiate the final response. Thus, the separate intracellular pathways need not be activated to the same extent but nevertheless have to be recruited simultaneously for a sufficient steroid production to follow.In spite of the interspecies differences and of the incongruences in some experimental data, it is probably safe to conclude that an adequate biological response necessitates the intricate interplay of parallel signaling pathways. That is to say that, e.g., in glomerulosa cells, sustained aldosterone response evoked by long-lasting tonic stimuli will be satisfactory only if the CaAS and GS compiled the literary data and wrote the manuscript. Data on the control of expression have been discussed with LH.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.


Despite modest improvements in survival over the last several decades, the treatment of AML continues to present a formidable challenge. Most patients are elderly, and these individuals, as well as those with secondary, therapy-related, or relapsed/refractory AML, are particularly difficult to treat, owing to both aggressive disease biology and the high toxicity of current chemotherapeutic regimens. It has become increasingly apparent in recent years that coordinated interruption of cooperative survival signaling pathways in malignant cells is necessary for optimal therapeutic results. The modest efficacy of monotherapy with both cytotoxic and targeted agents in AML testifies to this. As the complex biology of AML continues to be elucidated, many “synthetic lethal” strategies involving rational combinations of targeted agents have been developed. Unfortunately, relatively few of these have been tested clinically, although there is growing interest in this area. In this article, the preclinical and, where available, clinical data on some of the most promising rational combinations of targeted agents in AML are summarized. While new molecules should continue to be combined with conventional genotoxic drugs of proven efficacy, there is perhaps a need to rethink traditional philosophies of clinical trial development and regulatory approval with a focus on mechanism-based, synergistic strategies. Blood 2013; 122:360), etc.Despite significant progress in recent years in unraveling the genetic basis of AML , resultiMalignant cells are particularly vulnerable to the simultaneous disruption of multiple, cooperative survival signaling pathways ,16. In rA particularly popular combination has been that of DNA methyltransferase inhibitors (DNMTIs) with histone deacetylase inhibitors (HDACIs). That epigenetic processes play a fundamental role in cancer causation and progression has been recognized for over a decade now . ChromatFLT3-mutated AML and 0–7 (median 2) prior therapies, the ORR was 46%, including 27% CR/CRi [FLT3 mutations based on in vitro evidence of synergy against FLT3-internal tandem duplication (FLT3-ITD+) cells [DNMTIs have also been combined with many other classes of targeted agents in AML. Based on the ability of the proteasome inhibitor bortezomib to up-regulate miR-29b, resulting in loss of transcriptional activation of several genes relevant to myeloid leukemogenesis, including DNA methyltransferases and receptor tyrosine kinases, a phase I trial of bortezomib and decitabine was conducted . Seven o% CR/CRi . FLT3 li% CR/CRi . Decitab+) cells . Sequent+) cells . 57% of +) cells . Phase I+) cells  and with+) cells  in patie+) cells  and conc+) cells  administ+) cells ,37.In vitro, however, short interfering RNA (siRNA) silencing of the anti-apoptotic proteins Mcl-1 and Bcl-xL, but not Bcl-2, exhibited variable synergy with azacytidine [Given their efficacy and safety, and consequent widespread use as single agents for the treatment of patients with AML who are elderly and/or unfit for conventional chemotherapy, azacytidine and decitabine are increasingly being combined in clinical trials with a plethora of emerging, investigational targeted agents. Examples include volasertib (NCT02003573), a polo-like kinase 1 (PLK-1) inhibitor recently designated an “orphan drug” for AML , the fircytidine .FLT3-ITD+ AML cells and overcame FLT3 TKI resistance-conferring mutations such as F691L and D835V [A relatively recent development in the field of epigenetic targeting of chromatin networks in cancer has been the discovery of potent and specific small-molecule BET  inhibitors . The brond D835V . Furthernd D835V .Distinct from their role as epigenetic modifiers, HDACIs exert a plethora of other actions  in neoplAs noted above, the single-agent activity of HDACIs in AML is modest , and it 2 IV of bortezomib on days 1, 4, 8 and 11 and 1000 mg/m2 IV of belinostat on days 1–5 and 8–12 of a 3-week cycle. Of 35 response-evaluable subjects, one patient with mixed lineage leukemia (MLL)-rearranged biphenotypic acute leukemia refractory to “7 + 3” and FLAG-Ida  achieved a CR and went on to an allogeneic HSCT; 4 had a partial remission (PR), 14 achieved stable disease (SD), and 16 had disease progression. Four subjects discontinued study treatment due to adverse events (AEs). Overall, the regimen was very well-tolerated. One patient with Janus kinase 2 (JAK2)-mutated myelofibrosis transformed to AML remains on treatment beyond 31 cycles (manuscript in preparation). Efforts are currently underway to better characterize (at a genomic level) the leukemias of the two patients who did exceptionally well on this study.In pre-clinical studies, co-administration of sub-micromolar concentrations of the pan-HDACI belinostat with low nanomolar concentrations of bortezomib sharply increased apoptosis in AML and cell lines and primary blasts . Synergii.e., those that inhibit cyclin T/CDK9, additionally inhibit global cellular transcription [Cyclin-dependent kinase inhibitors (CDKIs) represent an interesting class of agents capable of inducing cell cycle arrest and apoptosis in malignant cells, particularly hematologic tumor types, which may be more susceptible to inhibition of cell cycling and apoptosis induction . Some ofcription , thus docription .WAF1/CIP1. These observations led to a phase I trial of the combination of alvocidib and vorinostat in patients with relapsed/refractory or poor prognosis newly diagnosed acute leukemia or higher risk MDS [2 IV load over 30 min followed by 20 mg/m2 infused over 4 h  on daysstration . No objestration .