{"text": "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\u00b0C 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."}
{"text": "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\u2013antigen 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\u2013antigen 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 \u2018fast screening\u2019 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\u2019s 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\u2005min at 277\u2005K, con\u00adcentrated to a final volume of 0.6\u2005ml using an Amicon Ultra 5\u2005kDa device (Millipore) and loaded onto a Superdex 200 10/300 column  equilibrated with 20\u2005mM HEPES pH 7.5, 0.1\u2005M NaCl. A shift in the elution profile (elution earlier than the free Fab) indicated complex formation. Three runs were performed, with 0.2\u2005ml protein solution applied each time to the column for each complex. Fractions corresponding to the main peak were pooled, concentrated to 6\u20139\u2005mg\u2005ml\u22121 in 20\u2005mM HEPES pH 7.5, 0.1\u2005M NaCl and used in crystallization trials.For complex formation, purified C836, H2L6 and M1295 Fab fragments were buffer-exchanged into 20\u2005m2.3.Crystallization of the complexes was carried out by the sitting-drop vapor-diffusion method at 293\u2005K. 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\u2005\u00b5l protein solution mixed with an equal volume of reservoir solution. The droplets were equilibrated against 90\u2005\u00b5l reservoir solution. Optimization screens were made using a Matrix Maker .2.4.Microcrystals used for seed-stock preparation were placed in 100\u2005\u00b5l reservoir solution, homogenized by vortexing for 3\u2005min with a Teflon Seed Bead  and stored at 253\u2005K. 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\u2005\u00b5l screening (reservoir) solution and 0.2\u2005\u00b5l microseeds were added to 0.8\u2005\u00b5l protein solution. The protein droplets were equilibrated over 500\u2005\u00b5l reservoir solution.3.3.1.M for ammonium sulfate versus a pH range of 3.5\u201310.5. Needle-like microcrystals of the H2L6 complex were observed in 28% PEG 8000, 0.1\u2005M MES pH 6.5 . The other two com\u00adplexes 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 \u00d7 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\u20053.2.M ammonium sulfate both in 0.1\u2005M 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\u201322% PEG 8000 or 1.6\u20132.4\u2005M lithium acetate pH 7.9 (condition No. 24), 0.2\u2005M ammonium tartrate pH 6.6 (No. 38), 0.2\u2005M ammonium phosphate pH 8.0 (No. 44) or 0.2\u2005M 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\u2005h from PEG/Ion Screen under several conditions, all of which contained 20% PEG 3350 plus one of the following salts: 0.2\u2005The 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\u2005M MES pH 6.5 and from 16% PEG 3350, 0.2\u2005M ammonium citrate, 0.1\u2005M MES pH 6.5. The crystals appeared within 2\u2005d and reached dimensions of 0.1 \u00d7 0.1 \u00d7 0.3\u2005mm . Both conditions produced the same crystal form. The crystals from ammonium tartrate diffracted to 1.9\u2005\u00c5 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\u00a0= 114.86\u2005\u00c5. The asymmetric unit contained one molecule of the complex.X-ray-quality crystals were obtained from 14% PEG 3350, 0.2\u20053.3.M ammonium citrate, 0.1\u2005M 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\u2005M lithium chloride pH 6.8 , 20% PEG 3350, 0.2\u2005M potassium chloride pH 7.0  and 20% PEG 3350, 0.2\u2005M sodium citrate pH 8.3 . In addition, X-ray-quality crystals grew from 25% PEG 8000, 0.1\u2005M sodium acetate pH 5.5 . The latter conditions were optimized (pH 4.5) to yield crystals of about 0.2 \u00d7 0.2 \u00d7 0.2\u2005mm in 2\u2005d . These crystals diffracted to 2.8\u2005\u00c5 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\u2005\u00c5. The asymmetric unit contains one molecule of the complex.The optimized H2L6 crystals obtained from 16% PEG 3350, 0.2\u20053.4.\u22121 protein concentration. Crystal formations of poor quality appeared in several drops in a range of conditions after 2\u2005d . A number of drops remained clear. A mixture of these crystals obtained in different conditions produced a \u2018self-seeding\u2019 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 \u2018fast screen\u2019 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\u2005mg\u2005ml\u22121. To minimize the number of experiments, the optimization screens included only one buffer, 0.1\u2005M HEPES buffer pH 7.5, and one of the four salts  at 0.2\u2005M concentration in the presence of 18\u201322% PEG 3350. X-ray-quality crystals were obtained in both the self-seeding and the cross-seeding experiments within 2\u2005d . 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 \u2018cross\u2019 and \u2018self\u2019 seeds were used in the MMS optimization, in which the protein concentration was 9\u2005mg\u2005mlM sodium tartrate, 0.1\u2005M 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\u2005\u00c5, \u03b2\u00a0=\u00a0107.02\u00b0. The best self-seeding conditions were 18% PEG 3350, 0.2\u2005M ammonium citrate, 0.1\u2005M HEPES pH 7.5 . These crystals were isomorphous to the cross-seeded crystals. Both types of crystals diffracted to 2\u2005\u00c5 resolution.The best cross-seeding conditions were 20% PEG 3350, 0.2\u20054.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 \u2018optimized\u2019 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 \u2018nucleation\u2019 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.\u22121 but hits were identified within 24\u2005h when the concentration was increased to 9\u2005mg\u2005ml\u22121. 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\u2005mg\u2005mlIn 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\u2013thaw 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\u2005\u00c53\u2005Da\u22121 for H2L6 and 2.35\u2005\u00c53\u2005Da\u22121 for C836. Analysis of intermolecular contacts in these crystals revealed one type of contact that is common to both crystal forms: a \u03b2-\u00adbridge 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 ortho\u00adrhombic (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 \u2018universal\u2019 seed stock can produce enough hits for a given protein or class of protein whose members have significant sequence homology, such as Fabs."}
{"text": "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 \u201csilent\u201d or \u201cregulatory\u201d 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\u20131 M\u2126.Whole cell two-electrode voltage clamp (TEV) currents were recorded using a Dagan CA\u20131B 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\u20131.5 M\u2126.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 \u00b1 S.E.M. with a simulated Boltzmann fit (G/Gmax\u200a=\u200a1/(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)\u200a=\u200aIi+Ae-t/\u03a4, where I(t) is the current at time t, Ii is the initial current, A is the amplitude of the fit and \u03a4 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)\u200a=\u200a. 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 \u221220 to \u221230 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\u20137 (ches 1\u20137 . 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\u201311) 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."}
{"text": "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 \u201cCHD composite.\u201d 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 \u201cCVD composite.\u201d 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 (\u03c72 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 \u22120.073 to \u22120.151) 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\u2014to achieve sustained smoking cessation and lower elevated serum cholesterol, weight, and BP by dietary/lifestyle means, supplemented as indicated by antihypertensive medication\u2014was effective in preventing clinically relevant CVD events. This was the case even though SI\u2013UC differences in major risk factors were less than expected during the trial period.16: Among men 40 to 49 years of age, a 5\u2013mm 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 \u201cevent-driven\u201d 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 ."}
{"text": "G\u03d5 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 G\u03d5 formation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH) (29 cycles/day for 5 days) completely abolished G\u03d5 formation, while acute IH had dose-dependent effects. Exposure to 24\u2009h (56 IH cycles) decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phox expression, whereas under 24\u2009h sustained hypoxia (SH) the size and expression of LC3B and gp91-phox/p22-phox resembled G\u03d5 formed in normoxia. Diphenyl iodide (DPI), a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished G\u03d5 formation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC) abrogated the effects of IH by inducing large CD66b+/LC3B+ G\u03d5 and increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in G\u03d5 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 \u20139. 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 \u03d5 within 5\u20137 days [\u03d5 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, G\u03d5 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 G\u03d5 development, demonstrating the importance of autophagy to G\u03d5 development [\u03d5 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\u20137 days . These Gelopment . However\u03d5 formation, we sought to investigate the effects of various hypoxic treatments on G\u03d5 development. To gain further insights into G\u03d5 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 [\u03d5 were evident in culture. Depending on the donor, from each 106 neutrophils plated, 100\u2013200\u2009G\u03d5 developed. As a control, in some experiments, the culture medium was supplemented with 30\u2009ng/mL granulocyte macrophage-stimulating factor (GM-CSF) and 30\u2009ng/mL IL-4 . GM-CSF/IL-4 was added at each medium change. The LPS content in FCS was lower than 1.0\u2009ng/mL and the addition of 1\u201310\u2009ng/mL LPS to the culture medium did not affect G\u03d5 formation [Blood samples were obtained from 28  healthy nonsmoker volunteers with a mean age of 28.0 \u00b1 6.6 years and BMI of 24.9 \u00b1 3.9\u2009Kg/mormation .\u03d5, 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, G\u03d5 did not develop. Thus, excluding the possibility that G\u03d5 arise from contaminating cells or cells other than mature PMN, we should also note that since the yield of G\u03d5 formed from neutrophils in culture is low (0.01\u20130.02% of cultured PMN in normoxia), biochemical and molecular measures are hard to implement [Of note, to validate the neutrophilic origin of Gmplement . Therefo6\u2009cells/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\u2009min, and this level of hypoxia was achieved after 15\u2009min of incubation. In the reoxygenation period, O2 levels reached normoxic levels (20%) after 10\u2009min 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\u2009h/day). Acute IH was induced by exposing PMN to 10 cycles (250\u2009min), 29 cycles (12\u2009h), or 56 cycles (approximately 24\u2009h), 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\u2009min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4\u00b0C using the following primary Abs (dilution 1\u2009:\u2009100) 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\u2032,6-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\u2009mL of PKH-26 or PKH-67 (1\u2009:\u2009500 in Diluent C) and incubated for 5\u2009min at room temperature. Labeling was stopped by adding 0.5\u2009mL FCS. Then, cells were washed three times with complete medium and cocultured.\u03d5 was determined on day 7 using fluorescent latex beads of 1.0\u2009\u03bcm in diameter . Briefly, G\u03d5 were incubated for 2\u2009h with carboxylate-modified fluorescent yellow-green latex beads  at a cell\u2009:\u2009bead ratio of 1\u2009:\u2009500 (because of the large G\u03d5 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 G\u03d5 were kept on ice for 15\u2009min and cytospins were prepared immediately or 2\u2009h after adding the latex beads. (2) To inhibit phagocytosis, G\u03d5 were preincubated with 10\u2009\u03bcM cytochalasin B  for 30\u2009min prior to adding the latex beads, and cytospins were prepared 2\u2009h after incubation with latex. (3) To ensure intracellular localization rather than adhesion, the intracellular localization of latex beads was confirmed in G\u03d5 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 G\u03d5 were incubated with 50\u2009nM of LysoTracker for 90\u2009min at 37\u00b0C in the dark. To identify mitochondria, viable cells were stained for 30\u2009min at 37\u00b0C in the dark with 100\u2009nM 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 ce\u03bcM diphenyl iodide (DPI); a ROS scavenger, 20\u2009\u03bcM N-acetylcysteine (NAC) ; and PI3K inhibitor LY-294002, 20\u2009\u03bcM  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\u2009h to IH, SH, or normoxia with or without various inhibitors. Each inhibitor was added 10\u2009min prior to the various oxygen treatments and remained throughout the treatments. The following inhibitors were used: a NADPH oxidase inhibitor, 10\u2009\u03d5 by NBT test. NBT (Sigma-Aldrich) was dissolved in RPMI 1640 without phenol red (0.2%). Cells were incubated without or with 100\u2009nM PMA at 37\u00b0C for 15\u2009min, then kept at room temperature for 10\u2009min, and assessed by light microscopy. In some experiments also DPI was added to G\u03d5, 2\u2009h prior to PMA stimulation. Cytoplasmic clumps of formazan deposits in G\u03d5 were considered positive for ROS.Intracellular ROS was determined in 7-day G4 cells/well in 100\u2009\u03bcL culture medium  and were incubated with the WST-1 reagents (10\u2009\u03bcL/well) for 1\u2009h. The formazan dye formed was determined with ELISA reader at 450/650\u2009nM. 