There is an urgent need to develop novel approaches to vaccination against the emerging, highly pathogenic avian influenza viruses. Here, we engineered influenza viral-like particles (Flu-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two highly pathogenic influenza viruses of either H7N1 or H5N1 antigenic subtype. We demonstrate that, upon recovery of viral RNAs from a field strain, one can easily generate expression vectors that encode the HA, NA and M2 surface proteins of either virus and prepare high-titre Flu-VLPs. We characterise these Flu-VLPs incorporating the HA, NA and M2 proteins and we show that they induce high-titre neutralising antibodies in mice. Influenza virus infects thousands of people each year, causing epidemics with severe mortality . MoreoveVaccination, so far, has been the best manner to protect individuals from influenza infection. Influenza vaccines have been used for ca. 50 years . CurrentAltogether, there is a strong need for developing novel immunogenic formulations that can rapidly be prepared as vaccines against the emerging highly pathogenic avian influenza virus. As a step along this road, here we describe a novel influenza virus immunogen using engineered viral-like particles (Flu-VLPs) that mimic the properties of the viral surface of two highly pathogenic influenza viruses of H7N1 and H5N1 subtypes.We used the surface proteins HA, NA and M2 of two highly pathogenic avian influenza viruses: A/Chicken/FPV/Rostock/1934 (H7N1)  and A/Th0 precursor and HA1/HA2 cleaved mature forms, was incorporated on the surface of the viral particles at high levels for both H7-VLPs and H5-VLPs, when expressed together with NA . For immunisation purposes, they consisted of empty "core" particles generated by the sole expression of MLV Gag proteins , whereas NA Fig. . The neu NA Fig. , yet at Vibrio cholerae induced efficient release of the viral particles (data not shown). This confirmed the essential role of NA to promote the release virus particles from the cell surface by removing sialic acid receptors from producer cells [The expression of M2 during Flu-VLP production did not influence the incorporation of HA or NA onto the viral particles Fig. . In conter cells ,26.To estimate the concentration of Flu-VLPs, we determined their 'transduction titres' by adding serial dilutions of viral particle preparations harbouring a GFP marker gene to TE671 human rhabdomyosarcoma cells. The medium was then replaced with normal culture medium and the transduction titre was deduced 72 hr later from the percentage of GFP-positive cells measured by fluorescence-activated cell sorter (FACS) analysis, as previously described .5 t.u./ml  and were decomplemented by heat inactivation at 56°C for 1 hr. We next determined the neutralising activity of the sera using the Flu-VLPs or the VSV-VLPs harbouring a GFP marker gene. The results of a typical experiment shown in Fig. vice-versa. Sera from mice injected with native Flu-VLPs neutralised more efficiently the homologous Flu-VLPs than sera from mice injected with acid pH-denatured Flu-VLPs; yet no cross-neutralisation was observed for the latter sera. Consistently, as tested on immunoblots of H7-VLPs vs. H5-VLPs, no cross-reactivity of H7- and H5-VLP sera could be observed for HA. Antibodies against M2 that detected M2 from either influenza virus strain were raised in some immunised mice  declare that they have no competing interests.JS, DK and FLC conceived the study. JS and FLC coordinated the research efforts and edited the manuscript. BB, PJ, and MM contributed to parts of the experimental work. MM, HDK, DK and VV provided guidance and analysed the data. All authors have read and approved the manuscript.


The application of microseed matrix screening to the crystallization of related antibodies in complex with IL-13 is described. Both self-seeding or cross-seeding helped promote nucleation and increase the hit rate. The application of microseed matrix screening to the crystallization of antibody–antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate. It is recognized that X-ray crystallography provides the most accurate and detailed data on protein conformation and interactions. This method depends on the production of well ordered single crystals of the macromolecule or complex of interest that diffract X-rays.et al., 1992et al., 2000et al., 2003et al., 1978et al., 2008The crystallization of macromolecules has advanced in recent years with the use of protein engineering to enhance the crystallizability of proteins  approach, in which seeds are systematically transferred into new conditions to promote crystal growth .We have applied the MMS method to the crystallization of antibody–antigen complexes and report here the successful crystallization of three IL-13 complexes with different but related Fab fragments: C836, H2L6 and M1295. C836 is a mouse hybridoma mAb against human IL-13. The C836 mAb binds IL-13 with high affinity and blocks the binding of IL-13 to its receptors. The variable (V) regions of this mAb were chimerized to human G1 and kappa constant regions. This antibody was further humanized by grafting the complementarity-determining regions (CDRs) into human variable framework segments to yield H2L6 mAb. H2L6 was subsequently affinity-matured by selection of CDR variants to produce M1295 mAb  conventional ‘fast screening’ with commercial kits, (ii) selection of hits and preparation of the seed stock, (iii) MMS in a subset of the initial screen and (iv) final optimization of conditions if needed. Both self-seeding and cross-seeding proved to be effective in producing diffraction-quality crystals. Application of the MMS method increased the hit rate and consequently reduced the number of experiments and the amount of protein needed.2.2.1.Recombinant human IL-13 was purchased from R&D Systems . The protein was reconstituted in phosphate-buffered saline (PBS) pH 7.4 according to the manufacturer’s protocol.et al., 2010His-tagged C836 Fab was expressed in CHO cells. The H2L6 and M1295 Fabs were prepared by papain cleavage of the corresponding mAbs. All Fab proteins were purified using affinity and size-exclusion chromatography as described previously . The mixture was incubated for 20 min at 277 K, concentrated to a final volume of 0.6 ml using an Amicon Ultra 5 kDa device (Millipore) and loaded onto a Superdex 200 10/300 column  equilibrated with 20 mM HEPES pH 7.5, 0.1 M NaCl. A shift in the elution profile (elution earlier than the free Fab) indicated complex formation. Three runs were performed, with 0.2 ml protein solution applied each time to the column for each complex. Fractions corresponding to the main peak were pooled, concentrated to 6–9 mg ml−1 in 20 mM HEPES pH 7.5, 0.1 M NaCl and used in crystallization trials.For complex formation, purified C836, H2L6 and M1295 Fab fragments were buffer-exchanged into 20 m2.3.Crystallization of the complexes was carried out by the sitting-drop vapor-diffusion method at 293 K. Screening for crystallization conditions was carried out using a Hydra II eDrop robot  to set up crystallization trials in 96-well Corning 3550 plates . The experiments were composed of 0.5 µl protein solution mixed with an equal volume of reservoir solution. The droplets were equilibrated against 90 µl reservoir solution. Optimization screens were made using a Matrix Maker .2.4.Microcrystals used for seed-stock preparation were placed in 100 µl reservoir solution, homogenized by vortexing for 3 min with a Teflon Seed Bead  and stored at 253 K. The MMS was set up manually using the hanging-drop vapor-diffusion method in 24-well VDX greased plates . In each crystallization drop, 0.6 µl screening (reservoir) solution and 0.2 µl microseeds were added to 0.8 µl protein solution. The protein droplets were equilibrated over 500 µl reservoir solution.3.3.1.M for ammonium sulfate versus a pH range of 3.5–10.5. Needle-like microcrystals of the H2L6 complex were observed in 28% PEG 8000, 0.1 M MES pH 6.5 . The other two complexes did not produce any hits. Optimization of the H2L6 complex crystallization conditions in a standard approach of refining the PEG 8000 concentration and using various additives did not improve the original needle-like crystals. Therefore, the H2L6 complex microcrystals were used as seeds in the second screening by the microseed matrix method  and in-house grid screens: 192 conditions in total. The in-house screens, PEG 8000/pH and ammonium sulfate/pH, each containing 24 conditions, were designed in a small 6 × 4 matrix format. In these screens the concentration of the precipitating agent varied from 18 to 34% for PEG 8000  and from 1.5 to 2.4 3.2.M ammonium sulfate both in 0.1 M MES pH 6.5, representing an optimization screen for the H2L6 complex microcrystals.MMS was performed with the Hampton Research PEG/Ion Screen (48 conditions). This screen was extended by the addition of eight conditions containing 14–22% PEG 8000 or 1.6–2.4 M lithium acetate pH 7.9 (condition No. 24), 0.2 M ammonium tartrate pH 6.6 (No. 38), 0.2 M ammonium phosphate pH 8.0 (No. 44) or 0.2 M ammonium citrate pH 5.1 (No. 48) . No crystals were observed in the experiments using the eight additional conditions.Small isometric crystals were observed after 24 h from PEG/Ion Screen under several conditions, all of which contained 20% PEG 3350 plus one of the following salts: 0.2 The new crystallization hits were optimized using a screen composed of the most promising salt/PEG 3350 combinations (24 conditions). The second MMS was performed with the same seeds, but the seed stock was diluted fivefold with 30% PEG 8000, MES pH 6.5 to minimize nucleation events.M ammonium tartrate, 0.1 M MES pH 6.5 and from 16% PEG 3350, 0.2 M ammonium citrate, 0.1 M MES pH 6.5. The crystals appeared within 2 d and reached dimensions of 0.1 × 0.1 × 0.3 mm . Both conditions produced the same crystal form. The crystals from ammonium tartrate diffracted to 1.9 Å resolution and were used for structure determination. They belonged to the orthorhombic space group P212121, with unit-cell parameters a = 63.78, b = 73.02, c = 114.86 Å. The asymmetric unit contained one molecule of the complex.X-ray-quality crystals were obtained from 14% PEG 3350, 0.2 3.3.M ammonium citrate, 0.1 M MES pH 6.5  were used to prepare seeds for the M1295 complex crystallization. The initial MMS included the 192 crystallization conditions described above. Crystals were obtained directly from this screen in the following conditions from PEG/Ion Screen: 20% PEG 3350, 0.2 M lithium chloride pH 6.8 , 20% PEG 3350, 0.2 M potassium chloride pH 7.0  and 20% PEG 3350, 0.2 M sodium citrate pH 8.3 . In addition, X-ray-quality crystals grew from 25% PEG 8000, 0.1 M sodium acetate pH 5.5 . The latter conditions were optimized (pH 4.5) to yield crystals of about 0.2 × 0.2 × 0.2 mm in 2 d . These crystals diffracted to 2.8 Å resolution and were used for structure determination. The crystals have the same space group and nearly identical unit-cell parameters as the H2L6 crystals that were used as seeds. The space group is P212121, with unit-cell parameters a = 63.37, b = 72.50, c = 114.20 Å. The asymmetric unit contains one molecule of the complex.The optimized H2L6 crystals obtained from 16% PEG 3350, 0.2 3.4.