+ cells, including those bearing the gatekeeper mutation T315I, as well as in AML cells, both FLT3-mutated and -wild type . Considering that mutations that confer resistance to one of the most promising FLT3 TKIs, quizartinib, have been described [As discussed above, HDACIs, especially those that inhibit HDAC6, acetylate and thereby interfere with the function of chaperone proteins, in particular Hsp90, consequently down-regulating several pro-growth and pro-survival Hsp90 “client” proteins of critical importance in myeloid leukemias, e.g., breakpoint cluster region-Abelson (Bcr-Abl), mutant FLT3, c-Raf and Akt . In the escribed , this stCell cycle checkpoints, part of the DNA damage response (DDR) network, are in-built safety mechanisms the activation of which helps preserve genomic integrity by halting cell division upon the occurrence of DNA damage and allowing time for DNA repair . If repap53-wild type and -deficient, as well as in those bearing FLT3-ITD [+CD38−CD123+) population [Given that HDACIs induce DNA damage  and inhiFLT3-ITD . Furtherpulation . However15 to inactivate it, and Wee1 inhibition causes forced activation of CDK1, premature mitotic entry and impairment of homologous recombination [i.e., Tyr15 and Thr14, and dephosphorylation of both sites is necessary for full cyclin B/CDK1 (cdc2) activation. Upon G2/M checkpoint activation, ATR/Chk1 phosphorylates (and thereby inhibits) CDC25A, -B and -C, thus preventing premature mitotic entry [The Wee1 kinase has recently emerged as a novel therapeutic target in AML ,117,118.bination . Activatbination . Notablyic entry .14 [p53 and FLT3 mutational status, including in “LIC”s, primary AML blasts and in a xenograft mouse (flank) model [15 dephosphorylation, it also induced Chk1 activation and Thr14 phosphorylation [15 and Thr14 dephosphorylation, and full cyclin B/CDK1 (cdc2) activation, accompanied by premature mitotic entry and DNA damage [While Chk1 is a positive regulator of Wee1 (through stimulatory phosphorylation), Wee1 inhibition results in compensatory activation of Chk1, leading to phosphorylation of cyclin B/CDK1 (cdc2) at Thr14 , a thera14 . While t14 ,126,127,k) model . Importarylation . HoweverA damage . These d+ cells, both in vitro and in vivo [Aside from the strategies discussed above, HDACIs may potentially be successfully combined with a number of other investigational agents in AML. The first-in-class polo-like kinase 1 (PLK1) inhibitor volasertib was recently granted first “breakthrough” , and the in vivo . Enhance in vivo . Given t in vivo , the comThe first-in-class inhibitor of protein “neddylation”, MLN4924 , is anot3°8/Ser473) variably occurs in 50–80% of patients [CIP1/WAF1 [The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is a cellular growth, proliferation, motility and survival signaling axis  that reppatients ,156; henpatients . Althougpatients ,159, haspatients ,161. ComIP1/WAF1 . Inactivi.e., they displace the apoptosis effectors from their association with the anti-apoptotic proteins [The Bcl-2 family of proteins stands at the crossroads of cellular survival and death, and the pro- and anti-apoptotic members of this family regulate the intrinsic, or mitochondrial, pathway of apoptosis . The semproteins , a functproteins .The discovery of ABT-737, a specific “BH3-mimetic” antagonist of Bcl-2 and Bcl-xL, demonstrated for the first time that specific protein-protein interactions could be targeted by small molecules, and ushered in a new era in cell death research . Subsequi.e., Mcl-1 and Bcl-2/-xL [in vitro (including in “LICs”) and in vivo, through the same mechanism, i.e., down-regulation of Mcl-1, which is induced by ABT-737 via ERK activation [Neither ABT-199 (GDC-0199) nor navitoclax inhibits Mcl-1, which is fundamental to the pathogenesis and maintenance of AML  and the cl-2/-xL ,184. Sincl-2/-xL  or transcl-2/-xL , respectcl-2/-xL . In the cl-2/-xL  and obatcl-2/-xL  have beecl-2/-xL . In thescl-2/-xL ,188. HDAcl-2/-xL  and obatcl-2/-xL . HDACI-icl-2/-xL . Both mocl-2/-xL . Finallytivation .FasL and Bim), Mdm2, glycogen synthase kinase 3 (GSK3) isoforms (which down-regulate cyclin D1 and Myc), procaspase 9, IκB kinase (the negative regulator of NF-κB), the endogenous CDKI p27KIP1 and Chk1 [A particularly strong rationale exists  for comband Chk1 ,194. Impand Chk1 , and conand Chk1 . Some ofand Chk1 ,198. Finand Chk1 ,161 and and Chk1 , in AML and Chk1 , throughand Chk1  and mTORand Chk1  demonstrand Chk1 . Indeed,and Chk1 . PI3K/mTand Chk1 . Respondand Chk1 . StudiesWAF1/CIP1 [The PI-CDKI combination of bortezomib and alvocidib is synergistic in myeloid leukemia cells ,205 but,AF1/CIP1 . In CML Dramatic potentiation of CDKI-induced apoptosis by inhibitors of PI3K has been demonstrated in AML cell lines and primary patient-derived blasts, accompanied by diminished Bad phosphorylation, induction of Bcl-2 cleavage, and down-regulation of XIAP and Mcl-1 . In contin vitro and in vivo, along with a requirement for ERK1/2 activation in progression across the G2/M boundary and through mitosis [p53 mutational status [The observation that inhibition of Chk1 triggers marked ERK1/2 activation, which can be blocked by MEK inhibitors  or Ras-t mitosis , as well mitosis ,215 prov mitosis . Furtherl status . Howeverl status ,211,212 l status , combinel status , could aThe number of rational combinations of targeted agents that are possible in AML and may be effective, at least in theory, is virtually limitless. The biggest challenge, therefore, is how to most judiciously choose the most promising combinations and bring them forward into clinical trials, which are costly and time-consuming. For these reasons, consideration should also be given to identifying the biologic subtypes of AML most likely to benefit from a given combination, as illustrated by the suggestion that basal Akt activation might predict for efficacy of a strategy simultaneously targeting Bcl-2/-xL nd PI3K/mTOR. Additionally, attention needs to be paid to better trial designs, e.g., adaptive designs, to get us answers to the biggest challenges confronting our patients in the most expeditious manner possible. Finally, the current paradigm for regulatory approval of new drugs in the United States discourages manufacturers from venturing into combinations of unapproved agents, and slows the pace of therapeutic progress. As a result, very few of the combinations discussed in this article have been tested in patients. While a complex problem, this is one that will require a concerted effort by lawmakers, researchers, industry and the concerned public to effect real change in the fight against cancer in general, and AML in particular.