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 . Briefly\u03bcg 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 \u03b2-actin and the ratio of LC3BII/LC3BI was also calculated.Cells were washed twice and extracts were prepared in lysis buffer containing 50\u2009mM Tris-HCl (pH 7.4), 150\u2009mM 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\u2009 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 \u00b1 SD for each experimental group. A two-tailed Student's\u03d5 under prolonged culture conditions [\u03d5 which developed in normoxic conditions had the same characteristics of G\u03d5 as previously shown . These G\u03d5 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 G\u03d5 [\u03d5, 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 G\u03d5 . Thus, w\u03d5, 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\u2009:\u20091 ratio and cocultured for 2\u2009h and cytospins were prepared. Notably, G\u03d5 formation is dependent on the local cytokine milieu, and culturing neutrophils with GM-CSF/IL-4 did not induce G\u03d5 formation but rather a different long-lived subpopulation. Since cells obtained in GM-CSF/IL-4 supplemented cultures did not resemble G\u03d5, 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\u201314 days in culture as previously described , 29. Unljections , and wer\u03d5 formation, but their development was evident in SH and in normoxic cultures. Thus, in the following experiments we focused on acute IH treatments for G\u03d5 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\u2009min), 29 cycles (approximately 12\u2009h), or 56 cycles (approximately 24\u2009h) 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.\u03d5 development  had no effect on Gelopment . However < 0.005 ) and the < 0.005 , althoug Figures . Moreove0\u2009min hadsibility , 15, 16,\u03d5 formation, it was chosen to further investigate G\u03d5 phenotype and functions. As shown in \u03d5 formed at all oxygen treatments were CD66b positive. The developed G\u03d5 were also neutrophil elastase (NE) positive, indicating that G\u03d5 express this important neutrophil specific protein after differentiation. However, the expression of NE was significantly lower in IH-treated G\u03d5 compared to G\u03d5 formed in normoxia or SH. Although in SH-treated G\u03d5 NE levels were also attenuated, they did not significantly differ from controls  had the strongest effect on Gcontrols . By staioTracker , a staineated G\u03d5 , indicat\u03d5 development under SH treatments basically resembled the development of normoxia-treated G\u03d5, mainly with regard to size, CD66b, and LysoTracker expression. Yet other measures as NE varied. However, under IH, G\u03d5 development was significantly attenuated in a severity-dependent manner.All in all, G\u03d5 by demonstrating the presence of LC3B-II as vesicular puncta associated with autophagosomes and that treatment with specific autophagy inhibitors abolished G\u03d5 formation [\u03d5 at the protein level as illustrated in \u03b2-actin and normalizing by LC3BII/LC3BI ratio revealed a high LC3BII expression in G\u03d5 compared to fresh PMN, clearly indicating active autophagy in G\u03d5. Examples of G\u03d5 stained for LC3B at the various oxygen treatments are depicted in \u03d5   . These f\u03d5 were noted (\u03d5 expressing LC3B positive structures containing mitochondria (mitophagy) were the predominant G\u03d5 type . These appeared as yellow spots with significant colocalization of LC3B and MitoTracker (MOC > 0.6). In SH, 34.8 \u00b1 2.8% expressed mitophagy, but the morphology was mixed (\u03d5 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-G\u03d5 and as expected was localized in the plasma and phagolysosome membranes as previously shown [\u03d5 the gp91-phox expression was on average lower than that of normoxia. However, in 24\u2009h IH-treated G\u03d5, the expression of gp91-phox was significantly lowered to 28.4 \u00b1 15.4 of normoxic-G\u03d5 (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 \u201337. We tly shown . In 24\u2009hphox subunit was also mainly localized in the plasma membranes and phagolysosomes in normoxic and in 24\u2009h SH-treated G\u03d5. However, while, in 24\u2009h IH-treated G\u03d5, the intensity of the p22-phox subunit expression was lowered to 45% of normoxic-G\u03d5, in 24\u2009h SH-treated G\u03d5 its intensity of expression was significantly increased to 165% compared to normoxic-G\u03d5 (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\u2009h SH-treated G\u03d5 . Equal volumes of DMSO were added as a negative control. Inhibiting the PI3K kinase pathway with LY-294002 abolished G\u03d5 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 G\u03d5 formation [\u03d5.The PI3K specific inhibitor LY-294002 was added at 20\u2009ormation . Jointlyphox, facilitating its membrane translocation and activation [\u03d5 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 \u201342. Akt tivation . Akt is tivation . Thus, i\u03d5 development, the potent antioxidant and glutathione precursor\u2014NAC\u2014was used [\u03bcM) was added to PMN cultures 10\u2009min prior to the exposure to 24\u2009h of normoxia, SH, or IH. Viability of the cells in the presence of NAC, measured after 24\u2009h, 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 G\u03d5 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 G\u03d5  exhibited a lower phagocytic activity (p = 0.015) compared to normoxic-G\u03d5 (\u03d5 to about 50% of the ability of normoxic-G\u03d5 (p = 0.001) . Additionally, the intracellular localization of latex beads was also validated in G\u03d5 by 3D z-stack images as illustrated in Gmoxic-G\u03d5 , 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 [\u03d5 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 G\u03d5, as already indicated by restoring the formation of G\u03d5 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, t\u03d5 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 [\u03b2 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 t\u03d5) derived from neutrophils maintained in prolonged culture conditions [\u03d5 development in a dose-dependent manner whereas sustained hypoxia had no  effect on their development. Intermittent hypoxia reduced G\u03d5 size, autophagy, neutrophil elastase and NADPH oxidase expression, and their phagocytic activity. Inhibiting NADPH oxidase or the PI3K/Akt signaling pathway completely abolished G\u03d5 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 [\u03d5 in carotid plaques from patients undergoing elective endarterectomy. Thus, a better understanding of G\u03d5 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 G\u03d5 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"}
{"text": "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\u2013DeCarli liquid diet model), chronic intragastric EtOH administration (Tsukamoto\u2013French 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\u2013French 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\u2013DeCarli ad libitum EtOH feeding (even for longer periods) usually causes hepatic steatosis with limited liver injury, inflammation and no fibrosis. The Tsukamoto\u2013French 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\u2019s 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\u2013French model). Dr. Tsukamoto\u2019s 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\u20135 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."}
{"text": "Upon publication of the original article , it was The second line of the subsection \u201cRead alignment\u201d currently reads \u201c--outSJfilterOverhangMin 12 12 12\u201d and should instead be \u201c--outSJfilterOverhangMin 12 12 12 12\u201d."}
{"text": "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-1\u03b1 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-1\u03b1 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\u201322-nucleotide non-coding RNA molecules that negatively regulate gene expression by binding to the 3\u2032-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.\u00a0. The resTo study the role of miR-30e in cancer carcinogenesis, stable cell lines were established Fig.\u00a0. 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.\u00a0. Our reswww.targetscan.org; www.microrna.org) were used to explore potential targets of miR-30e in BC. Figure\u00a0To 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.\u00a0. 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\u2032-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-1\u03b1 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\u2019an First People\u2019s 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\u2009T (HEK293T), Breast cancer cell lines , and mammary epithelia cell (MCF10A), were obtained from American Type Culture Collection. Cells were incubated at 37\u2009\u00b0C 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\u2019s 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.\u2212\u25b3\u25b3Ct).Total RNAs were isolated from harvested cells or human tissues using Trizol reagent according to the manufacturer\u2019s 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\u2019s instruction. ECL Detection System  was used for protein signal detection.3 per well in 96-well plate incubated at 37\u2009\u00b0C in 5% CO2 incubator. The cell proliferation was measured using a CCK-8 kit  according to the manufacturer\u2019s 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\u2009\u00d7\u200910Cells were transfected with miR-30e or miR-NC according to the manufacturer\u2019s instructions, and then cells were cultured to 95% confluence in 6-well plates. Cell monolayers were scratched using a 20\u2009\u00b5L tip to form wound gaps and washed twice with PBS to remove the detached cells. After 24\u2009h, 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\u2009\u03bcl of the DMEM medium with 10% FBS to act as the nutritional attraction. After incubation for 24\u2009h, 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\u2009nm). Three independent experiments were conducted in triplicate.Invasion assay was determined using 24-well BD Matrigel invasion chambers  in accordance with the manufacturer\u2019s instruction. Indicated cells were plated at a density of 5\u2009\u00d7\u200910For dual-luciferase assay, 3\u2032-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\u2009h 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\u2009h later, freshly prepared paclitaxel  was added with the final concentration ranging from 1 to 32\u2009nM. 48\u2009h later, cell viability was assayed by CCK8 kit.Apoptosis were measured by flow cytometry. For AnnexinV staining, 5\u2009\u03bcL phycoerythrin-Annexin V, 5\u2009\u03bcL propidium iodide (BD Pharmingen) and 300\u2009\u03bcL 1\u2009\u00d7\u2009binding buffer were added to the samples, which were incubated for 15\u2009min at room temperature in the dark. Then the samples were analyzed by flow cytometry  within 1\u2009h. 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\u2009\u03bcL protein of cell lysate per sample in 80\u2009\u03bcL reaction buffer containing 10\u2009\u03bcL caspase-3 substrate at 37\u2009\u00b0C for 2\u2009h according to the manufacturer\u2019s protocol. The reaction was then measured at 305\u2009nm for absorbance.6) were suspended in 150\u2009\u03bcl 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\u2009=\u20090.5\u2009\u00d7\u2009(Length\u2009\u00d7\u2009Width2). 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\u2019s rank test. Statistical evaluation for data analysis was determined by"}