−1 protein concentration. Crystal formations of poor quality appeared in several drops in a range of conditions after 2 d . A number of drops remained clear. A mixture of these crystals obtained in different conditions produced a ‘self-seeding’ stock.The same cross-seeding stock from the optimized H2L6 crystals was used for the C836 complex crystallization by MMS. The screen, a subset of the original 192 ‘fast screen’ conditions, included 24 selected conditions from each of the Hampton PEG/Ion Screen and in-house PEG 8000 grid screens and was performed at 6 mg ml−1. To minimize the number of experiments, the optimization screens included only one buffer, 0.1 M HEPES buffer pH 7.5, and one of the four salts  at 0.2 M concentration in the presence of 18–22% PEG 3350. X-ray-quality crystals were obtained in both the self-seeding and the cross-seeding experiments within 2 d . It is worth noting that the cross-seeds yielded crystals in all four salts, whereas the self-seeds only gave crystals in ammonium citrate. The seed quality may be one reason for this difference.Both ‘cross’ and ‘self’ seeds were used in the MMS optimization, in which the protein concentration was 9 mg mlM sodium tartrate, 0.1 M HEPES pH 7.5 . The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 76.62, b = 65.56, c = 118.74 Å, β = 107.02°. The best self-seeding conditions were 18% PEG 3350, 0.2 M ammonium citrate, 0.1 M HEPES pH 7.5 . These crystals were isomorphous to the cross-seeded crystals. Both types of crystals diffracted to 2 Å resolution.The best cross-seeding conditions were 20% PEG 3350, 0.2 4.M MES pH 6.5) with H2L6 produced needle-like crystals. Since a classical optimization of the conditions for improving these crystals proved fruitless, we faced a choice of either using these microcrystals as seeds or extending, perhaps significantly, the initial screening. The latter option would certainly require much more protein and would not after all guarantee the result. In contrast, the first option proved to be very efficient and given the variety of successful conditions appears to be more robust and reliable.The initial crystallization screening for all three complexes included 192 conditions from the commercial and in-house screens. Despite sequence similarities between the complexes, only one experiment , a number of hits were obtained that could be optimized. However, conditions that favored the growth of large crystals were not among the 48 conditions selected for the MMS screen. A different set of 12 ‘optimized’ conditions based primarily on the H2L6 results was used with the same seed stock as before and yielded X-ray-quality crystals. This experiment showed that although the MMS screen may be less extensive than the initial ‘nucleation’ screen, it still must contain a sufficient array of refined conditions to find crystal-growth conditions. From a practical perspective, to conserve protein and time we start with a limited set of conditions and then extend it, if necessary, through additional screens covering different pH, reagent concentrations or additives.−1 but hits were identified within 24 h when the concentration was increased to 9 mg ml−1. In the refinement step, high protein concentrations in the drop may trigger additional nucleation or even precipitation and should be avoided.Besides the reservoir composition, the protein and seed concentrations are other important parameters that affect crystal growth. For instance, in the C836 trials some drops remained clear after several days at a protein concentration of 6 mg mlIn the current application of MMS, we do not have much control over the seed concentration. However, under conditions where showers of crystals appeared, dilution of the seed stock was carried out in order to reduce the number of crystals in each drop. This was a key factor in H2L6 crystallization optimization. Conversely, the seed concentration in the first round of C836 MMS was not high enough. The desired concentration was achieved by combining and mixing seeds from different conditions. The use of the frozen seed stock ensured constant seed concentration and reproducibility of the experiments. We did not notice any decrease in seed concentration after repeated freeze–thaw cycles as judged by the number of crystals in the drop.The introduction of seeds into the crystallization droplet increased the hit rate in all three cases described in this report. The effect was particularly noticeable for M1295 and C836, for which no hits were obtained from the initial screening. After MMS, crystals of various quality were observed in 18 of 192 conditions for M1295 and in seven of 24 conditions for C836. The question remains whether the seeds themselves or the seed-stabilization solution cause the effect. In our experiments, the dilution of the seed stock with the same stabilization solution reproducibly decreased the number of crystals in the drops. This may be the strongest argument supporting the role of seeds in nucleation.et al., 2007et al., 2009et al., 2008Although published results with MMS have described the self-seeding protocol . The C836 crystals are monoclinic . Both forms have approximately the same packing density, with V            M values of 2.23 Å3 Da−1 for H2L6 and 2.35 Å3 Da−1 for C836. Analysis of intermolecular contacts in these crystals revealed one type of contact that is common to both crystal forms: a β-bridge between the light and heavy chains of contacting Fabs. This interaction yields a row of Fab molecules linked through their constant domains (Fig. 4H2L6 and C836 have identical constant domains but different variable domains. The H2L6 crystals are orthorhombic (space group ns Fig. 4. InteracIn conclusion, MMS proved to be a fast, easy and reliable method for refinement of crystallization conditions. Once the initial conditions have been established, the number of crystals in the drop may be controlled by dilution of the seed stock, which often is sufficient to obtain large crystals. In our experience, the MMS method promotes crystal nucleation and increases the hit rate, thus reducing the size of the initial crystallization screen and saving time and protein. In some cases, MMS produces crystal forms that differ from those of the seeds. Further experiments may determine whether a ‘universal’ seed stock can produce enough hits for a given protein or class of protein whose members have significant sequence homology, such as Fabs.


Nematostella vectensis (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. Nematostella has a surprisingly large and diverse set of neuronal signaling genes including paralogs of most neuronal signaling molecules found in higher metazoans. Several ion channel gene families are highly expanded in the sea anemone, including three subfamilies of the Shaker K+ channel gene family: Shaker (Kv1), Shaw (Kv3) and Shal (Kv4). In order to better understand the physiological significance of these voltage-gated K+ channel expansions, we analyzed the function of 18 members of the 20 gene Shaker subfamily in Nematostella. Six of the Nematostella Shaker genes express functional homotetrameric K+ channels in vitro. These include functional orthologs of bilaterian Shakers and channels with an unusually high threshold for voltage activation. We identified 11 Nematostella Shaker genes with a distinct “silent” or “regulatory” phenotype; these encode subunits that function only in heteromeric channels and serve to further diversify Nematostella Shaker channel gating properties. Subunits with the regulatory phenotype have not previously been found in the Shaker subfamily, but have evolved independently in the Shab (Kv2) family in vertebrates and the Shal family in a cnidarian. Phylogenetic analysis indicates that regulatory subunits were present in ancestral cnidarians, but have continued to diversity at a high rate after the split between anthozoans and hydrozoans. Comparison of Shaker family gene complements from diverse metazoan species reveals frequent, large scale duplication has produced highly unique sets of Shaker channels in the major metazoan lineages.The genome of the cnidarian  NematostellaDrosophila and Caenorhabditis elegans, voltage-gated channel families are often represented by a single or only a few extant genes + channel families The classical view of cnidarian nervous system as a simple, diffuse nerve net has been eroded by recent anatomical and genetic findings. Sequencing of the Nematostella vectensis (anthozoan) and Hydra magnipapillata (hydrozoan) genomes revealed that the molecular building blocks of the nervous system  are conserved between cnidarians and bilaterians Part of the answer to this question is undoubtedly that cnidarian nervous systems are more complex than originally thought. For instance, neural development in Nematostella appears to be driven by a complex cascade of highly conserved neuronal transcription factors Nematostella Shaker, or Kv1, potassium channel subfamily. The Drosophila Shaker gene was the first K+ channel cloned + channels which includes four closely related gene subfamilies (Shaker (Kv1), Shab (Kv2), Shaw (Kv3) and Shal (Kv4)) +-selective pore; Tetrameric assembly in Shaker family channels is promoted by a unique N-terminal cytoplasmic domain, T1 + channels in vitro. Bilaterian Shaker subfamily channels characteristically activate near action potential threshold with rapid kinetics and are thus well suited to regulate action potential threshold and repolarization + channel subunits function only in heteromeric channels and have previously been identified in the mammalian Shab and Polyorchis Shal subfamilies In order to better understand the physiological role of voltage-gated ion channel diversity in cnidarians, we functionally characterized the highly expanded Nematostella vectensis husbandry was carried out according to best practices developed in the Nematostella community to optimize animal health.Drosophila melanogaster and Caenorhabditis elegans have been well established and were downloaded from RefSeq Nematostella vectensis  Hydra magnipapillata  Trichoplax adhaerens (Placozoa) Caenorhabditis briggsae (Nematoda) Helobdella robusta (Annelida) Capitella teleta (Annelida) Lottia gigantea (Mollusca) Daphnia pulex (Arthropoda) Anopheles gambiae (Arthropoda) Strongylocentrotus purpuratus (Echinodermata) Branchiostoma floridiae  Polyorchis penicillatus (Hydrozoa) Shaker family sequences were also included in phylogenetic analysis  using standard techniques. Multiple independent clones were fully sequenced for each gene to verify the coding sequence. Predicted amino acid sequences from verified sequences are included in Clones of full coding sequences were shuttled to the pOX expression vector - solution , pH 7.2). Bath clamp circuitry was isolated using