Despite the widespread recognition of the importance of mentoring in medical education, valid and reliable instruments for evaluating the relationship of mentors and protégés are lacking. The aim of this study was to develop a feasible instrument to measure the satisfaction with mentoring relationships.Based on two existing questionnaires, the authors developed an instrument to evaluate the weighted satisfaction of mentoring relationships, emphasizing the protégés' individual expectations and needs. Protégés first define individual areas of interest in their mentoring relationship, then assign relative levels of personal importance to them and finally rate their individual level of satisfaction with their mentors' support in each area of interest. In order to evaluate psychometric properties as well as acceptance and feasibility the investigators conducted a multi-method-study.134 protégés were included in the study. The instrument was neither perceived as distressing nor time-consuming. The two scores of the questionnaire correlated closely with the overall satisfaction regarding mentoring relationships .The authors propose MEMeQ as a reliable, valid and flexible instrument for measuring the weighted satisfaction of protégés with their individual mentoring relationship in medical education. Further research is needed to evaluate the generalizability of MEMeQ across other institutions and mentoring programs to add to its validity.The online version of this article (doi:10.1186/s12909-015-0469-0) contains supplementary material, which is available to authorized users. Mentoring is key for a successful and satisfying career in the medical context –4. FormaHence, there is a broad consensus with regards to the importance of evaluation of mentoring relationships in formal programs to monitor and ensure the quality of these relationships , 12.Berk et al.  pointed Studies that tried to identify predictors for successful mentoring relationships while they were still active, found satisfaction with the mentoring relationship to be the most reliable predictor , 15. ProAlthough both studies were limited by the scales used to measure satisfaction and quality of mentoring, because they were not validated in this context, they underline the importance of measuring satisfaction with the mentoring relationship. The scales used by Ragings and Cotton  correlatAs a previous study showed, students have indeed a large variety of topics they wish to discuss with their future mentors . For a sThe aim of our study is to develop a valid and feasible instrument with emphasis on protégés’ individual expectations and needs in order to evaluate the weighted satisfaction of mentoring relationships in formal mentoring programs in undergraduate medical education.To clearly differentiate mentoring from concepts such as tutoring or coaching we based our work on the definition of mentoring by Berk et al. : “A mentEby et al.  identifyMentorship Effectiveness Scale by Berk et al. [The first part of MEMeQ is based on the k et al.  and dealk et al. . This pak et al. .The six selected items from Berk et al.  thereforSchedule for Meaning in Life Evaluation (SMiLE) [SMiLE-questionnaire, Brandstätter et al. [The second part of MEMeQ deals with the content aspect of mentoring relationships (CAM). This part includes protégés’ perceptions of instrumental support behaviors  consider (SMiLE) , 22 and By using a modified transformation , 22, ovei = 0) is set to w’i = 0 and „extremely important“(wi = 7) is set to w’i = 100 with the levels of 14.3, 28.6, 42.9, 57.2, 71.5 and 85.8 in between.Concerning the IoW, „not important“(wi = −3) is set to s’i = 0 and „very satisfied“(si = 3) is set to s’i = 100 with the levels of 16.7, 33.3, 50.0, 66.7, and 83.3 in between.Concerning the IoS, “very unsatisfied“, the ratings for importance and satisfaction are combined .The MEMeQ consists of two scores. First, a sum score for the personal aspects of the mentoring relationship , and second an overall index score for the contentual aspects of the mentoring relationship . Since it is not possible to weight personal and contentual aspects according to a fixed pattern we decided against a total score.In order to meet the specific requirements of all medical students  we conceived a two-tiered mentoring program in Munich . In the At the moment 501 mentors from 43 medical specialities are involved in our mentoring program. Out of the current 1761 students in the clinical year, 1005 (57.1 %) have a mentor.To evaluate psychometric properties as well as acceptance and feasibility we conducted a multi-method-study using an online questionnaire and cognitive interviews. We conducted the study in June 2014 among medical students at Ludwig-Maximilians-University (LMU) Munich, Germany. All participants had started a mentoring relationship between 2010 and 2014 in our formal mentoring program . We contTo be included in the study, protégés had to fully complete the first part (PAM) and the question about the overall satisfaction with the mentoring relationship (OSM). In addition, a minimum of one area of interest with corresponding rates of importance and satisfaction were required (CAM). We used only data from participants who met all inclusion criteria for the final analysis.MEMeQ, as explained above, was created by adapting questions and variables of two existing instruments based on a theoretical background. This way content validity was addressed.Additionally, to adress the face validity, we conducted protocol-based cognitive interviews with five students who were randomly selected from the included study subjects. These were based on the think-aloud method  and alsoSince there are no established instruments to measure weighted satisfaction with mentoring relationships, we correlated both the overall index of weighted satisfaction (IoWS = CAM) and the PAM-Score with an additional question on the overall satisfaction with the mentoring relationship  in order to determine the construct validity , 25–27. Participation in our study was voluntary and anonymous. The LMU ethics committee reviewed the research design and exempted the study from additional ethical approval.For statistical analysis, we used descriptive and inferential methods. We used Spearman’s correlation to test the validity of the MEMeQ. We set the statistical significance level at alpha equal to or less than 0.05. We performed statistical tests using the Statistical Package for Social Sciences (SPSS), version 21.0.Out of the 271 current and former protégés we contacted (average age 24.1 (3.4) years, 55 % female, representative of the population of students who had a mentor), 160 completed the questionnaire (response rate of 59.0 %). 26 of these participants (16.3 %) did not meet the inclusion criteria because of incomplete data sets and were not considered for further analysis.In the first part (PAM) of the questionnaire, the average rating regarding the personal aspects of the mentoring relationship was 21.1 (8.9) (0–30).n = 134) mentioned 486 areas of interest in total. On average, 3.6 (1.4) areas of interest were listed per protégé . We clustered the listed areas of interest into 15 categories. Table In the second part of the MEMeQ, participants n = 34 mentioOn average, the mean importance rate (IoW) was 80.8 (16.4), the mean satisfaction rate (IoS) was 73.0 (25.6) and the mean weighted satisfaction rate (IoWS = CAM) was 72.8 (25.8). The mean score for the overall satisfaction regarding the mentoring relationship (OSM) was 77.6 (26.0).Twenty-two  students completed an online questionnaire regarding the feasibility and acceptance and further five participants of the study  were interviewed. The interviews took on average 14.7 min (range 9.6–22.9). We asked all participants to complete the web-based version of the MEMeQ by using the think-aloud-method [Qualitative analysis of the interview transcripts and online open questions revealed appreciation of the clarity, short completion time and simplicity of the questions. Protégés particularly emphasized the concept of defining individual areas of interest. In their opinion, the instrument adequately evaluates the quality of mentoring relationships. Several students stated that they found the questionnaire succinct yet comprehensive. For example: “The questionnaire is short and concise. It contains all important aspects”. And another protégé wrote: “The request to define individual areas of interest is very good. Not everyone has equal areas of interest and requirements for the mentor.” Quotes are translated from German. Table n = 22) was 0.738 (p < .001), for the personal aspects of the mentoring relationship (PAM) 0.736 (p < .001) indicating a high test-retest reliability . 