{"text": "This paper presents a new approach for the automatic detection of galaxy morphology from datasets based on an image-retrieval approach. Currently, there are several classification methods proposed to detect galaxy types within an image. However, in some situations, the aim is not only to determine the type of galaxy within the queried image, but also to determine the most similar images for query image. Therefore, this paper proposes an image-retrieval method to detect the type of galaxies within an image and return with the most similar image. The proposed method consists of two stages, in the first stage, a set of features is extracted based on shape, color and texture descriptors, then a binary sine cosine algorithm selects the most relevant features. In the second stage, the similarity between the features of the queried galaxy image and the features of other galaxy images is computed. Our experiments were performed using the EFIGI catalogue, which contains about 5000 galaxies images with different types . We demonstrate that our proposed approach has better performance compared with the particle swarm optimization (PSO) and genetic algorithm (GA) methods. In turn, galaxy morphology can be used to provide an independent test of the two proposed scenarios for galaxy formation. Elliptical galaxies, for example, are believed to be formed through major mergers2, whereas disk-dominated galaxies cannot have undergone recent major mergers, as such mergers would have severely disrupted their shape3. Thus, the class of quenching models is sufficient to explain the full range of morphological types observed for quenched galaxies. For example, bars can be found in all types of disk galaxies, from the earliest to the latest stages of the Hubble sequence. Barred galaxies constitute a major fraction of all disk galaxies. A small number of galaxies that appear unbarred at visual wavelengths have actually been found to be barred when observed in the near infra-red. The three clearest cases are NGC 15664, NGC 10686 and NGC 3097. De Zeeuw and Franx8 surveyed the literature for the dynamics of these objects. We are still far from a complete understanding of the dynamical structure of galaxies. Here, we will be able to do no more than scratch the surface of the majority of these problems.Astronomy has become an immensely data-rich field. For example, the Sloan Digital Sky Survey (SDSS) will produce more than 50,000,000 images of galaxies in the near future10. In ref. HST/WFC3 image containing 1639 galaxies, which identified disturbed morphologies using multimode, intensity and deviation statistics. Additionally12, proposed a method that consists of two stages: first, feature extraction  of galaxy images from the SDSS DR7 spectroscopic sample, followed by the classification of these features using a support vector machine.The development of galaxy morphological schemes can be used to successfully determine galaxy morphology via classification or image-retrieval methods. For example, the Deep Neural Network (DNN) algorithm has been used to classify the Galaxy Zoo  approach is one of the most commonly used image retrieval methods15, which aims to avoid the use of textual descriptions and instead retrieves images based on similarities in their content. Relevant content can be information related to image patterns, colors, textures, shape and location16.The image-retrieval method is a computer system for browsing, searching, detecting and retrieving images from a large database of digital imagesSuch image content is obtained by using feature-extraction methods, which is then saved in a database. To answer a queried image, the similarity between stored features and the features of a queried image (extracted using the same method) is computed and used to determine the closest between the images. However, the CBIR approach is a challenging problem for galaxy images, because there is a large number of galaxy images and determining the most relevant images from a large database becomes a non-trivial task.18. Next, ref. Several methods have been applied to improve the quality of CBIR for galaxy images. Ref. K-NN classifier as measure of the quality of the features which selected by Sine Cosine algorithm (SCA).In general, the previous methods consider either the shape, the texture features or the color, or both of them , but not all of them. Moreover, not all of the extracted features are important: some may be redundant/irrelevant, which in turn reduce the quality of the classification or image-retrieval results. To address this, the aim of this paper is to introduce a new machine-learning approach for the retrieval of galaxy images. Our approach avoids the limitations of previous methods by extracting the shape, color and texture features from galaxy images, and then determining the most relevant features and ignoring other features by using the 20 that selects the most relevant features using the classification accuracy as a fitness function. In the second stage, similar images to the queried image are returned by using the Euclidean distance as a measure.The proposed approach consists of two stages: training and image retrieval. In the training stage there are two steps: the first is feature extraction, where the color, shape and texture features are extracted from a dataset of galaxy images. The second step is feature selection, which is performed based on the modified sine cosine algorithm15.In this section, visual features such as color, texture and shape are introduced21. Therefore, the aim of any color feature extraction method is to represent the main colors of the image content  and then use these color features to describe the image and distinguish it from other images. RGB colors used in this study were obtained by converting from the SDSS color system using the Maxim DL astronomical software22.The color of an image is one of the most widely used features in image retrieval and several other image-processing applications. It is a very important feature since it is invariant with respect to scaling, translation and rotation34, which denotes the joint probability of the intensity of an image. From probability theory, a probability distribution can be uniquely characterized by its moments. Thus, if we interpret the color distribution of an image as a probability distribution, moments can be used to characterize the color distribution. The moments of the color distribution are the features extracted from the images; if we denote the value of the ith color channel at the jth image pixel as Pij, then the color moments can be defined as refs The first-order moment (the mean):The second-order moment (the standard deviation):The third-order moment (skewedness):The color histogram is one of the most well-known color features used for image feature extraction25. Textures can be rough or smooth, vertical or horizontal. Generally, they capture patterns in the image data, such as repetitiveness and granularity.The texture descriptor is an important feature that provides properties such as smoothness, coarseness and regularity27. The Gray Level Co-Occurrence Matrix (GLCM) and Color Co-Occurrence Matrix (CCM) are the most commonly used statistical approaches used to extract the texture of an image28. These features include the contrast, correlation, entropy, energy and homogeneity, which are defined as:The contrast represents the amount of local variation in an image. This concept refers to pixel variance, and it is defined as:The correlation represents the relation between pixels in an image, which determines the linear dependency between two pixels and is defined as:En) represents the textural uniformity, where large values of En indicate a completely homogeneous image.The energy (ET) measures the randomness of the intensity distribution. It is inversely correlated to En, and is defined as:The entropy (H) is used to measure the closeness of the distribution, where large values H indicate that the image contrast is low. The definition of H is given in the following equation:The homogeneity , the discrete Fourier transform (DFT), discrete wavelet transform (DWT) and the Gabor filterz(i) be an ordered sequence that represents the Euclidean distance between the centroid and all N boundary pixels of the object. The rth contour sequence moment mr14 is defined as:Shape features were extracted by using the contour moments defined mathematically as follows. Let 20, this algorithms is a new meta-heuristic algorithm which used either the sine or cosine function to search about the best solution. Consider the current solution Xi, 20:20:r1, r2, r3 and r4 are random variables, P is the best solution, and |\u00b7| represents the absolute value20.In this section, the sine cosine algorithm (SCA) is illustratedr2 parameter defines the direction of Xi , while r3 gives random weights to P in order to stochastically emphasize (r3\u2009>\u20091) or deemphasize (r3\u2009<\u20091) its influence when defining the distance. Next, r4 is responsible for switching between the sine and cosine functions in equation , which could be either in the space between Xi and P or outside of this space, and it is also responsible for balancing between the exploration and exploitation to improve the convergence performance by updating its value as ref. t is the current iteration, tmax is the maximum number of iterations, and a is a constant. Figure\u00a0Following ref. equation \u200920. Finaze r3\u2009>\u2009 or deempmentclasspt{minimaIn this section, we investigate a new approach to galaxy image retrieval as illustrated in Algorithm 1. Our proposed approach consists of two stages: a training stage and the galaxy image retrieval stage.I, which are combined into a feature vector FVI, where I is the current image. The next step in the training stage is to reduce the size of FV through using the Binary SCA (BSCA) algorithm (see Algorithm 2) to select the most relevant features. This process is performed by maximizing the accuracy of the K-NN classifier, which is used as a fitness function.In the first stage, the input is the dataset of galaxy images. Then the shape, texture and color features are extracted for each galaxy image popsize, and the output is the best solution P that points to the selected features (SelFeat). The solution in the population of the BSCA algorithm is represented as a binary vector by using the sigmoid function which transforms a real number into a binary number as:Xi is the current solution .The BSCA starts by generating a random population of size NC is the number of correctly predicted samples, and NI represents the total number of images. The dataset is divided by using a 10-fold cross validation (CV), and then the K-NN algorithm predicts, using the label of the testing set, where the output from 10-fold CV is the average of accuracy through 10\u2009runs.After the solutions are converted to binary vectors, the fitness function is computed for each solution. The fitness function is defined according to the classification accuracy rate as:Xi is updated using equations   Xi is uFQ, and then the same features corresponding to SelFeat are selected. Then the Euclidian distance is used to compute the similarity between FQ and FVThe second stage starts by extracting the features of a queried image 29. We also compared the performance of our method with the particle swarm optimization (PSO)30 and genetic algorithm (GA)31 methods. The parameters used in each algorithm is given in Table\u00a0We tested our proposed approach using the EFIGI catalogue, which consists of 4458 galaxy images29 contains 16 morphological attributes that were measured by visual examination of the composite g, u, r color image of each galaxy, derived from the SDSS FITS images using29. The EFIGI catalogue merges data from standard surveys and catalogues . The bulge-to-disk ratio32 and the degree of azimuthal variation of the surface brightness were often used as discriminant parameters along the Hubble sequence. This is not surprising since the EFIGI classification scheme is very close to the RC3 system. The final EFIGI database is a large sub-sample of the local universe which densely samples. The EFIGI morphological sequence is based on the RC3 revised Hubble sequence (RHS), which we call the EFIGI morphological sequence (EMS).The EFIGI catalogue22, using the same intensity mapping for all RGB images.Finally, all colors of the original data were used to create composite, \u201ctrue color\u201d, RGB images in PNG format with the Maxim DL astronomical software28.The precision rate is defined as the ratio of the number of retrieved images similar to the queried image relative to the total number of retrieved images28.The recall rate is defined as the percentage of retrieved images similar to the query image among the total number of images similar to the queried image in the databasep, q and r are the number of relevant images retrieved, relevant images in the dataset which are not retrieved, and non-relevant images in the dataset which are retrieved, respectively.Two measurements were used to evaluate the performance of the proposed algorithm: the precision rate and the recall rate.33. As discussed previously, the 10-fold CV works by dividing the dataset into ten groups, and the experiment was performed ten times by selecting one group as the test set and the remaining groups were used as a training set during each run. The output is the average of accuracy of the ten runs.In order to assess the effectiveness of our approach, we used the leave-one-out cross-validation method, where each image in the dataset was considered as the queried image, and the process was repeated 4458 times. Also, we used the 1-NN method based on 10-fold cross-validation (CV), which was used to evaluate the subset of selected features. This classifier is a parameter-free feature and is easy to implementIn general, we used color, texture and shape feature vectors for galaxy image retrieval. The total number of extracted features was 30, where nine features were extracted from the three colors RGB (three moments for each color), 20 texture features (four rotations for each measure) and one shape feature. The extracted feature vectors were applied to the feature selection method  to determine the relevant features.The best selected features with their accuracy  are given in Table\u00a0The comparison results of our proposed method with other methods are illustrated in Figs\u00a0Moreover, from Table\u00a0Figures\u00a0In order to investigate the influence of the size of the training set when selecting the best features, the dataset was randomly divided into training and testing sets. The proposed method was then evaluated at three different sizes, i.e. 50%, 70% and 85% of dataset (the remaining is the test set). Our results are shown in Table\u00a0Finally, from the previous results, we can conclude on two things: first is that the proposed approach for galaxy image retrieval is better than the PSO and GA algorithms in terms of recall, precision, accuracy and the time complexity. The second is that the most suitable method used to split the dataset (when selecting the best-fitting features) is the 10-fold CV, however, if the dataset is divided randomly then the most suitable size for the training set is in the range 85% to 90%.In this study, we proposed a machine learning approach for galaxy image retrieval used for the automatic detection of galaxy morphological types from datasets of galaxies images. The automated detection of galaxies types is very important to understand the physical properties of the past, present, and future of the universe, while also offering a means for identifying and analyzing peculiar galaxies that cannot be associated with a defined morphological stage on the Hubble sequence.Our analysis was performed such that our approach automatically detected specific morphology types from different morphological classes without human guidance. The proposed algorithm was compared with the PSO and GA algorithms, and its performance was evaluated based on recall and precision. The results indicate the superior performance of our proposed approach.Based on the promising results of the algorithm, our future work will attempt to further investigate its application to other complex problems in astronomy by modifying the proposed method."}