a 1 M NaCl-agarose bridge. Electrodes were filled with 3 M KCl and had resistances of 0.4–1 MΩ.Whole cell two-electrode voltage clamp (TEV) currents were recorded using a Dagan CA–1B amplifier  and the pClamp acquisition suite . Currents were leak subtracted using P/N protocol to remove linear leak (less than 50 nA) and capacitive transients. Recordings were carried out under constant perfusion of a low Cl+, NaMES was replaced with KMES. Grounds were bridged as described for TEV. Currents were recorded using a Multiclamp 700B amplifier and the pClamp acquisition suite . Pipette capacitance and series resistance were compensated, and bath offsets were cancelled prior to seal formation. Patch pipettes were coated with Sticky Wax  to reduce capacitance and fire polished to a resistance of 0.6–1.5 MΩ.For excised inside out patch recordings, oocytes were first shrunk in a hypertonic stripping solution 1-A2)/(1+ e((V-V50)/s)) + A2, where g(V) is the conductance at voltage V, 50V is the half-maximal conductance value, s is the slope factor and 1A and 2A are the minimum and maximum, respectively. Normalized data are displayed as mean ± S.E.M. with a simulated Boltzmann fit (G/Gmax = 1/(1+ e-((V-V50)/s)) in which V50 and s are fixed to the mean values obtained from fits of data from individual cells. Single exponential fits of the late phase of activation (defined as the last 50% of current rise) and tail current decay used the equation I(t) = Ii+Ae-t/Τ, where I(t) is the current at time t, Ii is the initial current, A is the amplitude of the fit and Τ is the time constant. Sigmoidal delay of activation was quantified as the time between the start of the voltage pulse and the time at which an exponential fit of the late phase of activation intercepted the zero current line. Exponential fitting was carried out in Clampfit (Molecular Devices) and Boltzmann fitting was carried out in Origin .Boltzmann fits of voltage activation and steady state inactivation data from individual cells used the equation g(V) = . Sequences used and source citations (where applicable) are given in We identified 20 Nematostella Shaker subfamily genes using mouse Shaker amino acid sequences to BLAST query Nematostella Shaker ORFs revealed 14 intronless gene loci  + sensitive. This result suggests that NvShak4 may inactivate by both N-type and C-type mechanisms, similar to the Drosophila Shaker A splice variant + (data not shown).Intrinsic inactivation in Shaker channels depends on an N-terminal ball and chain mechanism (N-type) or a K+ to maximize inward tail currents and to reduce C-type inactivation in NvShak5. We observed two distinct activation phenotypes in the Nematostella channels. One group of channels, consisting of NvShak1 and NvShak4-6 had low activation thresholds and V50 values ranging from −20 to −30 mV; this range of voltage activation is similar to (but slightly more depolarized than) that of typical bilaterian Shaker channels such as mouse Kv1.2  family contains an expansion of 10 genes that do not functionally express as homomultimers We reasoned that the 12 Nematostella Shaker genes that did not express functional homomultimeric channels Five Nematostella Shakers  did indeed introduce an inactivating component when expressed with NvShak3 . Since wSeven of Nematostella Shaker regulatory subunits measurably altered activation and/or deactivation of NvShak3, including NvShakR8. Examples of whole cell currents recorded in response to depolarizing voltage steps  are shown for each heteromeric combination compared to homomeric NvShak3 in Only one of the putative regulatory subunits, NvShakR10, failed to produce a measurable alteration of NvShak3. Our experiments do not rule out a regulatory functional role for NvShakR10; it may co-assemble with other NvShak subunits or regulate functional properties that we could not easily measure in this biophysical assay. For instance, we would not have detected a shift of activation to more depolarized potentials because of contamination with homomeric NvShak3 currents. NvShakR subunits could alternatively also affect modulation, trafficking or subcellular localization or of Shaker channels. For instance, inclusion of the mammalian Shaker channel subunit Kv1.5 in heteromeric channels confers regulation by Src-family tyrosine kinases in vivo, as these will depend on which subunits are co-expressed and whether NvShakR subunits are able to functionally co-assemble with multiple NvShak1-6 partners. We cannot rule out that co-expression of multiple NvShakR channels together would result in functional channels, but mammalian Shab-related regulatory subunits do not co-assemble into functional channels (data not shown). Nevertheless, combinatorial expression of NvShak1-6 with regulatory subunits could clearly provide a huge diversity of Shaker channel phenotypes in Nematostella.These experiments do not address what heteromeric channel combinations may exist HTTV), NvShak5 (-QTSV) NvShak6 (-DGFV) and NvShakR10 (-EFTV) differ from this consensus by a single conservative substitution (underlined); each substitution has been observed in a few PDZ-binding motifs Most mammalian Shaker genes (Kv1.1-1.7) contain C-terminal PDZ-binding motifs that anchor the channels to synapses and axons through interaction with MAGUK (membrane-associated guanylate kinase) family PDZ proteins such as PSD-95 and PSD-93 in situ hybridization , Shab (Kv2), Shaw (Kv3) and Shal (Kv4) gene families) using amino acid sequences from mammals, amphioxus, sea urchin, insects, lophotrochozoans, nematodes, trichoplax, and the cnidarians Nematostella, Hydra and Polyorchis. The Shaker subfamily branch of the phylogeny is shown in + channels is conserved across cnidarian orders. However, we found only a few cases of clear orthology between Nematostella and Hydra channels suggesting that much of the Shaker diversity in these species might therefore derive from separate anthozoan and hydrozoan gene expansions. Only 7 strongly supported branches in the Shaker subfamily tree contain sequences from both species  diversification occurred after the anthozoan/hydrozoan split. We assumed for this analysis that NvShakR10 has a regulatory phenotype since it does not express as a homomultimer. We also assumed that NvShakR3 and NvShakR13, two incomplete and thus untested clones, also are most likely regulatory subunits because they group closely and exclusively with proven regulatory subunits in the phylogeny. Only two clades exclusively contain Nematostella regulatory subunits and also contain sequences from Hydra. This indicates that the cnidarian-specific regulatory phenotype was present in ancestral cnidarians, but that much of the elaboration of the Nematostella regulatory subunit set occurred after the divergence from Hydrozoa. Late divergence is strongly supported by several instances of separate clustering for Nematostella and Hydra regulatory subunits. However, it is possible that the fast evolutionary divergence observed for regulatory subunits obscures some ancestral relationships between clusters. Cnidarian-specific gene expansions observed in the Shaw and Shal subfamilies also show a high degree of separate duplication in Nematostella and Hydra , S3, andth putative ancestral channel in the Shal subfamily is indicated by the close grouping of one Nematostella and one hydrozoan branch with bilaterian sequences separate from other cnidarian sequences. The Polyorchis member of this group is required for functional expression of Polyorchis Shal channels and is functionally orthologous to bilaterian Shal channels Analysis of the entire Shaker family tree suggests the presence of at least 13 Shaker family genes  in ancestral cnidarians , S2, S3.The presence of Shaker, Shab and Shaw orthologs in the placozoan Trichoplax adhaerens suggests that the Shaker family may predate the origin of true nervous systems. The placozoan genome suggests that these simple animals belong to the eumetaozan clade despite the apparent absence of a nervous system de novo emergences of subunits with a regulatory phenotype in the cnidarian Shaker and Shal subfamilies and the vertebrate Shab subfamily. Thus the major metazoan phyla have very unique sets of Shaker family genes.The results presented here show that a high functional diversity of Shaker currents has been preserved throughout eumetazoan evolution. Fast activation, diverse inactivation rates, N-type and C-type inactivation mechanisms and PDZ-dependent clustering may be universal functional properties of the Shaker subfamily within Metazoa. Vertebrates, protostomes and cnidarians all have distinct molecular strategies for producing kinetically diverse Shaker currents. Surprisingly, functional diversity of the Shaker family appears to be highest in cnidarians. Cnidarians have functional orthologs of diverse bilaterian Shaker channels and have so far cnidarian-specific high threshold channels and regulatory subunits. Amphioxus and some lophotrochozoans also have large Shaker gene expansions , but the+ channels in cnidarians may be normal rather than an exception Nematostella could enable this question to be answered.Our results from studies of the Shaker family indicate that the surprising genomic diversity of voltage-gated ion channels in cnidarians does indeed translate into extensive functional diversity of voltage-gated currents. How this functional diversity contributes to cnidarian neurophysiology is not yet known, but is a topic of considerable interest. Whole genomic analysis suggests the genetic complexity we see in voltage-gated KFigure S1Expanded view of the Shab subfamily tree from the Shaker family phylogeny. The Shab subfamily clade is shown with Nematostella highlighted in red, Hydra in green and mammals in blue. The circled number labels a single ancestral cnidarian branch and is numbered consecutively relative to the Shaker subfamily clade which contains ancestral branches 1–7 (ches 1–7 . Bars at(PDF)Click here for additional data file.Figure S2Expanded view of the Shaw subfamily tree of the Shaker phylogeny. Three ancestral cnidarian branches (9–11) are labeled with circled numbers and 6 species-restricted expansions of >3 genes  are indicated with bars. Color schemes, branch labels, gene name species codes and scale bar are identical to those used in (PDF)Click here for additional data file.Figure S3Full Shal subfamily tree of the Shaker phylogeny. Two putative ancestral Cnidarian branches  are labeled with circled numbers and 4 species-restricted expansions of >3 genes are indicated with bars. Color schemes, scales, branch labels and gene names follow the conventions of previous phylogeny figures.(PDF)Click here for additional data file.Table S1Amino acid sequences for metazoan Shaker family channels.(XLSX)Click here for additional data file.Table S2DNA sequences for the coding regions of Nematostella Shaker subfamily channels.(XLSX)Click here for additional data file.Table S3Summary of Nematostella Shaker expression patterns.(PDF)Click here for additional data file.