20 out of 22 participants mentioned exactly the same areas of interest as in the first test (participants 21 and 22 mentioned four of five previously identified areas of interest).Spearman’s correlation for the weighted satisfaction ratings (IoWS = CAM) of both surveys  with the sum score for the personal aspects of the mentoring relationship (PAM) and the overall satisfaction regarding the mentoring relationship (OSM) Fig. . There wMEMeQ appears reliable in measuring the satisfaction with individual mentoring relationships and our study provides data for its validity. As our results showed, both parts of the instrument, the score for the weighted satisfaction (IoWS = CAM) as well as the total score for the personal aspects of the mentoring relationship (PAM) correlated strongly with the overall satisfaction regarding the mentoring relationship (OSM). As areas of interest were self-defined, protégés can evaluate their mentoring relationships and mentors on an individual basis. By weighing the satisfaction based on the importance, students were able to express how much support they effectively got through their mentoring relationship. This feature of the questionnaire thus helps overcome the deficiencies of previous instruments that could not account for the heterogeneity of areas of interest.In addition, MEMeQ provides a descriptive profile of each protégé’s areas of interest with regards to their mentoring relationships. Categorization of the mentioned areas of interest produced similar results as previous findings  but alsoAlthough data from our feasibility and acceptability study were promising, the low response rate and high percentage of dropouts must be addressed. The low response rate in our study could be explained on one hand by a general tendency of low response in online evaluations  and on tUnfortunately, 16.3 % of the participants in our study did not complete all parts of the questionnaire. Looking for the reasons, we found problems with some settings of our web-based version of the questionnaire. Participants were able to submit the questionnaire although they had not completed all parts. After changing these settings we expect a much lower dropout rate.SMiLE-questionnaire [The time needed to complete the questionnaire was with 5.9 min on average acceptable. It appears unlikely that the length of the questionnaire had a significant impact on the dropout rate. Even though we asked the participants to name three to seven areas of interest, similar to the instruction of the ionnaire , 22, 15.With regards to contentual aspects of the questionnaire, there seems to be a high rate of satisfaction with the mentoring relationships in our formal mentoring program in general. The extrapolation of our data for low weighted satisfaction scores (IoWS) shows a good validity of the MEMeQ also for low-performing mentoring relationships. Nevertheless further studies in programs with lower general satisfaction scores would be useful.The work at hand did not consider outcome parameters like the grade in the final examination, satisfaction and success in the course of study or career entry. The correlation between the results of the MEMeQ and these parameters might be part of further studies.Since only items applicable for all mentoring relationships were chosen for the first part of MEMeQ and protégés can define their own areas of interest in the second part, we believe that the instrument is applicable in any mentoring program, or comparison of mentoring programs, even though it was presently only tested in one formal mentoring program with a clear structure and definition for mentoring. Nevertheless, further research is needed to ascertain validity across institutions, languages (present results stem from the German version of the questionnaire) and cultural contexts.The evaluation of mentoring relationships in medical education is important. Satisfaction seems to be the most reliable predictor for the success of mentoring relationships. It is necessary to consider protégés expectations and needs. MEMeQ is a reliable, valid and flexible instrument for measuring the weighted satisfaction of protégés with their individual mentoring relationship in medical education.


Intracellular calcium stores control many neuronal functions such as excitability, gene expression, synaptic plasticity, and synaptic release. Although the existence of calcium stores along with calcium-induced calcium release (CICR) has been demonstrated in conventional and ribbon synapses, functional significance and the cellular mechanisms underlying this role remains unclear. This review summarizes recent experimental evidence identifying contribution of CICR to synaptic transmission and synaptic plasticity in the CNS, retina and inner ear. In addition, the potential role of CICR in the recruitment of vesicles to releasable pools in hair-cell ribbon synapses will be specifically discussed. The potential role of CICR in the recruitment of vesicles to releasable pools in hair-cell ribbon synapses will also be addressed.Sensory transduction in the inner ear relies on the mechanoelectrical capabilities of hair cells, the sensory receptors in auditory and vestibular organs. Hair cells contain two differentiated compartments with distinct physiological roles: the mechanosensitive hair bundle and the basolateral membrane that includes specialized synaptic zones. The hair bundle is responsible for translating a mechanical vibratory input into an electrical current that, coupled with the basolateral complement of voltage and calcium activated ion channels, creates a receptor potential. The receptor potential drives synaptic output from ribbon synapses. Ribbons are vesicle-associated structures implicated in the modulation of trafficking and fusion of synaptic vesicles at presynaptic terminals . Ribbons bundles  and regu bundles , Ca2+ lesynapses . Evoked 2+ from voltage-dependent Ca2+ channels allows the release of neurotransmitter through the recruitment and exocytosis of vesicles in the active zone of presynaptic terminals (2+ released from intracellular organelles containing ryanodine receptors (RyRs) or inositol triphosphate receptors (IP3Rs) by an amplificatory process termed CICR. In addition, recent evidences point to a role for Ca2+ released by endolysosomal vesicles containing NAADP-gated two-pore channels in central synaptic transmission , which can also modulate neuronal  as large as 1 nA in cerebellar Purkinje cells , a form of plasticity lasting hundreds of milliseconds to seconds. During PPF, the amplitude of a second excitatory postsynaptic potential (EPSP) becomes larger than a first EPSP, a phenomenon often attributed to residual Cand pulse . In pyrand pulse  suggestind pulse , furthernd pulse . Howeverl stores , suggestoversial , and mor2+ rise spread from the active zone across the synaptic terminal and could be blocked by ryanodine, suggesting that CICR has a presynaptic role in retinal synaptic transmission  inhibitors, decreased the magnitude of depolarization-evoked and caffeine-evoked presynaptic Ca2+ transients, pointing to the ER as the intracellular Ca2+ store involved in CICR at photoreceptors  applied to rods evoked the exocytosis of vesicles located far from synaptic ribbons  reduced depolarization-evoked