{"text": "Motivated by the hierarchical micro and nanoscale features in terms of porosity of diatomite, the production of ceramic-graded porous foams with tailored porosity, obtained by using it as raw material, has been proposed. The main challenge during the foam-production process has been the preservation of diatomite nanometric porosity and the addition of other levels of hierarchical porosity. The coupled use of two techniques of direct foaming , combined with the use of 3D printing inverse replica method, assured the achievement of porosity of, respectively, microscopic and macroscopic dimensions. Optical and scanning electron microscopies have been performed for an in-depth characterization of the final microstructure. XRD analysis has been carried out to check the influence of sacrificial templates on the matrix mineralogical composition. The porosity of the diatomite-based foams has been investigated by means of nitrogen-adsorption analysis and mercury-intrusion porosimetry. The experimental tests confirmed the presence of different porous architectures ranging over several orders of magnitudes, giving rise to complex systems, characterized by hierarchical levels of porosity. The presence of porosity of graded dimensions affects the final mechanical performances of the macroporous diatomite-based foams, while their mineralogical composition does not result to be affected by the addition of templates. For this reason, these materials find application in a broad range of high-tech fields such as energy, building, aerospace, filtration and bioengineering. In many of these applications, hierarchical structures with pores of distinct sizes are desired to achieve a proper balance between conflicting properties2. Generally, graded porous materials are materials in which the properties vary in a given direction 3 and this is the major advantage compared to other composite materials. Several processing routes have been explored to fabricate FGPMs  including templating, etching, freeze casting3, microwave sintering processes and compression-molding salt leaching4. In the last years, apart from the aforementioned conventional processes, the use of advanced techniques such as additive manufacturing, which permits the fabrication of complex geometries with high accuracy, to make graded porosity (with predicted distributed porosities) and compositions, still remains a challenge5. Furthermore, this is even more a challenge if the \u201cstarting material\u201d itself contains an intrinsic gradient porosity, obtained through physical and chemical blowing reactions and in specific environmental conditions7.The fabrication of foam materials with a porosity gradient  is widely desired from scientists in order to achieve an enhanced performance , mainly due to their low density, high strength and specific functional properties. FGPMs are innovative materials suitable for specific and advanced functions, in which a spatial gradation in structure and/or composition leads to tailored properties8. Accordingly, combining a new fabrication method (3D printing) with foaming techniques can be considered an innovative approach in the production of graded porous ceramic foams11. For example, the additive manufacturing (AM) of ceramic foams has become an attractive field of research that has the potential to disruptively change the way complex-shaped bodies are fabricated12. An even more enticing feature that has only recently started to be explored is the use of AM technologies to create materials with locally-tuned chemical compositions and intricate microstructures that are not accessible by conventional processing routes13. However, the use of this kind of processing technologies for ceramic materials is challenging because they are not easy to process considering their processing requirements (in terms of feedstock and/or sintering)12. Consequently, an indirect inverse-replica approach where (macro-) porous structures (templates) are printed and the ceramic slurry is cast into the template cavity, can overcome some of the limitationsin the physical, functional, and geometrical characteristics of components produced of the other AM techniques14 especially of the direct printing. In this way, in fact, templates and molds can be produced with high levels of accuracy using common polymers, taking advantage of freedom and shape possibilities provided by this technique. Moreover, the use of an indirect inverse-replica approach allows to avoid limitations, in terms of homogeneity and microstructure control of the final ceramic part, typical of other AM techniques, by filling directly the templates with suitable slurries15. Finally, in this case, the rheological properties required to the slurry are much less stringent compared to those required in the case of the direct printing in which the rheology of the inks definitely plays a key role. In particular, in this paper, stereolithography has been selected as 3D-printing technique for the production of the polymeric sacrificial templates needed to add macropores to diatomite based foams.Improvements in conventional processing methods and the development of innovative fabrication approaches are required because of the increasing specific demands on properties and morphology  for these materials, which strictly depend on the application considered17. Recently, diatomite was proposed as active support to obtain hierarchically porous nanocomposites with interesting application in environmental field20. The addition of other levels of porosity, with varying sizes, to the nano porosity already present and typical of diatomite22, leads to a bio-inspired final product with hierarchical and graded porosity that can be properly tuned according to the final properties desired for the material.So, taking inspiration from these considerations, the aim of this paper is the design and synthesis of sustainable functionally-graded porous ceramic materials with hierarchical and tailored porosity. These are obtained by starting from a natural raw material, diatomite, which is similar to seashells, trying to fully exploit its intrinsic features. Diatomites, opokas, tripolis, spongiolites, and radiolarites form a group of sedimentary silica rock consisting predominantly of opal and cristobalite. The interest shown in the study of siliceous rock is largely due to their useful properties26. CBCs are produced through a chemical reaction, consisting of a polycondensation of silica phases dissolved in a strong alkaline environment at low temperature, in contrast to traditional ceramic materials that are usually produced using fusion or sintering processes at elevated temperature. The complete production process of the diatomite-based foams is well-known and is accurately described in our previous works7, where it has also been demonstrated that the chemical\u2013physical properties and density of the resulted foams can be tailored using the proposed aproach. In this manner, the innovative manufacturing process proposed in the present paper guarantees the presence of both microscopic- and macroscopic-scale porosity, preserving at the same time the nanoscale porosity of the starting diatomite, leading to ceramic foams with graded and tunable porosity.For the design and the production of the solid ceramic matrix, starting from diatomite, the so-called alkali-bonded ceramics or chemically bonded ceramics (CBCs), have been selected as reference materials, considering their ever increasing number of possible applicationsThree different geometries of sacrificial templates have been selected and printed in order to investigate the influence of the template geometry on the final microstructure and porosity of the diatomite based foam Fig.\u00a0.Figure 1The printed models replicate exactly the computationally designed desired macro-scale pore sizes and architectures; the solids struts in the polymeric scaffold will constitute the macropores in the inverse replica foam structure. In particular, the template reported in Fig.\u00a0Each sacrificial template has been impregnated pouring the diatomite-based slurry in all their cavities Fig.\u00a0 and thenIt is possible to notice that no shrinkage and no evidence of swelling are present after the curing time Fig.\u00a0, indicatAll the lattices of the produced foams are effectively precise and accurate inverse replica of the 3D printed designed structures Fig.\u00a0. Lookinget al.7. It is also possible to identify the presence of a significant porous structure at nanometric scale ranging from 100 to 400\u2009nm. Moreover, looking at the highest magnifications .); at 38\u00b0 and 56\u00b0, there are two peaks related to sodium fluoride, NaF (JCPDS card n. 36\u20131455) that is originated from the reaction between catalyst and sodium. Finally, the peak at 47\u00b0 is related to the unreacted silicon (JCPDS card n. 75\u20130589).The XRD spectra of diatomite based foam without macroporosity addition and of the macroscopic foam sample STa_DHCF, are reported in Fig.\u00a0Looking at the spectra, it appears evident that the mineralogical composition of the diatomite based foam does not result to be affected at all by the template addition and the following removal process. The main crystalline phases present, in fact, resulted to be the same in both spectra and, consequently, it is not possible to identify any other new peak related to potential mineralogical phases due to the possible interaction between the polymeric template and the ceramic matrix.2 adsorption analyses performed on the STa_DHCF samples produced are reported in Fig.\u00a0The results obtained from the NAccording to the Brunauer classification of the adsorption isotherms, the foam sample showed isotherm belonging to the type II. Moreover, the STa_DHCF isotherm resulted to be characterized by the appearance of hysteresis, absent in the isotherm of the raw diatomite reported in Fig.\u00a0xp) obtained is equal to 82.05%.In order to characterize also porosity of bigger dimensions, the pore size distribution resulted from the Hg intrusion is reported in Supporting Information. It is possible to deduce that diatomite-based foam showed a monomodal pore distribution located at around 60\u2009\u03bcm. Moreover, the values of pores total volume fraction  within the diatomite-based matrix which was already characterized by the presence of nanoporosity due to the nanoporous nature of the same diatomite (2\u20135\u2009nm). The last level of hierarchical porosity (macroporosity) has been added to the diatomite-based foams using the 3D printing inverse replica method and choosing the stereolithography as the additive manufacturing technology for the production of the polymeric sacrificial templates needed. The indirect inverse replica approach allowed to overcome some of the limitations of other AM techniques, especially of the direct printing. In fact, in this process, templates and molds can be produced with high accuracy with common polymers, taking advantage of freedom and shape possibilities provided by this technique and avoiding limitations, in terms of homogeneity and microstructure control of the final ceramic part, filling the templates with suitable slurries. Moreover, in this case, the rheological requirements of the slurry are much less stringent compared to the case of the direct printing. The optical microscopy and SEM analysis confirmed the presence of different porous architectures and confirmed the absence of negative effects of the template addition on the foam microstructure. XRD analysis provided a further confirmation of the stability of the ceramic matrix and its final properties also after the removal of the sacrificial templates. Morever, it has been found out that the presence of this porosity of graded dimensions affects the mechanical properties of the macroporous diatomite based foams causing a toughening effect. Finally, ranging from three orders of magnitudes macro, micro and nano, it is possible to properly tailor the foam porosity according to the final properties