The Multiple Risk Factor Intervention Trial evaluated a multifactor intervention on coronary heart disease (CHD) in 12 866 men. A priori defined endpoints  did not differ significantly between the special intervention (SI) and usual care (UC) groups over an average follow-up period of 7 years. Event rates were lower than anticipated, reducing power. Other nonfatal CVD outcomes were prespecified but not considered in composite outcomes comparing SI with UC.P<0.001) with >2-fold risk of CVD death. A CHD composite outcome  was experienced by 520 SI and 602 UC men . A CVD composite outcome  was experienced by 581 SI and 652 UC men .Post-trial CVD mortality risks associated with nonfatal CVD events occurring during the trial were determined with Cox regression. Nonfatal outcomes associated with >2-fold risk of CVD death over the subsequent 20 years were combined with during-trial deaths to create 2 new composite outcomes. SI/UC hazard ratios and 95% confidence intervals were estimated for each composite outcome. Of 10 during-trial nonfatal events, 6 were associated (In post hoc analyses, composite fatal/nonfatal CHD and CVD rates over 7 years were significantly lower for SI than for UC. These findings reinforce recommendations for improved dietary/lifestyle practices, with pharmacological therapy as needed, to prevent and control major CVD risk factors. The trial was underpowered as a consequence of fewer deaths having been observed than anticipated, which resulted in a wide confidence interval (CI) for the SI/UC hazard ratio (HR) for fatal CHD after an average follow-up period of 7 years . In 1997, when this article was reprinted as a Landmark Report, the accompanying Landmark Perspective commentary underscored this problem of inadequate statistical power and the reasons for it.2 Subsequent work confirmed that pretrial exclusions made during screening had a substantial effect on the mortality rates observed during the trial.3 Other factors such as secular trends in CHD mortality rates and unanticipated changes made by UC participants likely also contributed to reduced mortality.1Results of the Multiple Risk Factor Intervention Trial (MRFIT) were reported in 1982.4 but the composites considered here were not. To inform construction of 2 composite outcomes and the clinical relevance of each, we take advantage of the long-term mortality follow-up of MRFIT participants after closure of the trial.To address the loss of power, we here construct, for the first time, a CHD composite outcome and a cardiovascular disease (CVD) composite outcome, which include nonfatal and fatal CVD events, for comparing the SI and UC groups. The nonfatal outcomes were prespecified,6 In all, 12 866 men assessed to be in the upper 10% to 15% of CHD risk on the basis of higher levels of serum cholesterol, diastolic blood pressure (BP), and cigarette use were randomized. The UC group (n=6438) was offered no intervention program; they were referred to their usual source of medical care and were examined annually. The SI group (n=6428) participated in an in-depth sustained multifactor intervention program aimed at lowering serum cholesterol and BP and at smoking cessation.10 Active follow-up of participants and the SI ceased in February 1982. Each participant was followed up for a minimum of 6 years; average follow-up was 7 years.MRFIT methods have been reported in detail.4 A resting ECG and laboratory tests were performed as part of a comprehensive physical examination. Hospital records were requested for cardiac diagnoses, and reviewers of these records were blinded to treatment group. Event criteria and numbers experiencing nonfatal CVD events have been reported.4 No information on post-trial nonfatal CHD and CVD events is available.A key purpose of annual examinations was ascertainment of interim CVD events.1 CHD death, the primary endpoint, included death from myocardial infarction (MI), sudden death, CHF, and coronary artery surgery. Other CVD deaths included deaths from stroke, hypertension with left ventricular failure, and pulmonary embolus, as well as CVD deaths not classifiable into one of the forgoing categories.1 After closure of the trial, post-trial deaths were ascertained by using National Death Index and Social Security Administration files.12 Causes of these deaths were coded according to death certificates and the International Classification of Diseases . Deaths were classified as CVD on the basis of ICD-9 (390-459) and ICD-10 (I00-I99) codes.During the trial and through to its conclusion on February 28, 1982, deaths were ascertained by clinical center staff. Cause of death was determined by a committee blinded to treatment group.1 3 additional deaths (2 UC and 1 SI) during the trial were identified. Two of these  were considered CHD deaths on the basis of the underlying cause of death on the death certificate. One CHD death was coded ICD-9 410.0 and the other ICD-9 429.2; these deaths were subclassified as MI and sudden death, respectively. The third death was due to stomach cancer (ICD-9 150.0).Subsequent to the primary trial report,13 For the former, cause of death based on the review committee's was used when available; for the latter, causes of death were based on death certificate codes.Cox models, stratified by clinical center (22 centers), were used to estimate HRs for a priori defined endpoints through the end of the trial  and through 20 years after the end of the trial .13As a first step in constructing CHD and CVD composites incorporating the nonfatal outcomes, associations of during-trial nonfatal events with 20-year post-trial CVD mortality rates were estimated for men alive at study closure . Cox models, stratified by clinical center (22 centers), were used to estimate age-adjusted CVD mortality HRs separately for each nonfatal event.This information was used to construct 2 composite outcomes  for comparing the SI and UC groups during the 7-year follow-up period after randomization in intention-to-treat analyses. Nonfatal events associated with a >2-fold increased risk of CVD death in the 20-year post-trial period were included in the CHD and CVD composite outcomes. SI/UC HRs for composite outcomes were estimated with stratified Cox models, as above. In these analyses, follow-up was censored at trial closure for participants who did not experience an event and at the time of death for those who died from causes not being considered in the outcome . Nonfatal events ascertained at annual visits were assumed to occur at intervals of 365.25 days after randomization. Logistic regression analyses that ignored event times gave nearly identical results.14 Cox models, incorporating multiple events per participant, were used to compute a pooled SI/UC HR for the CHD and CVD composite outcomes. Chi-square tests of homogeneity of HRs (whether the SI/UC HRs are similar for different types of events in the composite) also are presented. Cited P values are 2 sided. Analyses were performed in SAS, version 9.2 .Multivariate failure time analyses also were carried out.1 Results also have been reported for the primary endpoint, CHD death, and the 3 other major endpoints through trial closure4; they are summarized below, with inclusion of 3 additional deaths identified after the primary trial report. There were 116 SI and 125 UC CHD deaths through the closing date . For CHD death or nonfatal MI, there were 396 SI and 432 UC participants with an event . Death from any cause occurred in 266 SI and 262 UC men . These deaths were classified as CVD for 139 SI and 146 UC participants .Baseline characteristics and risk factor changes have been reported for the SI and UC groups.The numbers of deaths and HRs for CHD and CVD death and for all-cause death through February 28, 2002  were as follows: 885 SI and 942 UC CHD deaths ; 1295 SI and 1332 UC CVD deaths ; and 2713 SI and 2735 UC deaths from any cause .P<0.001).On the basis of these results, 2 composite outcomes were constructed. One includes fatal CHD, nonfatal MI, CHF, and surgery for coronary artery disease. We refer to this as the “CHD composite.” The second composite outcome includes fatal CVD, nonfatal MI, CHF, impaired renal function, surgery for coronary artery disease, and stroke. We refer to this as the “CVD composite.” In each composite outcome, nonfatal events associated with >2-fold increased risk of CVD death when considered singly during the 20-year post-trial period are included as components of the endpoint.P=0.01) for SI than for UC. The CHD composite then was broken into 3 separate smaller fatal/nonfatal composites: fatal or nonfatal MI (327 SI and 355 UC participants), fatal or nonfatal CHF (3 SI and 20 UC), and fatal or nonfatal coronary artery surgery (187 SI and 220 UC). These components of the overall CHD composite are not mutually exclusive . HRs for these 3 outcomes were 0.92 , 0.15 , and 0.85 . In a multiple-events analysis incorporating sudden death along with these 3 CHD outcomes . SI/UC HRs across these 4 outcomes varied (χ2 test P=0.03), largely a result of the more extreme HR for CHF. Most CHF events occurred among participants who had been hypertensive at randomization  (2 SI and 14 UC).15 The SI/UC HR for the first event analysis of this expanded CHD composite was 0.86 . The second modification excluded CHF to assess sensitivity of the overall results to this component, which strongly favored SI over UC. This resulted in an SI/UC HR of 0.88 .We also considered 2 modifications of our composite CHD outcome. We first modified it by including ECG left ventricular hypertrophy , previously shown to be significantly reduced in SI compared to UC hypertensive men.P=0.04) for SI than for UC (P=0.07). There were 49 SI and 41 UC participants who experienced stroke . Most strokes occurred among hypertensive men (40 SI and 34 UC) .Risk of the CVD composite outcome during the trial was 11% lower , and the P value assessing homogeneity of SI/UC HRs was 0.10.In a multiple-events analysis  and 11% , respectively. Analyses that consider multiple events for each participant, not just the first event, are consistent. These analyses also suggest heterogeneity of the effect of intervention on different outcomes. There was a striking benefit of the SI on CHF and modest benefit for other outcomes, with the exception of stroke, for which more SI than UC men had an event.1 We attribute this in large part to the improved power that resulted from using the composite outcome . The consequence of the larger number of CHD events was both a reduction in the loge HR (from −0.073 to −0.151) and a reduction in the CI width (0.51 to 0.24 after loge transformation). Although the CHD composite considered here was defined post hoc, the components were prespecified during the trial design, and >70% of participants with events had a CHD death or nonfatal MI event that was a prespecified major outcome. The nonfatal CHF and coronary artery surgery components of the CHD composite were associated with 5.2-fold  and 2.4-fold  increased 20-year risks of death. Furthermore, fatal events attributed to these 2 components were part of the a priori defined CHD death primary endpoint. Thus, their inclusion in the CHD composite, though post hoc, is logical on 2 counts.The significantly lower composite CHD incidence for SI than for UC contrasts with the previously reported nonsignificant difference for the primary endpoint CHD death.Likewise, the inclusion of nonfatal stroke along with fatal stroke in the CVD composite is a logical extension of the CVD death outcome. Impaired renal function, which occurred in only 20 participants, was included in the CVD composite outcome because of the high 20-year risk of death associated with it.The treatment difference favoring SI for the CHD and CVD composite outcomes during the 7-year trial period did not result in significant treatment differences after 27 years of mortality follow-up. However, there was no planned intervention during the 20 years after the close of the trial, and neither risk factor levels nor incidence of nonfatal events were assessed. Thus, we cannot assess the extent to which risk factor differences were maintained and whether during-trial differences in nonfatal events persisted.1With these considerations, the data on during-trial differences for the post hoc defined CHD and CVD composites reported here indicate that the multifactor intervention program—to achieve sustained smoking cessation and lower elevated serum cholesterol, weight, and BP by dietary/lifestyle means, supplemented as indicated by antihypertensive medication—was effective in preventing clinically relevant CVD events. This was the case even though SI–UC differences in major risk factors were less than expected during the trial period.16: Among men 40 to 49 years of age, a 5–mm Hg lower systolic BP, as observed between SI and UC participants,1 would be expected to result in a 24% reduction in deaths from stroke and a 15% reduction in deaths from heart failure.16 These differences in risk associated with BP differences accurately predicted results of a recent meta-analysis of BP-lowering trials.17The contrasting findings for CHF and stroke are unexpected. In an overview of cohort studies, both stroke and CHF were strongly related to elevated BP18 Thus, the predicted reduction in stroke risk for SI men in MRFIT is even greater than 24%.With regard to stroke, we have shown that among the men screened for MRFIT, cigarette smoking is an important risk factor for death from ischemic and hemorrhagic stroke and that risks associated with BP and smoking are additive.19 are the findings for the men who were hypertensive at baseline, among whom the BP differences between SI and UC at 6 years were 7/5 mm Hg for systolic/diastolic BP. For this subgroup, a very modest, nonsignificantly 12%  lower CVD composite outcome was observed for SI compared to UC.It is possible that the stroke and CHF findings are due to chance. For both outcomes the number of participants with an event was small. Arguing against this, and in favor of the possibility of a less-than-optimal hypertension intervention program in MRFIT (as previously reported),4 which lessens concerns about differential ascertainment of nonfatal events; ability to assess long-term risk of death associated with different nonfatal events, which allows assessment of the importance of each component; and ability to assess all events a participant developed during the trial, not just the first. MRFIT was not designed as an “event-driven” study, with follow-up continuing until a target number of primary events had occurred. Instead, MRFIT was designed to continue until all participants had been followed up for at least 6 years. Assumptions about key design parameters were incorrect, and this had an adverse effect on power. This analysis aims to overcome that limitation by using clinically relevant composite outcomes that occurred with much greater incidence than the primary endpoint, CHD death.Strengths of this analysis include prespecification of several nonfatal outcomes for which post-trial mortality rate could be used for rank-ordering. Other strengths are completeness of follow-up in both the SI and UC groups,20 For the CVD composite, the HR was significantly <1.0 even with inclusion of nonfatal stroke, for which the rate was higher in SI than UC. Secondly, MRFIT was a nonblinded trial. Thus, there is a greater risk of bias in ascertainment of endpoints, particularly nonfatal outcomes. In fact, the original endpoint considered for MRFIT, CHD death or nonfatal MI, was changed to CHD death before beginning the study because of concerns about differential ascertainment.6 Finally, these findings pertain to middle-aged men.There are several limitations to our new findings here: First, as noted above, the composite CHD and CVD outcomes were not a priori defined. Also, composites are difficult to interpret if components go in opposite directions.In summary, these new overall findings demonstrate that the SI program achieved significantly favorable reductions in CHD and CVD composite outcomes in middle-aged men at above-average risk of CHD. The findings support recommendations, repeatedly made to the public by expert groups, for improved dietary/lifestyle practices  to prevent and control established major CHD/CVD risk factors .