Ca2+ transients. In a minority of cells, caffeine (500 μM) also evoked membrane capacitance increases whereas ryanodine (40 μM) reduced voltage-dependent capacitance increases  increased intracellular Cae 40 μM; . Additioncreases . Interesncreases . In addismission . Postsynsmission . The com+ stores . Similarar canal .2+ stores. BHQ (50 μM), a specific blocker of ER CaATPase, and caffeine (20 mM) elevated Ca2+ levels throughout the cell .Hair cells of the basilar papilla, the auditory organ of reptiles, amphibians and birds also contain intracellular Cathe cell , confirmcurrents  and incrchannels . These r2+ stores were identified in IHCs of the mammalian cochlea. Inhibitors of ER Ca2+ uptake such as BHQ (100 μM), thapsigargin (200 nM), or CPA (30 μM) increased resting cytoplasmic Ca2+ levels and slowed the recovery time after brief depolarization in neonatal IHCs  or caffeine and thapsigargin (1 μM), demonstrating that the activity of RyRs located in the ER modulate Ca2+ levels around plasma membrane channels in mature IHCs (+ by cesium (Cs+) in the intracellular pipette solution is often used in patch-clamp experiments to block K+ conductances and isolate Ca2+ currents. Interestingly, Cs+ reduced Ca2+ increase in IHCs by an unknown intracellular mechanism, apparently blocking CICR . The onset of a later larger superlinear component was Ca2+-load dependent and resembled non-linear exocytic components reported in bipolar and chromaffin cells . The two-kinetics behavior observed in capacitance recordings was paralleled by high-speed Ca2+ imaging experiments, where a supralinear intracellular Ca2+ rise was initiated at the vicinity of the ribbon and subsequently spread throughout the hair-cell cytoplasm . These two mechanisms would work in parallel to allow both phasic and tonic transmitter release for prolonged periods of time. In fact, the reduction in Ca2+ transients and exocytosis in hair cells by ryanodine application was more apparent after repeated stimulation, pointing to a CICR effect on the reserve pool of vesicles (2+-dependent mechanism necessary to speed up the supply of new vesicles to the RRP during repetitive stimulation was proposed in neurosecretory cells (2+ and is modulated by synaptic activity (2+ influx, allowing the resupply of synaptic vesicles to active zones. Temporal regulation of vesicle recruitment may uncover new forms of presynaptic plasticity in hair-cell ribbon synapses (Figure 1B; 2+ influx allows hair-cell ribbon synapses to operate in a continuously facilitated mode at resting membrane potentials, thus optimizing the timing and size of postsynaptic responses in the auditory nerve. Future experiments are needed to address the nature and physiological relevance of both the exocytic superlinear component and the non-linear intracellular Ca2+ rises in the recruitment of vesicles mediated by CICR for hair-cell ribbon synaptic transmission.Although the existence of CICR in hair cells has been ratified in different studies, a unifying theory that enlightens its physiological relevance in synaptic transmission is still missing. The recent development of new technologies is opening new avenues for the understanding of the role of CICR in synaptic transmission at hair-cell ribbon synapses . Dual siin cells . This suin cells . Alternavesicles . Accordiry cells . Centralry cells  that plary cells . Moreoveactivity . These oThe 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.


Non-infection caused urticaria is a common ailment in adolescents. Its symptoms  may contribute to the development of depressive stress in adolescents; the potential link has not been well studied. This study aimed to investigate the risk of major depression after a first-attack and non-infection caused urticaria.This study used the Taiwan Longitudinal Health Insurance Database. A total of 5,755 adolescents hospitalized for a first-attack and non-infection caused urticaria from 2005 to 2009 were recruited as the study group, together with 17,265 matched non-urticarial enrollees who comprised the control group. Patients who had any history of urticaria or depression prior to the evaluation period were excluded. Each patient was followed for one year to identify the occurrence of depression. Cox proportional hazards models were generated to compute the risk of major depression, adjusting for the subjects’ sociodemographic characteristics. Depression-free survival curves were also analyzed.Thirty-four (0.6%) adolescents with non-infection caused urticaria and 59 (0.3%) non-urticarial control subjects suffered a new-onset episode of major depression during the study period. The stratified Cox proportional analysis showed that the crude hazard ratio (HR) of depression among adolescents with urticaria was 1.73 times  than that of the control subjects without urticaria. Moreover, the HR were higher in physical  and allergy chronic urticaria .Individuals who have a non-infection caused urticaria during adolescence are at a higher risk of developing major depression. Urticaria is a common disease in children and is estimated to affect 15-25% of people at some point in their lives . SymptomNon-infection caused urticaria may result in prolonged or recurrent episodes of urticaria. A first-attack episode of non-infection caused urticaria can impose limitations on the lifestyles of patients and their families. For example, patients who have suffered from food-induced urticaria in the past due to peanut allergies would subsequently need to eliminate their exposure to peanut-based products. Thus, an allergy-related event of this type is stressful to the patient and is also likely to have an impact on the entire family’s dietary choices. In addition to the unusual-looking rash, the adolescent’s interpersonal relationships with peers might result in limitations of daily activities as the severity of physical urticaria can be increased by exercise, skin contact and even sunlight ,10,16. OWe used the Longitudinal Health Insurance Database (LHID) as the data source for this study. The LHID is derived from medical claims data available to the Bureau of National Health Insurance and provided to scientists in Taiwan for research purposes. The government of Taiwan launched its National Health Insurance (NHI) program in 1995 to provide affordable health care for all residents of Taiwan. As of 2007, over 98% of Taiwan's population was enrolled in this program. The LHID includes original data from one million people. The data in this study were randomly sampled from the period between 2005 and 2009. There were no significant differences in the gender or age distributions or the average payroll-related insurance premium rate between the people in the LHID and all NHI enrollees. The LHID also provides a valuable opportunity for researchers to evaluate medical service use since 1995. The details regarding how the database was generated are published online by the Taiwan National Health Research Institutes.This study was exempt from a full review by the Institutional Review Board of Changhua Christian Hospital (permission code: 121007) because the data set consisted of de-identified secondary data that were released for research purposes without restrictions. In addition, this manuscript has adhered to the strengthening the reporting of observational studies in epidemiology (STROBE) guidelines.This is a retrospective cohort study. During the period from January 1, 2005, to December 31, 2009, data were collected from the LHID for two patient groups, the study group and the control group. The study group was defined as adolescents who suffered non-infection caused urticaria. The control group was defined as adolescents who did not suffer any urticaria. We designated the first hospitalization for urticaria treatment during this period as the index hospitalization. In this study, the study group (with non-infection caused urticaria) and the control group (without any urticaria) were both followed for one year. The likelihood of suffering