desired.Oligomer: Allnex Ebecryl 8210 39.776%, Sartomer SR 494 39.776%Photoinitiator: Esstech TPO\u2009+\u2009 0.400%Reactive diluent: Rahn Genomer 1122 19.888%UV blocker: Mayzo OB\u2009+\u2009bis(5-tertbutylbenzoxazole)) 0.160%.Stereolithography has been selected as AM technology for the production of sacrificial templates with a defined and precise geometry, which corresponds to the negative of the geometry desired in the foam sample. In particular, sacrificial templates were designed and converted into stereolitography data using AutoCAD as CAD/CAM software. The model was then imported into a software (Autodesk Print Studio) that sliced it into layers and converted to a code path for the direct 3D printing. The printer used is an Autodesk Ember 3D-Printer, in which the tray holds the liquid resin that the machine turns into 3D parts. It has a clear window made of glass coated with PDMS (a member of the silicone family), through which 405\u2009nm ultraviolet light shines and cures each layer of the print that remains stuck on the printing head. The standard tray of the Ember printer needs at least 50\u2013100\u2009ml resin so that it can be used for printing. The window must allow oxygen permeation in order to form a layer that inhibits polymerization which prevents the print to stick to the window. The resin used to print templates was a standard resin  and it was supplied by the printer producers, who provided the following chemical formulation :2SiF6) has been added in order to promote the gelification of the entire system. Then, two different techniques of direct foaming have been coupled, one based on chemical reactions with gas and the other one based on a mechanical foaming6. The Sodium Silicate water solution (SS)  was provided by Prochin Italia Srl and has the following chemical composition: Na2O 8.15%, SiO2 27.40%. Si metal powder  and Na2SiF6 were supplied by Merck and Sigma-Aldrich respectively. Vegetable surfactant  in water solution (pH\u2009=\u20097) was supplied by Isoltech s.r.l. Italia and used as received after mechanical whipping. Taking into consideration the experimental results already obtained in our previous works7 the diatomite-based foam has been produced using the following mixture (wt%): 70% of SS, 8.65% of Na2SiF6, 21.3% of diatomite and 0.05% of silicon powder. Based on 100 parts of the above mixture 12 part of a \u201cmeringue\u201d type foam was added. The diatomite-based foam was prepared using simultaneously the silicon powder and the whipped protein as foaming agents, adding both of them to the starting mixture obtained homogeneously dry mixing first the powdered materials (diatomite and Na2SiF6) and then adding the SS solution.The produced slurry was poured inside the 3D printed sacrificial template and then the so obtained system, namely ST_DHCF, was cured at 40\u2009\u00b0C and at room humidity for 24\u2009hours. After curing, the consolidated foam, was thermally treated in order to burn out the polymeric template. The samples were slowly (0.5\u2009\u00b0C/min) heated in a furnace (Nabertherm HTC 03/15) at 500\u2009\u00b0C for 6\u2009hours, in order to avoid the formation of cracks and fractures inside the foam matrix, caused by the polymer burning out.An aqueous sodium silicate solution has been used as alkali activator for the consolidation process of the starting diatomite , flux-calcined diatomaceous earth, was provided by Celite France) and a catalyst  to evaluate macroporosity. Furthermore, the evaluation of pores total volume fraction (xp) was determined as follows:0 is the real density and \u03c1 was the bulk density of the foam, calculated as the mass of the foam divided by its apparent volume. The apparent volume has been geometrically calculated using a caliper (accuracy\u2009\u00b1\u20090.05\u2009mm), while the real density has been obtained performing a He picnometry .In order to investigate the morphology and microstructure and to verify the presence of all the different levels of porosity, optical microscopy  and SEM analysis  have been performed on all the produced samples. Also XRD analysis has been carried out to check if the template addition, especially the thermal treatment performed because of the template removal process, affected the mineralogical composition of the starting diatomite based foam. Porosity and specific surface were also evaluated by nitrogen adsorption analysis using a Nova 1000 machine . Density functional theory (DFT) method was used for determining pore size distribution in the range from micropores to mesopores3) using a universal testing machine  equipped with a 1 kN load cell. Measurements were performed in displacement control mode at a rate of 2\u2009mm\u2009min\u22121 until the sample fracture was detected in the force\u2013displacement plot. Three cubic samples have been produced and tested.Finally, compressive strength measurements were conducted on cubic ceramic foam samples (10\u2009\u00d7\u200910\u2009\u00d7\u200910\u2009mmSupplementary Information"}
{"text": "In comparison to WPT smokers, a larger increase in POX activity  and a smaller decline in DPPH activity  were found in non-smokers compared with WPT smokers. While these changes were slowly compensated within 1 hour after exhaustion, the activity of both markers was different from the pre-exercise values (p < 0.001). There was also a trend for UA concentration in non-smokers to be higher during the recovery period, with no significant difference between the groups (p > 0.05). It seems that WPT smoking is associated with negative effects on important human antioxidants and a diminished antioxidative response following acute exercise.Waterpipe tobacco (WPT) smoking is a public health problem with similar or even stronger effects than cigarette smoking. Although it appears to be associated with extensive oxidative stress, there is a limited number of studies on the oxidative effects of WPT smoking in stressful conditions. We, therefore, compared the responses of salivary flow rate (SFR), uric acid (UA) concentration, and peroxidase (POX) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) activities between WPT smokers and non-smokers following a bout of exhaustive aerobic exercise (AE). Twenty-three sedentary young women (age: 22.95 \u00b1 2.83 years) participated in this study, including 11 smokers (7.00 \u00b1 1.41 uses/week) and 12 non-smokers. All participants were required to perform the Bruce protocol treadmill test at an initial gradient of 10% at 1.7 mph, with increases of these parameters every 3 min until exhaustion. Unstimulated saliva samples were collected before, immediately after, and 1 hour after AE. WPT smokers showed lower SFR compared with non-smokers at all time points ( Tobacco smoking is one of the most common leading causes of death in the world because it is associated with many types of non-communicable chronic disease such as cancers, cardiovascular diseases, and respiratory problems ,2. It haCigarette smoking and waterpipe tobacco (WPT) or hookah smoking are two major forms of tobacco consumption by the adult population , with IrOn the other hand, although regular exercise training increases the activity of DNA repair enzymes involved in ROS-mediated damage , acute pTwenty-three young women, including 11 WPT users (5 to 9 uses per week) and 12 non-users, were selected from a total of 27 volunteers according to the entry criteria, i.e., no regular exercise training in the last 3 months, no current pregnancy, lack of any oral infection and acute disease, and no signs, symptoms, or history of cardiovascular, respiratory, and musculoskeletal diseases. All participants did not use drugs in the past month and supplements known to affect the results  in the past six months. WPT smokers also did not smoke tobacco products other than hookah . The demographic characteristics of participants are shown in A familiarization session was considered one week before the test day to familiarize participants with the AE protocol and collection of saliva samples. Descriptive characteristics of each participant including height, weight, and body fat percentage were also measured at the end of this session. Participants were instructed to maintain their current dietary routines during the following week. However, to control energy intake, each participant received both written and verbal instructions to record the type and portion sizes of her daily food (two working days and one weekend day). Participants were instructed to eat their last meal at 11 am along with 500 mL of water to reach the same hydration level and refer to the gym at 2 pm. After arriving, they brushed their teeth and rinsed their mouth with distilled water. A list of foods with high antioxidant properties  was provided to all participants, and they were not permitted to use any of these foods at least 24 h before the exercise test. Pretest saliva samples were collected before the exercise during the final 5 min of a 15 min rest on a comfortable chair. The second and third whole saliva samples were also obtained immediately and 1 h after the exercise, respectively. Participants did not eat or drink between the samplings. WPT smokers were also instructed to avoid smoking on the day before the testing. It should be noted that all the study procedures including data collection and familiarization were time-matched between the groups and performed in a temperature-controlled room (22\u201324 \u00b0C) to avoid circadian variations in salivary antioxidant status and exercise performance.Initially, all participants completed a 10 min warm-up period including 5 min walking on a treadmill with no gradient followed by lower limb stretching exercises. The graded AE protocol was according to the Bruce protocol treadmill test. The test started by walking at 1.7 mph (2.74 km/h) at a gradient of 10% for the first 3 min. Because the exercise workload was increased gradually, the AE protocol proceeded with running during next stages. The gradient of the treadmill was increased by 2% every 3 min, and the speed was 2.5, 3.4, 4.2, 5, 5.5, and 6 mph in the subsequent six stages . The proSaliva is the first biological fluid containing different antioxidant molecules and enzymes that protect the body cells against free radical oxidation . Therefo\u22124 mg/mL in 0.3 M phosphate buffer, pH 7.4) was then added, and alterations in absorption were recorded at 510 nm (\u0394A/min). One unit of enzyme activity was defined as an absorbance change of 0.001 per min under standard conditions . In WPT smokers, the flow rate immediately after AE was significantly lower than before exercise . However, the changes in non-smokers did not indicate a time effect (p = 0.068). Between-group comparisons revealed that the pre-exercise differences between WPT smokers and non-smokers (p = 0.013) as well as the differences immediately (p = 0.028) and 1 h (p = 0.014) after exercise were significant statistically. The changes in salivary flow rate during the 1 h recovery period after exhaustive AE are shown in p < 0.001; \u03b72 = 0.79]. On the basis of pairwise comparisons using the Bonferroni test, POX activity immediately after exercise was higher than before exercise in both WPT smokers  and non-smokers . These values then decreased near to pre-exercise levels, as POX activity 1 h after exercise was lower than immediately after exercise in both WPT smokers  and non-smokers . Moreover, in between-group comparisons, no statistically significant differences were found before exercise (p = 0.142) and 1 h after that (p = 0.085), whereas a significant difference was only observed immediately after exercise (p = 0.009). Alterations in salivary POX activity following exhaustive AE are shown in p < 0.001; \u03b72 = 0.51]. However, no significant differences were found between WPT smokers and non-smokers before exercise (p = 0.854), immediately after exercise (p = 0.615), or 1 h after exercise (p = 0.771). Although the mean concentration of UA was higher immediately and 1 h after AE in both groups compared to pre-exercise levels, these changes were only significant in non-smokers .In relation to salivary UA changes indicated in p < 0.001; \u03b72 = 0.76]. DPPH activity was decreased significantly in both WPT smokers  and non-smokers . However, the observed differences between the groups were found to be statistically significant only immediately after exercise (p = 0.004). As shown in As it has been well documented in previous research ,15,19,21We observed that saliva FR in WPT smokers before exercise and immediately and 1 h after an exhaustive AE was lower than in non-smoker women. These findings confirm that the presence of various toxic chemicals in hookah smoke may decrease saliva FR in smokers . As obseThe results obtained in the present study revealed that the responses of salivary POX activity and DPPH radical scavenging activity in WPT smokers were weaker than in non-smokers, particularly immediately after exercise. It was also observed that the responses of salivary UA could differ between WPT smokers and non-smokers during the recovery period after an exhaustive AE. In comparison to WPT smokers, non-smokers experienced a larger increase in POX activity (around 23% vs. 14%) and a smaller reduction in DPPH activity (around \u22128% vs. \u221215%) immediately after exercise. Moreover, UA concentrations in non-smokers tended to be higher than in WPT smokers following exhaustive AE, but the differences were not significant. 2O2 that are produced after acute physical exercise [Enzymatic and non-enzymatic antioxidants protect the body against the adverse effects of OS. Overall, the antioxidant capacity is determined by these antioxidative compounds . The salexercise ,28. Thusexercise . Likewisexercise . The imp2O2 and ROS) as a result of increasing exercise intensity to exhaustion may induce a vigorous stress on the salivary antioxidant defense system, specifically in WPT smokers, because UA concentrations were significantly increased only in non-smokers, and POX activity in non-smokers after exercise were significantly higher than in WPT smokers. These findings are in line with those of our previous experimental study [It is well established that extreme physical exercise is associated with high free radicals production, including that of superoxide, hydrogen peroxide, and hydroxyl radical, as a result of high oxygen consumption . In the al study  that revIn our experiment, DPPH analysis was used to assess peroxy radical clearance based on available studies ,31. DPPHIn conclusion, the present results allow us to assert that a bout of exhaustive AE on a treadmill induces OS in both non-smoker and WPT smoker young women. However, it seems that the responses of salivary antioxidative markers including POX activity and the percent of scavenging activity against DPPH radical after the exercise were weaker in WPT smokers. Therefore, contrary to popular belief, these findings provide experimental evidence that WPT smoking presents health risks similar to those of cigarette smoking. Further research in this field is needed to compare the effects of different forms of tobacco smoking on the responses of human antioxidants following acute exercise."}