Gϕ are CD66b+/CD63+/MPO+/LC3B+ and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS) production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated Gϕ formation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH) (29 cycles/day for 5 days) completely abolished Gϕ formation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles) decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phox expression, whereas under 24 h sustained hypoxia (SH) the size and expression of LC3B and gp91-phox/p22-phox resembled Gϕ formed in normoxia. Diphenyl iodide (DPI), a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished Gϕ formation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC) abrogated the effects of IH by inducing large CD66b+/LC3B+ Gϕ and increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in Gϕ development.Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (G Yet, increased neutrophil survival within tissues or in the circulation can promote persistent inflammation resulting in tissue injury and dysfunction . During eveloped . Also treveloped , 6. Murieveloped –9. Notabnditions . Specifinditions , 12. Botnditions , 14. Mornditions , 16.To adapt to hypoxia, cells undergo a metabolic shift by increasing the cellular dependency on anaerobic metabolism and activate autophagy for degrading damaged or unnecessary proteins and organelles. In neutrophils, autophagy plays a cell death role in inflammatory/infectious conditions and in tumors which are characterized by hypoxia. Of note, autophagy is vital in sensing oxidative stress and removing oxidatively damaged cellular components and is activated by stress or triggered by cytoplasmic overload of these proteins or organelles. It may also play a role in neutrophil differentiation  and has ϕ within 5–7 days [ϕ are characterized by unique morphology, phenotype, and functions. They are vastly enlarged due to autophagocytosis of dead neutrophil remnants, are vacuolated, and contain phagolysosomes. They express a marker of specific neutrophil granules CD66b, a marker of azurophilic granules CD63, CD15, CD11b, and MPO, the gp91-phox subunit of NADPH, and autophagy markers (LC3B). Functionally, they actively take up particles as latex and opsonized zymosan and generate ROS in response to these particulate stimuli and to PMA. Interestingly, unlike fresh PMN, Gϕ which also intensively expressed CD68 scavenger receptor took up oxidized LDL (oxLDL) and generated ROS in response to stimulation with oxLDL. Additionally, specific autophagy inhibitors as 3-methyladenine (3-MA) or bafilomycin (BafA1) abolished Gϕ development, demonstrating the importance of autophagy to Gϕ development [ϕ formation remain to be unveiled.In a previous study we have shown, for the first time, that freshly isolated purified PMN from healthy subjects maintained in prolonged culture conditions without additional growth factors or cytokines give rise to a small subpopulation of G5–7 days . These Gelopment . Howeverϕ formation, we sought to investigate the effects of various hypoxic treatments on Gϕ development. To gain further insights into Gϕ development in hypoxic conditions, PMN were also treated with various pharmacologic inhibitors for ROS generation and signalling pathways.To further elucidate the conditions and mechanisms involved in G2. Sleep studies were performed on all subjects using the WatchPAT-200 device [ϕ were evident in culture. Depending on the donor, from each 106 neutrophils plated, 100–200 Gϕ developed. As a control, in some experiments, the culture medium was supplemented with 30 ng/mL granulocyte macrophage-stimulating factor (GM-CSF) and 30 ng/mL IL-4 . GM-CSF/IL-4 was added at each medium change. The LPS content in FCS was lower than 1.0 ng/mL and the addition of 1–10 ng/mL LPS to the culture medium did not affect Gϕ formation [Blood samples were obtained from 28  healthy nonsmoker volunteers with a mean age of 28.0 ± 6.6 years and BMI of 24.9 ± 3.9 Kg/mormation .ϕ, in earlier experiments we also prepared a FACS-purified population of CD15/CD11b/CD63/CD66b neutrophils. Their development in culture was similar to that obtained from PMN isolated by Ficoll only. Moreover, by coculturing PMN with autologous monocytes, Gϕ did not develop. Thus, excluding the possibility that Gϕ arise from contaminating cells or cells other than mature PMN, we should also note that since the yield of Gϕ formed from neutrophils in culture is low (0.01–0.02% of cultured PMN in normoxia), biochemical and molecular measures are hard to implement [Of note, to validate the neutrophilic origin of Gmplement . Therefo6 cells/mL) were plated into 24-well plates after which they were exposed to normoxia, SH, or IH in custom-designed incubation chambers attached to an external O2-CO2-N2 computer-driven controller using BioSpherix-OxyCycler-C42 system . This system enables creating periodic changes in external O2 concentrations that control air gas levels in each chamber individually as previously described [2 in the medium dropped to 5% during the hypoxic period for about 1.5 min, and this level of hypoxia was achieved after 15 min of incubation. In the reoxygenation period, O2 levels reached normoxic levels (20%) after 10 min of incubation. Carbon dioxide was held constant (5%) at all treatments. For modeling chronic IH, the purified PMN were exposed for 5 consecutive days to 29 IH cycles/day (approximately 12 h/day). Acute IH was induced by exposing PMN to 10 cycles (250 min), 29 cycles (12 h), or 56 cycles (approximately 24 h), each in the first day in culture. SH was employed for comparable times at 5% actual oxygen in the medium for the entire periods . Thereafter, the hypoxia treated cells were transferred to normoxia for additional six days, after which various measures were performed. Control cells were maintained in normoxia for the entire period.Purified PMN  in PBS, at room temperature for 10 min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4°C using the following primary Abs (dilution 1 : 100) or the corresponding isotype controls: mouse monoclonal anti-CD66b Abs  and anti-cytochrome b-245 light chain , rabbit polyclonal anti-neutrophil elastase (NE) , anti-LC3B Abs , and anti-Nox2/gp91-phox Abs . Isotype controls included purified mouse IgG1 (clone MG1-45) and IgG2  and rabbit IgG . Then, the cells were washed and incubated with 1/400 secondary antibodies CF 488A or CF 647 goat anti-rabbit IgG and/or CF 647 goat anti-mouse IgG . After washing, slides were mounted with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining . Slides were analyzed by a confocal laser scanning system (LSM 700) using Nikon E600 (Japan) fluorescence microscope and Plan Apo X40 immersion oil objective. Cells' area and fluorescent intensities (FI) were integrated with Image J 1.49k Software . Data are presented as FI = Raw integrated density /Area of cells.Cytospins prepared from 7-day GThe fluorescence membrane stains PKH-26 (red) and PKH-67 (green) (Sigma-Aldrich) were used to label freshly isolated neutrophils according to manufacturer's instructions. The labeling vehicle provided by the kits (Diluent C) is an aqueous solution designed to maintain cell viability. Cells were washed in serum-free medium; the pellets were resuspended in 0.5 mL of PKH-26 or PKH-67 (1 : 500 in Diluent C) and incubated for 5 min at room temperature. Labeling was stopped by adding 0.5 mL FCS. Then, cells were washed three times with complete medium and cocultured.ϕ was determined on day 7 using fluorescent latex beads of 1.0 μm in diameter . Briefly, Gϕ were incubated for 2 h with carboxylate-modified fluorescent yellow-green latex beads  at a cell : bead ratio of 1 : 500 (because of the large Gϕ cell size). Cytospins were prepared and fixed as described above and analyzed by confocal microscopy. Three types of controls were performed to ensure intracellular localization of the beads. (1) Control Gϕ were kept on ice for 15 min and cytospins were prepared immediately or 2 h after adding the latex beads. (2) To inhibit phagocytosis, Gϕ were preincubated with 10 μM cytochalasin B  for 30 min prior to adding the latex beads, and cytospins were prepared 2 h after incubation with latex. (3) To ensure intracellular localization rather than adhesion, the intracellular localization of latex beads was confirmed in Gϕ by 3D images  using 3D reconstructing software IMARIS z-stack analysis , and only latex beads in the plane of the nucleus were considered positive for phagocytosis.The phagocytic activity of Gϕ were incubated with 50 nM of LysoTracker for 90 min at 37°C in the dark. To identify mitochondria, viable cells were stained for 30 min at 37°C in the dark with 100 nM MitoTracker Orange CMTMRos . To further detect mitophagy, fixed cytospins were stained with LC3B, as described above.LysoTracker  was used to detect acidified endosomes. Viable GColocalization was quantified by ZEN 2010 (version 6.0) Carl Zeiss MicroImaging GmbH, Germany using Manders Overlap Coefficient (MOC) . Only ceμM diphenyl iodide (DPI); a ROS scavenger, 20 μM N-acetylcysteine (NAC) ; and PI3K inhibitor LY-294002, 20 μM  which is indicated to act at this concentration as a PI3K inhibitor not affecting TLR signaling cascade. Equal volumes of DMSO were used as a negative control.Freshly isolated PMN were exposed for 24 h to IH, SH, or normoxia with or without various inhibitors. Each inhibitor was added 10 min prior to the various oxygen treatments and remained throughout the treatments. The following inhibitors were used: a NADPH oxidase inhibitor, 10 ϕ by NBT test. NBT (Sigma-Aldrich) was dissolved in RPMI 1640 without phenol red (0.2%). Cells were incubated without or with 100 nM PMA at 37°C for 15 min, then kept at room temperature for 10 min, and assessed by light microscopy. In some experiments also DPI was added to Gϕ, 2 h prior to PMA stimulation. Cytoplasmic clumps of formazan deposits in Gϕ were considered