a new-onset episode of major depression during the one-year follow-up period was analyzed for the two groups.Patients who were diagnosed with a principal diagnosis of urticaria using the International Classification of Diseases, 9th Revision, Clinical Modification codes  were included in the study provided they did not have a co-diagnosis of infection (using ICD-9 codes) ,20 and dPatients were included in the study if they were diagnosed by a psychiatrist with major depression as the principal diagnosis using the ICD-9-CM codes 296.2 and 296.3. The diagnosis of major depression adhered to the definitions and criteria in the Diagnostic and Statistical Manual of Mental Disorders (DSM) - IV published by the American Psychiatric Association . BipolarPatients who had been diagnosed with any form of urticaria or depression prior to their index hospitalization (first-attack of non-infection caused urticaria) were not included.To ensure that the medical resources provided by the government-supported NHI program were not over-used by the treating hospitals or patients, the diagnosis, treatments, and medications for each patient were randomly and routinely inspected by specialists. Over-treatment or over-coding in ICD-9 was not permitted and can result in fines.Our study group included 5,755 non-infection caused urticaria patients (adolescents 13 to 18 years of age). The control group was selected from the remaining NHI beneficiaries registered in the LHID. We then randomly selected 17,265 control patients (three control patients for each urticaria patient) who were matched to the study group by gender, age and years of index healthcare use for further analysis. A total of 23,020 adolescents were included in this study.X2 test was used to compare the differences between the study and control groups with regard to demographics, including socioeconomic level , the degree of urbanization in their cities of residence (levels 1 to 4), the geographical location of the patient’s residence , and the personal history of allergic diseases . The degree of urbanization was defined by population and certain development-related conditions. Level 1 urbanization was defined as a population over 1,250,000 people with specific political, economic, cultural, and metropolitan development. Level 2 urbanization was defined as a population between 500,000 and 1,250,000 with political, economic and cultural development serving an important role. Levels 3 and 4 were defined as a population between 150,000 and 500,000 and less than 150,000 people, respectively.The SAS 9.2 statistical package was used to perform the study analyses . We used the SAS program to select the study and control groups. Each patient  was tracked for one year after his/her index hospitalization to identify subjects who developed new-onset major depression. The results of the descriptive analyses of the independent variables including patient characteristics, demographics, personal allergy histories, family history (parents/brothers/sisters) of affective disorders are reported as percentages or as the mean ± standard deviation (SD). The X2 test. These variables included gender, age, socioeconomic level, the urbanization level in the city of residence, geographic regions, history of allergic diseases, family history of affective disorders, urticaria treatment with corticosteroids  and the mean number of hospital visits (for urticaria treatment). Age groups and the causes of chronic urticaria were also analyzed to identify interactions between these parameters  with a Cox proportional hazards model. The adjusted HR was analyzed after adjusting for allergic rhinitis, asthma, atopic dermatitis, family history of affective disorders, urticaria treatment with corticosteroids , geographic regions, socioeconomic level, and the urbanization level of their cities of residence. We used the Kaplan-Meier method and the log-rank test to estimate survival curves and to compare the one-year depression-free survival rate among urticaria patients versus patients in the control group. Finally, in the control group, chronic conditions  were identified that might increase the risk for affective or anxious disorders [Furthermore, the crude hazard ratio (HR) was calculated by creating stratified Cox's proportional hazards models (stratified by age), which were implemented in the study and control groups to analyze the risk of experiencing a new-onset of depression. In addition, the variables that were related and unrelated to the occurrence of depression among the urticarial patients were further analyzed using the isorders -31.p < 0.05).The characteristics and personal histories of allergic diseases of the study patients  and controls  are presented in Table p < 0.05). The chronic conditions that might the increase risk of affective or anxious disorders in the control group included allergic rhinitis , asthma , attention deficit , atopic dermatitis , hypertension , epilepsy , diabetes , congenital heart diseases , cerebral palsy  and cancer .During the one-year follow-up period, the incidence of major depression was significantly higher among the urticaria patients than among the control patients. In this study, 0.6% (n = 34) of patients suffered a new onset of major depression after an episode of urticaria, whereas the corresponding percentage was only 0.3% (n = 59) in the control group. The population attributable risk for major depression between non-infection caused urticaria exposed and non-exposed was 0.3% (0.6% - 0.3% =0.3%). The stratified Cox proportional hazard analysis showed that the study group had a crude HR that was 1.73-times greater than that of the control group . Moreover, in the adjusted model, the urticarial patients who had ever received corticosteroids were more likely to experience new-onset major depression compared with the control group patients   compared with the group without corticosteroids  (p < 0.05)  Table . Gender 5) Table . Of all,p <0.05).The depression-free survival curves for the urticaria and control patients during the study period are shown in Figure In this one-year follow-up study, we noted that non-infection caused urticaria significantly caused the subsequent major depression. The stratified Cox proportional analysis showed that the crude hazard ratio of depression among adolescents with urticaria was 1.73 times  than that of the control subjects without urticaria (p < 0.05). Although the sample size of chronic urticaria is small, we still noted that physical  or allergy caused  chronic urticaria have a higher risk of suffering depression than control group. Some factors that might potentially influence the occurrence of depression, including baseline personal allergic histories, economic conditions of the family, and geographic regions were adjusted. Non-infection caused urticaria still significantly increased the hazard ratio of major depression. Finally, the age groups and the causes of chronic urticaria did not exhibit significant interactions with the case/control groups, demographics or allergy histories.The incidence of major depression occurrence of adolescents with non-infection caused urticaria was low (0.6%); however, it was still significantly higher than adolescents without any urticaria 0.3%). Among these adolescents with non-infection caused urticaria, two patient characteristics that were most likely to associate a subsequent episode of depression. First, the older adolescent group (aged 16 to 18 years) was more predominant than the younger adolescents (aged 13 to 15 years). Second, a history of asthma was the other factor that associates with a subsequent episode of depression. Data of the Table %. Among In addition, affective disorders have been demonstrated to be increased by chronic autoimmune/allergy conditions that increase life stress, social phobia or even chronic central nerve inflammatory reactions -42. For Potential ICD-9 over- or miscoding was an inherent limitation of this database study. The codes sent to the