{"text": "Scientific Reports 10.1038/s41598-018-37017-4, published online 10 January 2019Correction to: This Article contains an error in Figure 4, where the key was omitted. The correct Figure 4 appears below as Fig."}
{"text": "Scientific Reports 10.1038/s41598-018-25967-8, published online 15 May 2018Correction to: This Article contains an error in Figure 9, where Figure 9b is a duplication of Figure 9a. The correct Figure 9 appears below as Figure"}
{"text": "Scientific Reports 10.1038/s41598-018-29865-x, published online 20 August 2018Correction to: In Figure 1C, Sites 1 and 2 are incorrectly labelled. The correct Figure"}
{"text": "Scientific Reports 10.1038/s41598-018-22504-5, published online 09 March 2018Correction to: 18O (blue) curves. The correct Figure 4 appears below as Figure\u00a0In Figure 4 the Ostracod curve does not align with \u03b4D (orange) and \u03b4"}
{"text": "Scientific Reports 10.1038/s41598-018-19421-y, published online 05 February 2018Correction to: In Figure 4c, the heading \u201cAutophagic ac\u201dvity analysis\u201d should read \u201cAutophagic activity analysis\u201d.Additionally, the \u201c+\u201d and \u201c\u2212\u201d symbols are reversed in the displayed Western blots and graphs.The correct Figure 4 appears below as Figure"}
{"text": "Anxiety disorders are common among children and adolescents; almost one-third of this population has an anxiety disorder. The most common anxiety disorders in this population are specific phobia (19.3%), social anxiety disorder/ social phobia (9.1 %), and separation anxiety disorder (7.6 %). Pediatric anxiety disorders are often associated with poor psychosocial functioning, academic underachievement, learning difficulties, substance abuse, relationship problems, and suicide behaviors. Psychotherapy, particularly cognitive behavioral\u00a0therapy as a stand-alone treatment or in combination with medication, is found to be efficacious in the treatment of various anxiety disorders. The early recognition and treatment of anxiety disorders result in better long-term outcomes in children and adolescents. This article summarizes the evidence-based pharmacologic treatments for anxiety disorders in youth, including social anxiety disorder generalized anxiety disorder, separation anxiety disorder, and panic disorder. Anxiety disorders are the most common psychiatric disorders in adolescents and young adults with a lifetime prevalence of up to 30%. In a national survey, lifetime prevalence rates for adolescents were 31.9 % for any anxiety disorder, 19.3 % for specific phobia, 9.1 % for social anxiety disorder (SAD), 7.6 % for separation anxiety disorder, 2.3 % for panic disorder, 2.4 % for agoraphobia, and 2.2 % for generalized anxiety disorder\u00a0(GAD). In adolescents, anxiety disorders are more prevalent among girls than boys, with an estimated ratio of 2:1 [Anxiety disorders usually have a chronic and persistent course as suggested by the prevalence rates throughout the life span . The perAnxiety disorders are associated with significant functional impairment and the development of the co-morbid psychiatric disorder in adulthood. They also result in a higher risk of cognitive impairment, academic under-achievement, and social dysfunction . MoreoveIn this review article, we have included the pharmacological treatment for the disorders that are currently recognized under the umbrella of anxiety disorder in the current Diagnostic and Statistical Manual of Mental Disorders (DSM)- 5. The existing literature has been assessed for the efficacy and safety of existing treatment options for SAD, GAD, panic disorder, and separation anxiety disorder. Selective serotonin reuptake inhibitors (SSRIs) are frequently prescribed for the treatment of anxiety disorders in youth. Only a few medications are approved by the Food and Drug Administration (FDA) to treat anxiety disorders in children and adolescents. Table A combination of treatments is often more efficacious. The results of the Child/ Adolescent Multimodal Study (CAMS) show that a combination of CBT and sertraline (80.7%) was better than CBT alone (59.7%) or sertraline alone ( 54.9%) to treat non-OCD anxiety disorder  .\u00a0MethodsIn August 2018, the PubMed and Scopus were systematically searched for relevant publications focused on pharmacological treatments of SAD, GAD, panic disorder and separation anxiety disorder in humans. Two independent reviewers performed the manual search of references and relevant articles for included studies. Search results from these databases were imported to Endnote \u00d77  to remove any duplicates. Two independent reviewers performed title and abstract screening (when available) followed by the full-text screening of the articles by selecting case reports, case series, open-label trials, and randomized controlled trials (RCTs). The abstract-only articles, conference papers without original data, reviews, theses, posters, book chapters, editorials, letters, and commentaries were excluded. No restrictions on language, country, publication year, age, gender, or ethnicity of patients were applied. This article provides a summary of the evidence-based pharmacologic treatments for different anxiety disorders in children and adolescents based on the available data.\u00a0Social anxiety disorder1. SertralineIn an open-label trial of 8 weeks, the efficacy and safety of sertraline were assessed among 14 participants, ages 10-17 (eight boys and six girls), with SAD. Sertraline was initiated at 50 mg/day with dinner, and it was titrated at 50-mg increments every other week to reach a maximum dose of 200 mg/day by week 6, depending on the clinical response and tolerability. At the end of the study, the doses were 200 mg/day in one, 150 mg/day in five, 125 mg/day in one, and 100 mg/day in four participants. The average daily dose of sertraline was 123.21 \u00b1 37.29 mg/day at the end of the study. The Clinical Global Impression-Improvement scale (CGI-I) was used to measure improvement relative to baseline\u00a0and the Clinical Global Impression - Severity scale (CGI-S) was used to measure severity. The Social Phobia and Anxiety Inventory for Children and a standardized behavioral avoidance test were used to assess for anxiety. By the end of the eight weeks, the independent evaluator reported that 5/14 (36%) subjects were classified as treatment responders or partial responders 4/14 (29%). However, psychiatrists rated 3/14 (21%) subjects as treatment responders and 7/14 (50%) as partial responders. The most common side effects were drowsiness, nausea, diarrhea, jitteriness, restlessness, headache, trouble sleeping, aggression, and skin rash in descending order of frequency. Most of these side effects were transient and resolved with a reduction in the dose of sertraline. Sertraline was discontinued in one patient due to severe gastrointestinal side effects [2. EscitalopramThe efficacy and safety of escitalopram were assessed in a 12-week open-label trial among 20 youth (10-17 years old) with SAD. Escitalopram was started at 5 mg/day in the first week, and then it was increased to 10 mg/day in the second week. The dose was maximized to 20 mg/day at 5-mg increments depending on the clinical response and tolerability. At the end of the study, 12 (60%) patients were taking 10 mg/day of escitalopram, four (20%) were taking 15 mg/day, and another four (20%) were taking 20 mg/day. The clinical efficacy was assessed by a change in CGI-I scores indicating that 13 of 20 (65%) patients achieved a score of 2 indicating a response to treatment. All secondary outcomes and quality of life measures showed improvements from baseline to week twelve, with effect sizes ranging from 0.9 to 1.9. The average daily dose of escitalopram over the 12-week open-label was 12.65 \u00b1 2.08 and at the endpoint was 13 \u00b1 4.1 mg. Somnolence, insomnia, flu symptoms, and appetite change were frequent, but transient\u00a0side effects. Tremors were reported in one patient and were resolved on discontinuation [3. ParoxetineIn a 16-week long, double-blind RCT, the efficacy and tolerability of paroxetine were evaluated for SAD among 322 children and adolescents (ages 8-17 years old). Participants were randomized to receive paroxetine (10-50 mg/day) or placebo. Paroxetine was started at 10 mg/day and was titrated to a maximum dose of 50 mg/day at 10 mg-increments per week. The average dose for paroxetine was 32.6 mg/day for all patients (26.5 mg/day for children and 35.0 mg/day for adolescents) at the end of the study. The CGI-I was used to assess for efficacy at the endpoint, a score of \u2264 2 indicating a response. Safety was evaluated at each visit by monitoring vital signs and adverse effects. About 95.6% of participants were categorized as high risk. Further breakdown yielded 45.1% were \u201cmoderately ill\u201d, another 38.5% were \u201cmarkedly ill\u201d, and 12% were \u201cseverely ill\u201d by using CGI-S with higher comorbidity among the paroxetine group. About 77.6% of participants receiving paroxetine were defined as responders compared to 38.3% receiving a placebo. The clinical benefits of paroxetine were noticeable within the first four weeks of treatment. At the endpoint, participants receiving paroxetine (47.2%) were more likely to achieve remission compared to placebo (13.3%). The most common adverse effects were insomnia, decreased appetite, and vomiting. These side effects were mild and transient. Behavioral side effects included nervousness, hyperkinesia, asthenia, hostility, somnolence, insomnia, agitation, manic reaction, and emotional liability. Four patients receiving paroxetine reported suicidal ideations with one patient displaying self-harm behaviors .4. Tandospirone1A receptor with anxiolytic properties. Tandospirone was started at a fixed dose of 10 mg twice daily for the first week with a gradual increase to three times/day. The maintenance dose was between 20 to 60 mg/day depending on the efficacy and safety. Sertraline was initiated at 25 mg/day during the first week and was titrated to 50 mg/day in the second week. The mean dose was 35.14\u00b17.75 mg for tandospirone and 89.58\u00b130.47 mg for sertraline at the end of the study with a maintenance dose of 25-200 mg/day. The efficacy was assessed by Hamilton Anxiety (HAM)-A scores and change from baseline in CGI-I scores. Vital signs and adverse events were assessed at baseline and at weeks two, four, six, and eight by using the Treatment-Emergent Scale. The response rates were 48.6% for tandospirone and 55.6% for sertraline on the CGI-I scale. By using the HAM-A scale, the rate of response was 37.1% for tandospirone and 41.7% for sertraline. The most common side effect was drowsiness (11.4%), fatigue, and decreased appetite (8.6%) for tandospirone and decreased appetite (13.9%) followed by nausea (11.1%) for sertraline. The study demonstrates the efficacy and safety of tandospirone for adolescents, showing it to be as effective as sertraline for treating SAD [Huang and colleagues examined the efficacy and safety of tandospirone compared to sertraline in an open-label trial of eight weeks among 71 adolescents (ages 14-21 years) with SAD. Tandospirone is a partial agonist of serotonin ting SAD .5. CitalopramChavira and colleague assessed the efficacy of citalopram in a 12-week long, open-label trial along with psychoeducational treatment. All 12 participants (ages 8-17 years old) received 12 weeks of citalopram treatment and eight brief CBT-oriented counseling sessions (15 minutes each). Of these 12 participants, two participants dropped out due to nausea, lightheadedness, or concentration problems. Citalopram was started at 10 mg/day, and it was increased at 10-mg\u00a0increments with a maximum dose of 40 mg/day over 12 weeks. About 83% of the participants were reported as