positive for ROS.Intracellular ROS was determined in 7-day G4 cells/well in 100 μL culture medium  and were incubated with the WST-1 reagents (10 μL/well) for 1 h. The formazan dye formed was determined with ELISA reader at 450/650 nM. The measured absorbance directly correlates to the number of viable cells.Cell viability was monitored in the various oxygen and inhibitor treatments by using a commercial reagent WST-1 , according to manufacturer's instructions . Brieflyμg of protein was loaded onto SDS-PAGE under reducing conditions. After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) and probed with rabbit polyclonal antibody to LC3B , followed by horseradish peroxidase-conjugated secondary antibody  and an enhanced chemiluminescent substrate . Densitometric analysis was performed using TotalLab TL100 v.2006c software . Data were normalized over β-actin and the ratio of LC3BII/LC3BI was also calculated.Cells were washed twice and extracts were prepared in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1% NP-40 supplemented with a mixture of protease inhibitors (Roche Applied Science). Protein concentration was determined using the Bradford reagent (Bio-Rad), and 30  t-test with Bonferroni correction was used for multiple comparisons. Therefore, only values of p < 0.008 were considered significant. The NCSS 2004 statistical package  was used.Data are expressed as mean ± SD for each experimental group. A two-tailed Student'sϕ under prolonged culture conditions [ϕ which developed in normoxic conditions had the same characteristics of Gϕ as previously shown . These Gϕ avidly phagocytosed neutrophil remnants, including granules and microparticles, suggesting that this active phagocytosis of neutrophil remnants may induce activation of some neutrophil subsets or precursors resulting in their transformation into Gϕ [ϕ, indicating the importance of autophagocytosis to their development.Previously we have shown that, under normoxic conditions, a new neutrophil-derived subpopulation of cells characterized by neutrophilic markers CD15/CD11b/CD63/CD66b, as well as a unique morphology and functions, spontaneously develop into Gnditions . Accordi into Gϕ . Thus, wϕ, the cells which developed in GM-CSF/IL-4 supplemented medium were mostly smaller in size, showed widespread cytoplasmic projections  dye and cultured in cytokine-free medium for 7 days, while the other half of PMN were labeled with PKH-67 (green) dye and cultured in GM-CSF/IL-4 supplemented medium for 7 days. Then, both cell types were mixed in a 1 : 1 ratio and cocultured for 2 h and cytospins were prepared. Notably, Gϕ formation is dependent on the local cytokine milieu, and culturing neutrophils with GM-CSF/IL-4 did not induce Gϕ formation but rather a different long-lived subpopulation. Since cells obtained in GM-CSF/IL-4 supplemented cultures did not resemble Gϕ, we did not follow their phenotypic and functional characteristics.In addition, for comparison, we also followed PMN cultures supplemented with GM-CSF/IL-4. These cells were shown to develop into various cell types after 7–14 days in culture as previously described , 29. Unljections , and werϕ formation, but their development was evident in SH and in normoxic cultures. Thus, in the following experiments we focused on acute IH treatments for Gϕ development.Hypoxic environments are common in inflammatory and other pathological conditions. Neutrophils adapt to such hypoxic-pathological environments for the resolution of inflammation by relying on their unique molecular features such as preferential glycolysis as an ATP source over the mitochondria and a potent NADPH oxidase-dependent machinery producing massive amounts of ROS . Thus, fIn the acute IH protocol, purified PMN were exposed to 10 cycles (250 min), 29 cycles (approximately 12 h), or 56 cycles (approximately 24 h) of IH, each on the first day in culture. In parallel cells were also exposed to corresponding times of SH . Subsequently, PMN were cultured during the following 6 days at normoxic conditions. Control cells were maintained in normoxia for the entire durations.ϕ development  had no effect on Gelopment . However < 0.005 ) and the < 0.005 , althoug Figures . Moreove0 min hadsibility , 15, 16,ϕ formation, it was chosen to further investigate Gϕ phenotype and functions. As shown in ϕ formed at all oxygen treatments were CD66b positive. The developed Gϕ were also neutrophil elastase (NE) positive, indicating that Gϕ express this important neutrophil specific protein after differentiation. However, the expression of NE was significantly lower in IH-treated Gϕ compared to Gϕ formed in normoxia or SH. Although in SH-treated Gϕ NE levels were also attenuated, they did not significantly differ from controls  had the strongest effect on Gcontrols . By staioTracker , a staineated Gϕ , indicatϕ development under SH treatments basically resembled the development of normoxia-treated Gϕ, mainly with regard to size, CD66b, and LysoTracker expression. Yet other measures as NE varied. However, under IH, Gϕ development was significantly attenuated in a severity-dependent manner.All in all, Gϕ by demonstrating the presence of LC3B-II as vesicular puncta associated with autophagosomes and that treatment with specific autophagy inhibitors abolished Gϕ formation [ϕ at the protein level as illustrated in β-actin and normalizing by LC3BII/LC3BI ratio revealed a high LC3BII expression in Gϕ compared to fresh PMN, clearly indicating active autophagy in Gϕ. Examples of Gϕ stained for LC3B at the various oxygen treatments are depicted in ϕ   . These fϕ were noted (ϕ expressing LC3B positive structures containing mitochondria (mitophagy) were the predominant Gϕ type . These appeared as yellow spots with significant colocalization of LC3B and MitoTracker (MOC > 0.6). In SH, 34.8 ± 2.8% expressed mitophagy, but the morphology was mixed (ϕ expressed mitophagy). Thus, in IH, mitochondria and LC3B were mostly not colocalized  mediates the selective elimination of dysfunctional or unwanted mitochondria . Therefore noted . In normotential . These fphox, p22-phox, p-47-phox, p67-phox, p40-phox, and Rac2) are assembled as the functional NADPH oxidase at the phagosomes and/or the plasma membrane [phox and p22-phox. The intracellular localization of gp91-phox (green) and p22-phox (red) was determined by double immunofluorescence staining, as depicted in phox subunit was highly expressed in normoxic-Gϕ and as expected was localized in the plasma and phagolysosome membranes as previously shown [ϕ the gp91-phox expression was on average lower than that of normoxia. However, in 24 h IH-treated Gϕ, the expression of gp91-phox was significantly lowered to 28.4 ± 15.4 of normoxic-Gϕ (n = 5). Typical examples of gp91-phox fluorescence intensity of expression at different oxygen conditions are depicted in NADPH oxidase-derived ROS are a key signal for autophagy through LC3 recruitment to phagosomes . In restmembrane –37. We tly shown . In 24 hphox subunit was also mainly localized in the plasma membranes and phagolysosomes in normoxic and in 24 h SH-treated Gϕ. However, while, in 24 h IH-treated Gϕ, the intensity of the p22-phox subunit expression was lowered to 45% of normoxic-Gϕ, in 24 h SH-treated Gϕ its intensity of expression was significantly increased to 165% compared to normoxic-Gϕ (p < 0.01). Typical examples of p22-phox fluorescence intensity of expression at different oxygen conditions are depicted in phox with p22-phox was evident in the cell membranes of normoxic- and 24 h SH-treated Gϕ . Equal volumes of DMSO were added as a negative control. Inhibiting the PI3K kinase pathway with LY-294002 abolished Gϕ formation at all oxygen conditions studied (data not shown). Also, inhibition of class III PI3K by 3-methyladenine (3-MA), a commonly used autophagy inhibitor, was previously shown to inhibit Gϕ formation [ϕ.The PI3K specific inhibitor LY-294002 was added at 20 ormation . Jointlyphox, facilitating its membrane translocation and activation [ϕ formation via controlling NADPH oxidase activation.The PI3K/Akt signaling pathway is essential for several neutrophil functions, including migration, degranulation, and superoxide production by controlling NADPH oxidase activation –42. Akt tivation . Akt is tivation . Thus, iϕ development, the potent antioxidant and glutathione precursor—NAC—was used [μM) was added to PMN cultures 10 min prior to the exposure to 24 h of normoxia, SH, or IH. Viability of the cells in the presence of NAC, measured after 24 h, was unaffected at all three oxygen conditions . It is thus suggested that the presence of NAC can restore the cellular ROS balance by replenishing the intracellular glutathione levels during IH and therefore facilitate the development of Gϕ with similar characteristics to those obtained in normoxia with regard to size, LC3B, and NADPH oxidase expression.To further probe the potential involvement of ROS in Gwas used . NAC  more avidly than freshly isolated neutrophils. Yet, the IH-treated Gϕ  exhibited a lower phagocytic activity (p = 0.015) compared to normoxic-Gϕ (ϕ to about 50% of the ability of normoxic-Gϕ (p = 0.001) . Additionally, the intracellular localization of latex beads was also validated in Gϕ by 3D z-stack images as illustrated in Gmoxic-Gϕ , which w Figures . Treatme Figures . To excl in vitro of human neutrophils with NAC enhanced their phagocytic ability [ in vivo to healthy individuals increased this ability [ϕ under IH similarly to increasing their size, the expression of LC3B, and that of NADPH oxidase subunits gp91-phox and p22-phox. This may indicate that phagocytosis is one of the mechanisms contributing to the formation of Gϕ, as already indicated by restoring the formation of Gϕ under IH in the presence of NAC.Enhanced phagocytosis in NAC-treated neutrophils was previously shown in a number of studies. While treatment ability , also or ability . Similar ability . Thus, tϕ phenotype and functions in IH-treated cells may have clinical relevance to conditions associated with cyclic-intermittent hypoxia. NAC that is best known for treating glutathione deficiency [β cells were protected from apoptosis [The importance of NAC in restoring, at least partially, Gficiency  was alsoficiency . Treatmeficiency . For insficiency , pancreapoptosis , pharyngpoptosis , and diapoptosis . In addipoptosis . Taken tϕ) derived from neutrophils maintained in prolonged culture conditions [ϕ development in a dose-dependent manner whereas sustained hypoxia had no  effect on their development. Intermittent hypoxia reduced Gϕ size, autophagy, neutrophil elastase and NADPH oxidase expression, and their phagocytic activity. Inhibiting NADPH oxidase or the PI3K/Akt signaling pathway completely abolished Gϕ development at all oxygen conditions investigated, indicating their importance for this process. Conversely, the antioxidant N-acetylcysteine (NAC) abrogated the IH-associated effects and partially restored a control like phenotype and functions.Previously we have described for the first time a new subpopulation of giant phagocytes  and to generate ROS in response to oxLDL uptake, unlike fresh neutrophils [ϕ in carotid plaques from patients undergoing elective endarterectomy. Thus, a better understanding of Gϕ formation may provide insights into basic neutrophil biology in inflammatory and atherogenic conditions or in the resolution of neutrophilic inflammation. Moreover, the a priori low yield of Gϕ may indicate that they have a unique function and may represent a subgroup of progenitor cells. However, their identification and roles in vivo warrant intensive investigation.The physiological/pathophysiological significance of Gtrophils . In acco


Both chronic and acute (binge) alcohol drinking are important health and economic concerns worldwide and prominent risk factors for the development of alcoholic liver disease (ALD). There are no FDA-approved medications to prevent or to treat any stage of ALD. Therefore, discovery of novel therapeutic strategies remains a critical need for patients with ALD. Relevant experimental animal models that simulate human drinking patterns and mimic the spectrum and severity of alcohol-induced liver pathology in humans are critical to our ability to identify new mechanisms and therapeutic targets. There are several animal models currently in use, including the most widely utilized chronic ad libitum ethanol (EtOH) feeding (Lieber–DeCarli liquid diet model), chronic intragastric EtOH administration (Tsukamoto–French model), and chronic-plus-binge EtOH challenge  model). This review provides an overview of recent advances in rodent models of binge EtOH administration which help to recapitulate different features and etiologies of progressive ALD. These models include EtOH binge alone, and EtOH binge coupled with chronic EtOH intake, a high fat diet, or endotoxin challenge. We analyze the strengths, limitations, and translational relevance of these models, as well as summarize the liver injury outcomes and mechanistic insights. We further discuss the application(s) of binge EtOH models in examining alcohol-induced multi-organ pathology, sex- and age-related differences, as well as circadian rhythm disruption. Alcohol consumption, both acute and chronic, is an important social, economic, and clinical problem. The harmful use of alcohol ranks as the fifth leading global cause of preventable morbidity and mortality ,2,3. AccExcessive alcohol intake is a causal factor in a wide range of multi-organ pathology, including alcoholic liver disease (ALD). ALD is manifested as a spectrum of clinical disorders ranging from steatosis (fatty liver) to alcoholic hepatitis , and may progress further to the more severe forms, cirrhosis and hepatocellular carcinoma. AH is associated with high mortality; up to 40% of severe AH patients die within six months . Howeverad libitum EtOH feeding , chronic intragastric (IG) EtOH administration model (Tsukamoto–French model), and second-hit models that combine EtOH administration with an additional hit(s) , lipopolysaccharide, genetic knockout/overexpression, and others) to facilitate progression to advanced ALD. However, current animal models do not recapitulate the full spectrum of human ALD. Lieber–DeCarli ad libitum EtOH feeding (even for longer periods) usually causes hepatic steatosis with limited liver injury, inflammation and no fibrosis. The Tsukamoto–French IG EtOH feeding causes severe steatosis with inflammation and mild fibrosis, however, it is a technically complicated and labor-intensive model. The chronic-plus-binge EtOH administration paradigm recently developed at the National Institute on Alcohol Abuse and Alcoholism (NIAAA) of the National Institutes of Health (NIH) by Dr. Bin Gao simulates the drinking pattern(s) of heavy drinkers who indulge in chronic-binge-drinking and induces a robust neutrophil-mediated liver injury  or the NIAAA model) was developed by Dr. Bin Gao’s group . In this 5 g/kg, % EtOH oroduction . Chronicoduction . Althougoduction . Notablyoduction . Mechani 5 g/kg, % EtOH oroduction ,83,84,85Longer-term variations of the 10 d + 1 B NIAAA model combined with single or multiple binges have recently been reported. For example, Xu et al. showed that combining single or multiple EtOH binges with long-term chronic EtOH feeding in mice recapitulated certain histological and molecular features of advanced clinical alcoholic steatohepatitis (ASH) . In thisw/v). This paradigm causes greater plasma ALT levels, hepatic macro-vesicular steatosis, inflammation, and neutrophil infiltration compared to chronic EtOH feeding alone or multiple EtOH binges [Chronic binge EtOH models have also been developed in rats. For example, multiple EtOH binges  were administered to rats after 4 weeks of chronic EtOH feeding  to direct continuous IG EtOH infusion (the Tsukamoto–French model). Dr. Tsukamoto’s group has recently modified the model by incorporating EtOH binges into the paradigm. They developed a hybrid model where animals were fed a Western diet (high in cholesterol and saturated fat) for two weeks followed by IG infusion of EtOH (up to 27 g/kg/day) with a high fat liquid diet (corn oil-enriched) for eight weeks and weekly binges of EtOH (~4–5 g/kg) from the second week of IG infusion [As highlighted previously, greater liver injury has been achieved by modifying the route of EtOH delivery from infusion . This prThe initiation and progression of ALD is strongly affected by the presence of comorbid conditions . Obese alcoholic subjects have increased serum ALT levels and higher risk of developing steatohepatitis, cirrhosis, and hepatocellular carcinoma as compared to non-obese alcoholics or obese non-alcoholics ,119,120.Chang et al. described a simple two-hit model to show that short- or long-term HFD feeding plus a single EtOH binge synergistically induces marked elevation of serum ALT and produces features of severe steatohepatitis in mice . In thisfa/fa Zucker rats compared to EtOH-binged lean littermates [Binge EtOH administration is also detrimental in animal models of genetic obesity. For instance, Carmiel-Haggai et al. showed that an EtOH binge of 4 g/kg every 12 h for three days induced serum ALT activity, hepatic steatosis, and inflammation in genetically obese termates . Liver iBoth chronic and acute EtOH consumption causes endotoxemia  in humans and experimental animals ,66,121. There are several critical factors that may influence the effects of EtOH binge administration, including, for example, the dose of EtOH, the number of binges, the concentration of EtOH in the solution used for the gavage, and the time point(s) of sample collection. The control solution for EtOH bolus is also an important consideration. Maltodextrin remains the more widely-used control rather than water or saline. It enables the maintenance of isocaloric intake in all animals (controls and EtOH-binged) by substituting the calories gained from EtOH intake by those provided by maltodextrin. However, maltodextrin administration might cause distinct metabolic abnormalities , which cad libitum chronic EtOH feeding) causes more severe liver pathology. It closely resembles the drinking behavior in humans, and produces many of the histological and molecular features of alcohol-induced liver injury seen in patients with ALD. Several binge EtOH animal models have been recently developed, including administration of single or repeated EtOH binges alone or in combination with chronic EtOH exposure, HFD feeding, or LPS challenge. In particular, the recent NIH NIAAA paradigm (single or multiple EtOH binges combined with chronic EtOH) has been extensively utilized by numerous investigators to examine alcohol-induced liver and multi-organ pathology. Alcohol has been administered to rodents by a variety of different paradigms, each having their respective strengths, limitations, and applications. Incorporating binge EtOH administration into traditional animal models of ALD  and mild fibrosis. However, this model can be long-term and laborious. The recapitulation of alcohol-induced fibrosis in rodent models is more challenging than steatosis and inflammation. The hybrid model of HFD and IG EtOH feeding with weekly EtOH binges produces liver fibrosis, further supporting the utility of binge EtOH paradigms. Binge EtOH-plus-HFD model allows to examine the effect of comorbidities such as obesity on the progression of ALD. Therefore, the careful choice of experimental model is vital, and should be based on the specific scientific inquiry (acute or chronic EtOH exposure), the human liver injury feature to be simulated , and the technical expertise available. Taken together, binge EtOH models are attractive tools for identifying possible novel mechanisms, diagnostic or prognostic biomarkers, and therapeutic targets for ALD.