National Health Database were only made by attending physicians in outpatient/emergency departments. According to the Taiwan’s law, all codes must be made and confirmed by the treating physicians. Because major depression requires more clearly defined diagnostic criteria, which we discussed in the methods section, all cases of major depression were only diagnosed by psychiatrists. Urticaria is a common disease in dermatology, pediatric and even rheumatology outpatient departments; therefore, we did not limit the urticaria diagnosis to diagnoses made by dermatologists. Our procedure for separating the different divisions of treating physicians was to trace who required treatment payments from the government. Excluding patients who had a co-diagnosis of infection 7 days before or after their urticaria attack might represent an over exclusion. However, because upper airway infection and acute gastroenteritis are the most commonly reported etiologies of infection caused urticaria , the resIndividuals who have a non-infection caused urticaria during adolescence are at a higher risk of developing major depression than those without urticaria.HR: Hazard ratio; LHID: Longitudinal Health Insurance Database; NHI: National Health Insurance; STROBE: Strengthening the reporting of observational studies in epidemiology ICD-9-CM, International Classification of Diseases, 9th Revision, Clinical Modification codes; DSM: Diagnostic and Statistical Manual of Mental Disorders; SD: Standard deviation; CI: Confidence interval.There are no conflicts of interest related to this study.C-LK, C-YC, H-PW and Y-RL conceived the study. Y-RL, W-LC, H-CL, C-YC and C-CC managed the data, including quality control. S-YH, Y-RL, W-LC and H-LH provided statistical advice on study design and analyzed the data. H-PW, Y-RL and S-YH chaired the data oversight committee. C-LK and C-YC drafted the manuscript, and all authors contributed substantially to its revision. H-PW, S-YH and Y-RL take responsibility for the paper as a whole. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/14/181/prepub


In this paper, the performance of precoded bit-interleaved coded modulation (BICM) spatial multiplexing multiple-input multiple-output (MIMO) system with spatial component interleaver is investigated. For the ideal precoded spatial multiplexing MIMO system with spatial component interleaver based on singular value decomposition (SVD) of the MIMO channel, the average pairwise error probability (PEP) of coded bits is derived. Based on the PEP analysis, the optimum spatial Q-component interleaver design criterion is provided to achieve the minimum error probability. For the limited feedback precoded proposed scheme with linear zero forcing (ZF) receiver, in order to minimize a bound on the average probability of a symbol vector error, a novel effective signal-to-noise ratio (SNR)-based precoding matrix selection criterion and a simplified criterion are proposed. Based on the average mutual information (AMI)-maximization criterion, the optimal constellation rotation angles are investigated. Simulation results indicate that the optimized spatial multiplexing MIMO system with spatial component interleaver can achieve significant performance advantages compared to the conventional spatial multiplexing MIMO system. Coded modulation is one of the pivotal techniques of wireless communications. High spectral efficiency and link reliability are always the challenges and goals of wireless communication systems. Nowadays, bit-interleaved coded modulation (BICM) multiple-input multiple-output (MIMO) technology has become one of the fundamental technologies and has been widely used in current wireless communication standards, such as IEEE 802.11ac, 3GPP LTE .M independent single-input single-output (SISO) channels, where M is the number of the transmit layers , assume that the code sequence cl is transmitted and jth coded bits ith bit of kth symbol where x∈χ whose label has the value α∈{0,1} in position i. According to the LLRs, the information bits are decoded via channel decoder.where H is decomposed as Based on knowledge of the full CSI at the transmitter, the ideal precoding matrix can be obtained by SVD of the MIMO channel , 8. The NR×NR matrix U and the NT×NT matrix V are unitary matrices. Λ is a NR×NT diagonal matrix with singular values H on the main diagonal in decreasing order. We denote M column vectors of U and V, respectively. As introduced in [M layer spatial multiplexing MIMO system, the precoding and detection process can be expressed as linear transformations where the duced in , 40, 45,kth received symbol on lth layer, the fading coefficients of I-component and Q-component are different that can be expressed as After the spatial Q-component deinterleaving, for the lth layer, the PEP of cl and where cl and dfree. Without loss of generality, we assume j except for dfree distinct values of j.In this paper, convolutional code is employed as the channel code. The Hamming distance between dfree bits, we define For the duced in , the PEPdfree terms with index j. ξ is a Gaussian random variable with zero mean and variance where (Q)2·σ2.  can be ex≥0). dImin, and dQmin, are minimum Euclidean distance of the I-component and Q-component of two symbols, respectively. For the rotated QAM constellation, it is easy to get that dImin,=dQmin,=dcpmin,. Using an upper bound for the Q function where lth layer is related to λl and λf(l). For the spatial multiplexing MIMO system, the performance is dominated by the weakest subchannel. That means the performance of proposed scheme should improve as the smallest λf(l)|l=1,2,…,M} can be viewed as a rearrangement of sequence {λ1,λ2,…,λM}. In order to obtain the best performance, we should find an optimum spatial Q-component interleaved sequence from the full permutation of all elements in sequence {λ1,λ2,…,λM}. The design criterion of optimum spatial Q-component interleaver can be written as From , we can M layer spatial multiplexing MIMO system with spatial Q-component interleaver, the descending-order eigenvalues of MIMO channel matrix are λ1>λ2>⋯>λM. The optimum Q-component interleaving rule is f(l)=M−l+1, l=1,2,…,M, which brings For the SVD-precoded See Appendix. □From Lemma 1, we prove that the component interleaver used in  is optimThe most serious drawback of SVD precoding is the requirement of complete CSI at both the transmitter and receiver. To reduce the data rate requirement for the feedback channel, LF precoding has drawn much attention and has been widely used in practical systems.NT×M precoding matrix F shall be selected in a codebook set For the proposed scheme with LF precoding, the Hp=HF.where SNRmin must be maximized. A close approximation to maximizing the minimum SNR for ZF receiver is also provided under the assumption of M<NT. These two error rate-based percoding matrix selection criteria (SC) are summarized as follows.For the spatial multiplexing MIMO system without spatial Q-component interleaver, the optimal precoder selection criteria from a codebook were proposed in , 14. It SC 1 - Maximum minimum SNR. l corresponding to the precoding matrix Fk.where SC 2 - Maximum minimum singular value. λmin(HFk) is the minimum singular value of HFk.where lth layer after spatial Q-component deinterleaving can be simplified to However, the selection criteria above are unsuitable for the proposed scheme with ZF receiver because spatial Q-component interleaving readjusts the SNR of each layer. Due to the orthogonal modulation, the transmit powers of the I-component and that of the Q-component on each layer are both shown in , the effl,l) of A−1. Assume that the probability of symbol error for the lth layer is Pl and where puted as , 47  and λk(A) are the kth eigenvalue and singular value of A, respectively. Therefore, 19), for Hp. We define that Through the above analysis, we find a simple method to calculate the minimum effective SNR SC 4 - Minimum singular value function. g(Fk) is chosen.For each precoding matrix