being very much improved (41.7%) and much improved (41.7%) on the CGI-I scale. The treatment was well-tolerated without any side effects during the course of study .6. CannabidiolA double-blind randomized trial tracked the effects of a simulation public speaking test (SPST) among 12 healthy control participants and 24 patients with SAD who received a single dose of Cannabidiol (CBD) or placebo before the test. After careful screening, a total of 24 SAD subjects who never received any treatment for SAD were randomized to either CBD 600 mg (n=12) and placebo (n=12). The control subjects were assigned to perform the SPST without any medical intervention. CBD was given before the test. Psychological assessment was conducted by using the Visual Analogue Mood Scale (VAMS) and Negative Self-Statement scale (SSPS-N), and physiological measures including blood pressure, heart rate, and skin conductance. This study reported that pretreatment with CBD resulted in improvement in anxiety, cognitive impairment, and discomfort in their speech performance. It also resulted in a significant reduction of alertness in their anticipatory speech, compared to the placebo group. There was no significant difference observed among the healthy control participants .7. Several Serotonergic AgentsMancini and colleagues reported the efficacy of different serotonergic agents for social phobia among seven participants. The age range for these participants was 7 to 18 years  with a mean age of 14.4 years. Of the seven patients, four had comorbid anxiety and mood disorder. The mean age of onset for social phobia was six years. These patients were treated with paroxetine (n=5), sertraline (n=1), and nefazodone (n=1). The response to treatment was assessed by retrospective chart review performed by treating physicians. All patients responded to serotonergic agents, and their anxiety improved with minimal side effects. The most common side effects reported by three participants were transient diarrhea, minor visual accommodation difficulty, and mild somnolence. The dose range was 5-80 mg for paroxetine, 175 mg for sertraline, and 400 mg for nefazodone [8. FluoxetineIn an RCT, the effectiveness of fluoxetine, placebo, and social effectiveness therapy (SET-C) was studied among children and adolescents (age 7-17 years) with social phobias . In this9. Venlafaxine Extended-Release (ER)A 16-week placebo-controlled RCT evaluated the efficacy, safety, and tolerability of venlafaxine ER in 293 youth aged 8-17 years with SAD. Participants were randomized at baseline visit to venlafaxine ER or placebo in a double-blind manner. Venlafaxine ER was started at 37.5 mg /day and titrated up to 75 mg/day depending upon weight and clinical symptoms. The efficacy was assessed by Social Anxiety Scale-child or adolescent version (SAS-CA), and responder assessment was performed by CGI-I scores at each visit. Safety was assessed by measuring weight, heart rate, temperature, and electrocardiogram (EKG) during the visits. About 35% of participants dropped out of the study due to lack of efficacy and 4% due to the adverse effects. The average daily dose of venlafaxine ER during the active treatment phase was 141.5\u00b147.5 mg for > 0 days of treatment among 140 participants and 155\u00b139.0 mg for > 112 days of treatment among 54 participants. The average daily dose was given at 2.6 mg/kg to 3 mg/kg.A statistically significant benefit was observed for venlafaxine ER on the SAS-CA compared to placebo with a number needed to treat of five. About 56 % of venlafaxine ER was scored 1 (very much improved) or 2 (much improved) on the CGI-I scale compared to 37% among the placebo group. About 90% of participants reported treatment-emergent adverse effects which were mild-to-moderate in severity. These side effects resolve over the course of study with continued treatment. The most common side effects were anorexia, nausea, weight loss, dizziness, and nervousness. Three patients developed suicidality with no completed suicide and one patient developed an episode of hypomania with venlafaxine .10. BuspironeIn an open-label study of treatment of 25 subjects with pediatric social phobia, buspirone  showed some efficacy in the treatment of the phobia. Only three children improved enough to continue the medication after study completion (nine weeks) .Generalized anxiety disorder1. SertralineRyan et al. evaluated the efficacy of sertraline (n=11) compared to placebo (n=11) in children and adolescents (ages 5-17 years with mean age 11.7+3.9 years) with GAD in a 9-week long, double-blind RCT. Primary outcome measures were evaluated by the HAM, and the CGI-I and -S scales. Sertraline was started at 25 mg/day, and then it was titrated to 50 mg/day from week two to nine. The HAM total, psychic and somatic factor scores showed a significant benefit for sertraline from week four to the end of the study. About 90% of patients taking sertraline improved on the CGI scale compared to 10% in placebo. However, only two patients taking sertraline markedly improved. Dry mouth, drowsiness, leg spasm, and restlessness were the adverse effects common in the sertraline group while dizziness, nausea, and stomach pain were observed in the placebo group .2. DuloxetineThis 10-week long double-blind RCT of duloxetine  and placebo (n=137) was followed by an 18-week open-label phase (dose 30-120 mg). This study included children and adolescents (ages 7-17 years) with GAD who received duloxetine or placebo in a flexible-dose manner depending on the tolerability and response. The mean daily dose was 53.6 mg during the initial phase, followed by 66 mg (continued on duloxetine) and 60.4 mg (transition from placebo) during the open-label phase. Efficacy measures included the Pediatric Anxiety Rating Scale (PARS), CGI-S, and Children\u2019s Global Assessment Scale (CGAS), while safety was assessed by the Columbia Suicide Severity Rating Scale (C-SSRS). The duloxetine group showed significant improvement in PARS, CGI-Severity, and CGAS in terms of symptomatic response, remission and functional remission (CGAS >70% improvement). The mean improvement was statistically significant in the duloxetine group, while the placebo group showed significant mean improvement in adolescents. During the second phase, there was a significant mean improvement in GAD symptoms in the group of duloxetine who transitioned from placebo.Severe adverse events in the acute phase included suicidal ideation and self-injurious behaviors in one subject with a history of depression and suicidal ideations. In the open-label phase, the duloxetine group manifested acute psychosis, bipolar disorder, suicidal ideations, tonsillitis, and adenoiditis. A patient who suffered from apathy and later developed suicidal in the tapering phase was eliminated from the study. In the acute phase, seven patients in duloxetine and six in placebo discontinued due to the adverse events which include nausea. Duloxetine group exhibited side effects in a 28-week period resulting in discontinuation of the treatment included nausea, suicidal ideation, vomiting, abdominal pain, upper dyspepsia, self-harming thoughts, bipolar disorder, abnormal behavior, irritability, fatigue, initial insomnia, somnolence, apathy, vaginal bleeding, blepharospasm, pharyngeal inflammation, and severe adenoiditis. When duloxetine was tapered, headache and upper abdominal pain were observed. Duloxetine caused a three-fold increase in alanine transaminase which became normal after discontinuing it.During the open-label phase, suicidal ideations with uncompleted suicidal attempts were reported\u00a0seen in two patients. In comparison to baseline , three patients in each group (receiving duloxetine from the start and one switched from placebo to duloxetine) manifested treatment-emergent suicidal ideations. Duloxetine group exhibited more adverse events like nausea, vomiting, decreased appetite, oropharyngeal pain, cough, dizziness, and palpitations. In the acute phase, duloxetine showed a greater increase in pulse rate than the placebo group; also weight loss was seen in the duloxetine group, while weight gain was observed in the placebo group. In the second phase, the duloxetine group (receiving duloxetine from the beginning) showed a decrease in systolic and diastolic blood pressure, pulse, and increase in weight. The other group, which shifted from placebo to duloxetine, showed a mild increase in pulse, blood pressure, and weight .3. Venlafaxine ERRynn et al. reported the results of two RCTs of eight weeks (each and tapering period of 14 days) evaluating the efficacy of venlafaxine ER (n=157) compared placebo (n=163) in children and adolescents of age 6-17 years with GAD . The priAsthenia, anorexia, pain, and somnolence were common adverse effects in the venlafaxine group, twice as high as for placebo. Somnolence was common among adolescents. Changes in height, weight, pulse rate, blood pressure, and cholesterol level were significant with venlafaxine. Initially, 25% of placebo and 24% of the venlafaxine group dropped prematurely. Two subjects in the venlafaxine group discontinued treatment due to aggressive behaviors. On subgroup analysis, primary outcome measure improvement from baseline was observed both in children and adolescents. No gender difference in baseline severity existed. Tapering-related adverse events were 32% in the venlafaxine group and 16.5 % in the placebo group with dizziness more common with venlafaxine. Serious adverse events were observed in the first study with two participants dropping out of the study: one boy had suicidal ideation 48 hours after the last full dose, and one girl in the placebo group attempted suicide. In the second study, a girl showed agitation, and confusion resulted after venlafaxine taper. EKG changes like abnormal right atrial rhythm with short PR interval; and left atrial rhythm, rightward axis, and non-specific ST abnormality were observed in the venlafaxine group. These EKG changes were not significant enough to cause the withdrawal of participants.4. AlprazolamIn an RCT of 30 children with the overanxious disorder (n=21) and avoidant disorder (n=9) one week of placebo\u00a0administration was followed by either placebo or alprazolam. The medication was tapered with placebo substitution in week five, and all the subjects received placebo on week six. There was a strong treatment effect in both the alprazolam and placebo group. Alprazolam was superior to placebo only on clinical global ratings, and the difference was not statistically significant .Panic disorder1. ParoxetineIn a retrospective chart review of 18 participants ages 7-14 years , Masi et al. reviewed the efficacy and safety of paroxetine for panic disorder. Participants with medical causes of panic disorder were excluded from the study. Paroxetine was started with an initial minimal dose of 8.9\u00b1 2.1 mg/day and was gradually titrated up to 40 mg/day depending upon clinical response and tolerability. CGI-I scale was used to assess clinical response among participants and adverse effects were monitored retrospectively at each follow-up visit. These patients were followed for a period of 11.7\u00b1 8.3 months (range 2-24 months). About 83.3% (15 out of 18 patients) reported an improvement in symptoms of panic disorder on the CGI scale (scores of \u2264 2). It took 21.87\u00b1 7.1 days (range 10-30 days) for these participants to respond, with the average dose at the initial response of 22.00\u00b1 7.75 mg/day. The average dose for participants with a response was 22.67\u00b1 9.61 mg/day (range 10-40 mg) at the end of the study. Paroxetine was well-tolerated with mild and transient side effects. The most common adverse effects were nausea, tension, agitation, sedation, insomnia, palpitations, and headache .2. ClonazepamBiederman investigated the efficacy of clonazepam for anxiety disorder with panic-like symptoms among three pre-pubertal children . Two of Separation anxiety disorder1. ImipramineIn the second phase of a six-week-long double-blind RCT, children (n=21) with age range of 6-11 years (mean age 9.5\u00b10.8 years) were assigned to either imipramine (n=11) or placebo (n=10) group. The participants were diagnosed with a separation anxiety disorder who failed to respond to an initial psychotherapeutic intervention. Of these, only half of the subjects improved. The imipramine was started at 25 mg/ day for three days, after which it was increased to 50 mg for next four days, and subsequently increased gradually with the maximum dose of 5 mg/kg/day. By the end of the study, the dose of imipramine was 75-275 mg /day with a mean dose of 153 mg/ day (4.67 mg/kg). EKG was obtained at the initial visit, and then with each dose increment after the first increase to 50 mg, and no significant changes were observed. Pre-study assessments included the Wechsler Intelligence Scale for Children-Revised, Diagnostic Interview Schedule, problem list, Conner\u2019s Questionnaire by parents and teachers, and Children\u2019s Manifest Anxiety Scale for parents and youth. The global rating of improvement, which was rated by the child psychiatrists, parents, and teachers, was also performed. The assessment measures performed by parents, teachers and children self-rating failed to show any significant differences between the two groups. About 40-60 % of subjects improved overall without any difference between the two groups. Side effects were reported by 8/11 subjects in the imipramine group and 2/9 in the placebo group. Imipramine group had 12 moderate to severe complaints out of 18 total complaints, with the irritability and anger most frequent side effect. None of the side effects were serious enough to warrant a dropout .2. ClonazepamIn a double-blind cross-over study of 15 patients with separation anxiety disorder, subjects were treated with clonazepam (up to 2 mg /day ) and placebo. There were no significant treatment differences between the two groups. The authors did not support the use of clonazepam for separation anxiety disorder .DiscussionCurrent literature suggests a role of pharmacotherapy in the management of moderate to severe anxiety. An SSRI is considered as the first-line of treatment with an option of switching to another SSRI or Serotonin and norepinephrine reuptake inhibitors (SNRIs) in patients with persistent anxiety . HoweverA complete bio-psycho-social assessment, a working diagnosis, physical examination, and a comprehensive treatment plan that includes tangible goals is necessary before starting medications for anxiety disorders. SSRIs are the most common and efficacious treatment for anxiety disorders in youth. A combination of psychotherapy and medications usually results in better treatment outcomes in several anxiety disorders."}