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MicroRNA-30e (miR-30e) is downregulated in various tumor types. However, its mechanism in inhibiting tumor growth of breast cancer remains to be elucidated. In this study, we found that miR-30e was significantly downregulated in tumor tissues of breast cancer (BC) patients and cell lines, and overexpression of miR-30e inhibited cell proliferation, migration and invasion. To understand the potential mechanism of miR-30e in inhibiting tumor growth, we showed that miR-30e blocked the activation of AKT and ERK1/2 pathways, and the expression of HIF-1α and VEGF via directly targeting IRS1. Moreover, miR-30e regulates cell proliferation, migration, invasion and increases chemosensitivity of MDA-MB-231 cells to paclitaxel by inhibiting its target IRS1. MiR-30e also inhibited tumor growth and suppressed expression of IRS1, AKT, ERK1/2 and HIF-1α in mouse xenograft tumors. To test the clinical relevance of these results, we used 40 pairs of BC tissues and adjacent normal tissues, analyzed the levels of miR-30e and IRS1 expression in these tissues, and found that miR-30e levels were significantly inversely correlated with IRS1 levels in these BC tissues, suggesting the important implication of our findings in translational application for BC diagnostics and treatment in the future. Neoadjuvant systemic treatment before surgery for advanced breast cancer is considered one of the most crucial factors in reducing mortality5. Invasion and metastasis remain the main obstacles in the treatment of breast cancer. Thus, research on the molecular mechanisms of breast cancer is receiving increased interest.Breast cancer (BC) is the most common malignancy in women in the world. The mortality of breast cancer over the last several decades has decreased, because of a combination of mammographic screening and improvements in systemic therapy7. It has been found that miRNAs regulate various pathological behaviors of cancer cells, such as cell proliferation, motility and sensitivity to chemotherapy14.MicroRNAs (miRNAs) are 20–22-nucleotide non-coding RNA molecules that negatively regulate gene expression by binding to the 3′-untranslated region (UTR) of their target genes with partial complementarity, leading to degradation of the target mRNAs, inhibition of their translation or both17. Previous studies reported the downregulation of miR-30 family members during osteoblast differentiation from mouse preosteoblast cell lines19. miR-30a/b/c/d were demonstrated to be able to negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad119. In contrast, miR-30 family members were upregulated during adipogenic differentiation of adipose tissue-derived stem cells, and miR-30a and miR-30d contributed to adipocyte formation20. To date, some genes have been identified as target genes of miR-30e, including Ubc9, Bmi1, P4HA1, ABL and ATG525. Furthermore, our present work provides novel evidences which demonstrating that miR-30e inhibits tumor growth and chemoresistance via targeting IRS1 in breast cancer.The miR-30 family members include miR-30a, miR-30b, miR-30c, miR-30d and miR-30e. The miR-30 family is associated with cell differentiation, cellular senescence, apoptosis, and involved in the pathogenesis of tumors and other disorders of the nervous, genital, circulatory, alimentary and respiratory systemsvia this direct target. These results will provide new insights into the molecular mechanism of breast cancer as well as provide potential new therapeutic strategy for breast cancer treatment in the future.In this study, we demonstrated that miR-30e levels were downregulated in human breast cancer specimens using 40 pairs of normal and cancer tissues. Then, we will investigate: (1) what is the role of miR-30e in breast cancer cell growth, migration and invasion; (2) what is the direct target of miR-30e that is associated with cancer development; and (3) whether forced miR-30e expression inhibits cell growth, migration, invasion and chemoresistance To evaluate the role of miR-30e in breast cancer, we first investigated the expression levels of miR-30e in normal tissues and breast cancer tissues by qRT-PCR Fig. . The resTo study the role of miR-30e in cancer carcinogenesis, stable cell lines were established Fig. . miR-30eSince cell migration and invasion are key characteristics of malignant tumor, next, we investigated the effects of miR-30e on migration and invasion. Over-expression of miR-30e dramatically inhibited the normally strong migration and invasive capacity of breast cancer cells Fig. . Our reswww.targetscan.org; www.microrna.org) were used to explore potential targets of miR-30e in BC. Figure To analyze the underlying molecular mechanism of miR-30e in BC, TargetScan and miRanda  and vascular endothelial growth factor (VEGF) have been linked to the PI3K/AKT and MAPK/ERK pathways. Cellular levels of p-AKT and p-ERK1/2 were significantly decreased in cells stably expressing miR-30e compared with miR-NC, while no statistically significant reduction of AKT and ERK1/2 were detected Fig. . Here, win vitro. Over-expression of miR-30e dramatically inhibited the normally strong invasive capacity of MDA-MB-231 cells  mRNA21. Granot G et al. confirmed that miR-30e induces apoptosis and sensitizes K562 cells to imatinib treatment via regulation of the BCR-ABL protein22, and miR-30e regulated Ubc9 expression in cancer cells24. Among others, miR-30e expression was upregulated in hepatoma patients who did not respond to cisplatin-therapy30. It is not surprising that miRNA can modulate different target expressions depending upon disease and cellular contest. In this study, the miR-30e expression was down-regulated in human BC, and over-expression of miR-30e inhibits the ability of cell proliferation, migration and invasion in BC cells.In the present study, miR-30e expression was significantly reduced in BC tissues and cell lines when compared with normal controls, respectively. Previous studies have shown that miR-30e is down-regulated in several cancer types. Feng 32. In our study, IRS1 oncogene was validated as the novel target of miR-30e. Firstly, luciferase reporter assay found that miR-30e directly recognized the 3′-UTR of IRS1 transcripts. Secondly, the IRS1 expression was significantly abolished in BC cells expressing miR-30e. Finally, miR-30e suppressed constitutive phosphorylation of AKT and ERK1/2, and inhibited expression of HIF-1α and VEGF via targeting IRS1. Taken together, our study provided the first evidence that miR-30e played a significant role in suppressing BC cell growth through inhibition of IRS1. Although we confirmed that miR-30e could inhibit the phenotype of BC by targeting IRS1, there might be other targets of miR-30e, which could also affect the growth of BC cells. Therefore, future studies are required to identify additional targets and pathways of miR-30e.IRS1 transmits signals from insulin/IGF receptor to activate PI3K/AKT and MAPK pathways, both of which are critical in mitogenesis and oncogenesisTaken together, our study indicated that IRS1 was a direct downstream target of miR-30e and was involved in the miR-30e-induced suppression of the activity of cell migration and invasion in breast cancer cell. The miR-30e/IRS1 link may play a role in breast cancer as markers of metastasis and prognostic factors. Furthermore, the therapeutic value of miR-30e in reducing cancer invasion, metastasis and chemoresistance should be further validated by independent cohorts and prospective trials.Human breast cancer specimens (30 pairs) and adjacent normal tissues were obtained from Nanjing Medical University and Department of Breast and Thyroid Surgery, Huai’an First People’s Hospital. All tissue samples were snap-frozen in liquid nitrogen immediately after surgery and stored in liquid nitrogen. All samples were histologically classified by clinical pathologist. Samples used for molecular analysis were initially grinded into powder in liquid nitrogen and then collected separately for RNA analyses. This study was approved by the institutional review board and the ethics committee of Nanjing Medical University. Informed consents were obtained from all subjects participating in the study.Human embryonic kidney cell line 293 T (HEK293T), Breast cancer cell lines , and mammary epithelia cell (MCF10A), were obtained from American Type Culture Collection. Cells were incubated at 37 °C in a humidified chamber supplemented with 5% CO2. Antibodies against IRS1, p-AKT, AKT, p-ERK1/2 and ERK1/2 were purchased from Cell Signaling Technology . Antibody against GAPDH was from Bioworld Technology . The growth factor reduced Matrigel was from BD Biosciences .To stably overexpress miR-30e in breast cancer cells, the lentiviral packaging kit was used (Thermo Fisher Scientific). Lentivirus carrying miR-30e or negative control (miR-NC) was packaged using HEK293T cells following the manufacturer’s manual. The lentiviral vector has red fluorescent protein (RFP) tag which can be used to check the efficiency of packaging using microscope. Cells were infected by lentivirus carrying miR-30e or miR-NC in the presence of polybrene (Sigma-Aldrich) and selected by puromycin (Sigma-Aldrich) for two week to obtain stable cell lines.−△△Ct).Total RNAs were isolated from harvested cells or human tissues using Trizol reagent according to the manufacturer’s instruction . To measure the expression levels of miR-30e, qPCR assay was performed and the U6 was used as an endogenous control. To determine the mRNA levels of IRS1, total RNAs were reversely transcribed by oligodT primer using PrimeScript RT Reagent Kit . Housekeeping gene GAPDH was used as internal control. The cDNAs were amplified by real-time PCR using SYBR Premix DimerEraser  on a 7900HT system, and fold changes were calculated by relative quantification .The supernatants were collected and protein concentrations were determined using BCA assay . Tumor tissues from human and nude mice were grinded into powder in liquid nitrogen with RIPA buffer, and the total tissue proteins were extracted as described above. Aliquots of protein lysates were fractionated by SDS-PAGE, transferred to a PVDF membrane , and subjected to immunoblotting analysis according to the manufacturer’s instruction. ECL Detection System  was used for protein signal detection.3 per well in 96-well plate incubated at 37 °C in 5% CO2 incubator. The cell proliferation was measured using a CCK-8 kit  according to the manufacturer’s instruction. Data were from three separate experiments with six replications per experiment.To evaluate the proliferation effect of miR-30e in breast cancer cells, stable cell lines were plated at a density of 2 × 10Cells were transfected with miR-30e or miR-NC according to the manufacturer’s instructions, and then cells were cultured to 95% confluence in 6-well plates. Cell monolayers were scratched using a 20 µL tip to form wound gaps and washed twice with PBS to remove the detached cells. After 24 h, the wound healing was photographed at different time points. The cell migration distances were measured in three different areas to indicate the migration ability of different treated cells.4 per well in the upper chamber without serum. The lower chamber was filled with 600 μl of the DMEM medium with 10% FBS to act as the nutritional attraction. After incubation for 24 h, noninvading cells were removed from the top well with a cotton swab, while the bottom cells were fixed with 3% paraformaldehyde, stained with 0.1% crystal violet, and photographed in three independent fields for each well. They were finally extracted with 33% acetic acid and detected quantitatively using a standard microplate reader (OD at 570 nm). Three independent experiments were conducted in triplicate.Invasion assay was determined using 24-well BD Matrigel invasion chambers  in accordance with the manufacturer’s instruction. Indicated cells were plated at a density of 5 × 10For dual-luciferase assay, 3′-UTRs of IRS1 containing predicted miR-30e seed-matching sites and corresponding mutant sites were amplified by PCR using human cDNA template, and inserted into the SacI and HindIII restriction enzyme sites by pMIR-REPORTER vector . These constructs were validated by the DNA sequencing. Cells were seeded in a 24-well plate and co-transfected with the wild type or mutant reporter plasmid, pRL-TK plasmids, and miR-30e or miR-NC. Luciferase activities were analyzed 24 h after transfection using the Dual Luciferase Reporter Assay System . Experiments were performed in three independent replicates.Cancer cells were seeded at a density of 4,000 cells per well in a 96-well plate. 24 h later, freshly prepared paclitaxel  was added with the final concentration ranging from 1 to 32 nM. 48 h later, cell viability was assayed by CCK8 kit.Apoptosis were measured by flow cytometry. For AnnexinV staining, 5 μL phycoerythrin-Annexin V, 5 μL propidium iodide (BD Pharmingen) and 300 μL 1 × binding buffer were added to the samples, which were incubated for 15 min at room temperature in the dark. Then the samples were analyzed by flow cytometry  within 1 h. The data were analyzed using FlowJo software. Three experiments were performed in triplicate.The activity of caspase-3 was determined using the Beyotime caspase-3 activity kit. Cell lysates were prepared and incubated with reaction buffer containing caspase-3 substrate (Ac-DEVDpNA) after the treatment as indicated. Caspase-3 activity assay were performed on 96-well plates by incubating 10 μL protein of cell lysate per sample in 80 μL reaction buffer containing 10 μL caspase-3 substrate at 37 °C for 2 h according to the manufacturer’s protocol. The reaction was then measured at 305 nm for absorbance.6) were suspended in 150 μl of serum-free DMEM medium, and injected subcutaneously into each side of the posterior flank of the same nude mouse. Tumor sizes were measured every three days from the 14th day. Tumor volumes were calculated using vernier caliper using the formula: volume = 0.5 × (Length × Width2). Mice were sacrificed 30 days after implantation, and tumors were dissected. Total proteins were extracted and used for immunoblotting. All animal experiments were approved by the Committee of Laboratory Animal Experimentation of Nanjing Medical University.Male nude mice  were purchased from Shanghai Laboratory Animal Center , and bred in special pathogen-free (SPF) condition. Cells . The correlations between miR-30e expression levels and IRS1 levels in human breast cancer tissues were analyzed using Spearman’s rank test. Statistical evaluation for data analysis was determined by