M=2, SC 4 are equal to SC 3.It is worth noting that for the proposed scheme with In the practical communication system, the transmitted signal mentclass2pt{minimAccording to Lemma 1 in , an effewhere For the proposed scheme, the optimal constellation rotation angle is one of the most important problems for SSD. For the coded MIMO system, a number of factors  will impact the optimal angle. As a result, the selection of the optimum angle becomes much more challenging. Maximizing BICM-AMI is an effective way to optimize the system performance. In , the BICIBICM varies with the rotation angle. Some examples are presented in Figs. NR=NT=4 and NR=NT=8 MIMO schemes are based on [The AMI reflects the maximum information rate that can be reliably transmitted at a given SNR. Therefore, The larger AMI value indicates a better system performance. For a given SNR, the BICM-AMI value based on , 48, resNR=NT=8, M=4, in order to verify the performance advantage of the optimal Q-component interleaver, the commonly used cyclic Q-component interleaver is also investigated for comparison purpose, which can be expressed as For the proposed MIMO system based on SVD precoding with The numeric results are shown in Fig. NR=NT=4, M=2 are different at SNR = −2.6 dB, which are about 45° and 31° for SC 1 and SC 4, respectively. For NR=NT=8, M=4 MIMO system based on LF precoding with the cyclic Q-component interleaver at SNR = −3 dB, the optimal angles for SC 1 and SC 4 are both 0°. From Fig. For the LF precoded MIMO system, the AMIs of proposed systems with SC 1 and SC 4 are plotted in the same figure. As can be seen from the Fig. 3NR=NT=4, M=2 MIMO scheme, the BICM AMIs corresponding to the proposed schemes with optimal rotation angles and without rotation are plotted in Fig. For the purpose of studying the effect of constellation rotation on the system performance, for the proposed θopt and corresponding IBICM can be obtained at a given SNR. We can get the relationship between IBICM and θopt by making various adjustments of SNR. For NR=NT=4, M=2 MIMO system, the the curve of θopt versus IBICM is shown in Fig. By the same method introduced in , based oθopt can be viewed as a function of IBICM, i.e., θopt=κ(IBICM). It provides a good reference for selecting the optimal angle. For the proposed M-layer BICM MIMO system with code rate R and 2m-ary modulation, the theoretical maximum information rate that can be transmitted is NR=NT=4, M=2 MIMO system with 0.5 code rate and QPSK modulation, From Fig. mentclass2pt{minim8 is used as the channel code. The high code rate R=3/4 is derived from it by employing “puncturing” as introduced in [N=1200. The decoding of BCC is the standard Bahl-Cocke-Jelinek-Raviv (BCJR) algorithm. The i.i.d. Rayleigh MIMO fading channel is employed and the channel is changed independently from each block of N coded bits.In this section, simulation results are provided to illustrate the performance of the proposed spatial multiplexing MIMO system with spatial component interleaver. In the simulation, 1/2-rate 64-state BCC with the generator of duced in . The codNR=NT=8 3/4-rate BCC-coded MIMO systems. It is observed in Fig. M=8, 4.8 dB for M=6 and 1.5 dB for M=4 at BER = 10−4 than the proposed scheme with cyclic Q-component interleaver. From the simulation results, an observation can be made that the SNR gains are greater for more layers.In order to verify the effectiveness of the proposed optimum spatial Q-component interleaver, the BER performance compared with commonly used cyclic Q-component interleaver proposed in  is preseNR=NT=4, M=2 MIMO systems with 3GPP LTE precoding codebook [−4. For the conventional spatial multiplexing MIMO scheme without Q-component interleaver, SC 1 has the best performance and achieves about 0.27 dB SNR gain compared with SC 2 for both code rate at BER = 10−4. In contrast, the performance of proposed scheme with SC 1 is worse than that with SC 3 and SC4 about 0.5 dB. This observation indicates that the proposed SC 3 and SC 4 are more suitable for the proposed LF MIMO system with spatial Q-component interleaver. For the 3/4-rate BCC-coded proposed scheme, the optimal rotation angle is 45°. However, the performance improvement caused by rotation is very limited, which is consistent with the AMI analysis in Fig. M=2 and just has a little performance loss compared with SC 2 for the conventional spatial multiplexing MIMO scheme without Q-component interleaver.For the proposed scheme with LF precoding and ZF receiver, simulations are carried out to compare the performance of different percoding matrix selection criteria. Figure codebook . WithoutM>2, the proposed LF precoded schemes with the proposed criteria SC 3 and SC4 always have the effective SNR gains. Figure NR=NT=8 MIMO systems with precoding codebook introduced in [M=2, M=4, and M=6, respectively. For M=2, the proposed scheme with SC 3 and SC 4 have the same performance, while the performance of SC 4 is about 0.15 dB worse than that of SC 3 for M>2. The simulation results illustrate the effectiveness of the proposed system.For the MIMO system with duced in . CompareM>2. Moreover, the method of determining the optimal rotation angles for the proposed schemes via maximizing the BICM AMI is also presented. The AMI analysis also illustrates that the optimal rotation angle is more crucial for SVD-precoded MIMO scheme with spatial component interleaver. For the proposed scheme with LF precoding, performance gain brought by constellation rotation is very limited. The simulation results validate all the theoretical analyses. Through the above optimization, the performance of the proposed precoded BICM spatial multiplexing MIMO system with spatial component interleaver outperforms that of conventional precoded spatial multiplexing MIMO system.In this paper, precoded BICM spatial multiplexing MIMO system with spatial component interleaver is discussed. For the SVD-precoded spatial multiplexing MIMO system with spatial component interleaver, the PEP of coded bits is analyzed. Based on the bound of PEP, the optimum spatial Q-component interleaver design criterion and a optimum spatial Q-component interleaving rule are proposed. The performance of conventional spatial multiplexing MIMO system is dominated by the smallest singular value of the channel matrix (the weakest layer), while the proposed scheme can improve the performance of the weakest layer through the spatial component interleaving. In addition, the LF precoded BICM spatial multiplexing MIMO system with spatial component interleaver and ZF receiver is studied. Based on the minimum average probability of symbol vector error criteria, the optimal effective SNR-based precoding matrix selection criterion and a simplified criterion are proposed. The simplified criterion has the same performance for two-layer transmission and small performance loss for λi2=ρi is the ith  largest eigenvalue of H·HH. Correspondingly, ρ1>ρ2>⋯>ρM. According to Eq. (ρf(1),ρf(2),…,ρf(M)} is another new permutation of {ρ1,ρ2,…,ρM}. The interleaving rule of proposed optimum spatial Q-component interleaver in Lemma 1 can be list in Table Assume that g to Eq. , the optnth (nth column) is equal to that of (M−n+1)th pair of eigenvalues (the (M−n+1)th column). Without loss of generality, we assume that the sum of the ith =ρM−i+1 that corresponding to the minimum value ηf must be changed. In fact, the changing of ρf(i) can be viewed as exchanging position with another eigenvalue ρk, (k≠M−i+1). Correspondingly, f′(i)=k, and f′(f(k))=M−i+1. The eigenvalue sequences for new spatial Q-component interleaver rule f′ is listed in Table Suppose Lemma 1 is not tenable, we can find a new interleaved sequence corresponding to the new interleaving rule k>M−i+1, we can get In the case of k<M−i+1, M−k+1>i. For the new spatial Q-component interleaver rule f′, If f′ which has f(l)=M−l+1 is the optimum Q-component interleaving rule to assure the maximum value of In a word, we cannot find a new spatial Q-component interleaver rule