{"text": "Recognizing a person in a crowded environment is a challenging, yet critical, visual-search task for both humans and machine-vision algorithms. This paper explores the possibility of combining a residual neural network (ResNet), brain-computer interfaces (BCIs) and human participants to create \u201ccyborgs\u201d that improve decision making. Human participants and a ResNet undertook the same face-recognition experiment. BCIs were used to decode the decision confidence of humans from their EEG signals. Different types of cyborg groups were created, including either only humans (with or without the BCI) or groups of humans and the ResNet. Cyborg groups decisions were obtained weighing individual decisions by confidence estimates. Results show that groups of cyborgs are significantly more accurate (up to 35%) than the ResNet, the average participant, and equally-sized groups of humans not assisted by technology. These results suggest that melding humans, BCI, and machine-vision technology could significantly improve decision-making in realistic scenarios. Visual search is the process of looking for an item of interest in a scene. Everyone engages in visual search many times every day . For exaThese difficulties are present also when searching for a target face in pictures of crowded environments, a critical task in security and surveillance . This chface detection) [face identification) [Searching for a target face in a crowded environment is a challenging task also for fully-automated computer-vision systems , albeit tection) . Secondlication) . If the ication) . In prinication) , especiaication) , 15.worse than individual ones. For example, when the opinions of group members are too correlated [To partially address the limitations of the human visual system, people can work together to search a scene. When making perceptual decisions, groups are generally more accurate than their members . This \u201cwrrelated , when a rrelated , or whenrrelated , 23.The strategy used to aggregate individual opinions critically influences performance in group decision making. In the case of binary judgements, standard majority is the most straightforward strategy to use. However, in many circumstances it is suboptimal . A well-In principle, decision confidence could be more objectively assessed via means other than self-reporting. There is ample evidence that physiological correlates of decision confidence exist. For instance, several brain-activity patterns as recorded via electroencephalography (EEG) are known to provide information about decision confidence  and the error related negativity , 32), anhybrid Brain-Computer Interfaces (hBCIs) to test whether it is indeed possible to decode the decision confidence of single users from their EEG signals, RTs and other physiological measurements. In all cases, the hBCI relies heavily on machine-learning technology, which is used to specialise the interface to each particular user and to learn to predict the probability that, in a certain trial, the user made a correct decision. The confidence estimates provided by the hBCI were then used to weigh individual opinions and obtain group decisions. That is, our hBCI did not make automated decisions, it only produced weights for integrating behavioural decisions provided by the operators. These hBCI-assisted group decisions were then compared to decisions obtained by other forms of opinion aggregation. After initial tests of our system with a simple visual-matching task [In recent research, we developed a series of ing task , which sing task , 40, wheing task , which iing task . While tcombine these together. At least in principle, within such \u201ccyborgs\u201d, automated algorithms could compensate for the weaknesses of humans and vice versa, thereby producing more reliable and accurate decisions. For example, in a situation where most users in a group missed a target face , both traditional and hBCI-assisted groups would likely make a wrong decision. Conversely, a machine-vision algorithm might still identify the target with confidence. So, if it was included as a member in a group of humans, the machine-vision algorithm could potentially swing the group decision towards a correct decision.Given advantages and limitations of both humans  and computer-vision systems, one may wonder whether we could usefully search an image of a crowded, indoor environment and decide whether or not it contained a target face, a more challenging task than the face identification task used in [This study explores the benefits and drawbacks of this idea. Prior research has shown that aggregating decisions from a superrecognizer and a deep convolutional neural network in a face identification task led to significant more accurate decisions than two superrecognizers . We used used in .This research received UK Ministry of Defence Research Ethics Committee  and University of Essex ethical approval in July 2014. All participants signed an informed consent form before taking part in the experiment.Ten healthy participants  with normal or corrected-to-normal vision and no reported history of epilepsy took part in the experiment. Each participant was paid GBP 16 for taking part in the experiment plus an additional amount of up to GBP 4 proportional to the participant\u2019s percentage of correct decisions. The latter was used to encourage participants to focus on the task and achieve maximum performance.Participants were comfortably seated at about 80 cm from a LCD screen (refresh rate 60 Hz) in an experimental room with normal white illumination .The experiment consisted of six blocks of 48 trials. At the beginning of each block, participants were shown a cropped face image of the person to be considered as a target for that block and were asked to memorise it before starting the presentation of the trials in the block. Each trial  started The images used as stimuli were obtained from the sequences P2L_S5 and P2E_S5 of the ChokePoint video dataset . The twoIn each block of the experiment, the images selected from a given sequence (1 or 2) and viewpoint  were presented. All six possible combinations of sequences and viewpoints have been tested. The order of presentation of the images in a particular block was the same for all participants. However, the order of presentation of the blocks was pseudo-randomly chosen for each participant, for counterbalancing reasons. In each block 25% of the images contained the target.Briefing, preparation and task familiarisation  took about 45 minutes, while the experiment took roughly 25 minutes. The images used as stimuli during familiarisation were pseudo-randomly selected for each participant from the dataset used in the experiment.Neural data were recorded at 2048 Hz from 64 electrode locations with a BioSemi ActiveTwo EEG system. Each channel was referenced to the average voltage recorded from the electrodes placed on each earlobe and band-pass filtered between 0.15 and 40 Hz. Artefacts caused by ocular movements were removed with a standard correlation-based subtraction algorithm. From each trial, response-locked epochs starting 1 s before the user\u2019s response and lasting 1.5 s were extracted from the EEG data, baseline corrected with the average voltage recorded in the 200 ms before the stimulus onset, and downsampled to 32 Hz. Each epoch was then associated to the class \u201ccorrect\u201d (confident) or \u201cincorrect\u201d (not confident) depending on whether the decision made by the participant in that trial was correct or not, respectively.Common Spatial Pattern (CSP)  filterinp, we trained a logistic regression classifier with L2 penalty to predict the decision confidence wp, i.e., the probability of a particular decision being correct. Hence, each participant had his/her own BCI able to assess the objective decision confidence of the decision maker. We used 8-fold cross-validation to ensure that the results were not unfairly affected by overfitting. In each fold, 252 trials were used as training set and the remaining 36 as test set, to evaluate the groups\u2019 performance.For each participant face_recognition Python library v1.2.3 available at https://github.com/ageitgey/face_recognition) to decide whether or not the target person was present in each image used as a stimulus in our experiment. The ResNet was formed of 29 convolutional layers  , and hae et al. , where iface_recognition library. The image of each face was then mapped to a 128-dimensional vector space where images of the same person are likely to be near to each other . Then, the ResNet computed the difference between the encoding of each face and the encoding of the target face, normalised using the Frobenius norm. If any extracted face had a difference smaller than a threshold, t, the stimulus was labelled as \u201ctarget\u201d, otherwise it was labelled as \u201cnontarget\u201d. The threshold was set to the value that maximised the accuracy on the training set using 8-fold cross-validation .In each trial of our experiment, the ResNet firstly scanned the input image to identify and extract individual faces using a pre-trained face-detection model integrated in the wResNet, was computed as follows:d is the difference between the encodings of the face extracted from the image and the target face . If more than one face was extracted from an image, only the face with the minimum d was considered. t. This formula ensures that the ResNet has substantially bigger confidence weights when the difference between the target face and the stimulus is very small or very big (confident decisions) than when it is close to the threshold (not confident decisions).In each trial, the confidence weight of the ResNet, wResNet were set to 0, so that, in cyborg groups, the ResNet vote would be ignored and only the humans influenced the decision.Trials where the ResNet did not identify any face in the image were labelled as \u201cnontarget\u201d, given the prevalence of nontarget stimuli in the training set . In such trials, the corresponding weights m = 2, \u2026, 10 were formed off-line by combining the N = 10 participants in all possible Groups of size m in a trial was given by:dp = \u22121 if user p decided the trial represented a \u201ctarget\u201d and dp = + 1 for a \u201cnontarget\u201d, wp is the corresponding weight, and rsign a randomising sign operator which returns +1 (\u201cnontarget\u201d) if its argument is positive, \u22121 (\u201ctarget\u201d) if it is negative, and randomly chooses between +1 and \u22121 if its argument is 0. Traditional group decisions were obtained using standard majority, i.e., wp = 1 for all trials and users, while BCI-assisted group decisions used the decision confidence estimated by the BCI from the brain signals as weight wp.Traditional and BCI-assisted groups were composed only by human participants  of 12.0%. We have also looked at the ability of each participant of identifying nontargets and targets by computing their specificity and sensitivity as:The participants\u2019 average specificity was 77.4% (SD = 15.8%) and their average sensitivity was 56.9% (SD = 10.9%). Hence, they recognised nontarget stimuli much better than target ones.t , but quite weak in detecting targets  compared to the average participant. Since target stimuli appeared only in 25% of the trials, the ResNet could still achieve high accuracy, which was the metrics used to choose the threshold t see . In two t see . Hence, t could achieve better sensitivity. When choosing t in order to maximise the F1 score in cross-validation instead of the accuracy, the ResNet reduced its accuracy (82.6%) and specificity (94.0%), and increased its sensitivity (48.6%). The average value of the threshold t over the eight folds was 0.559, while it was 0.526 when maximising accuracy. Similar results were obtained when maximising Cohen\u2019s kappa to optimise t . In all cases, the sensitivity of the ResNet was worse than that of the average participant.One may wonder whether a ResNet using a different metrics to optimise the choice of the threshold To exhibit error-correction capability, groups require their members to make errors in different trials . In factParticipants had performance better than random and, therefore, their decisions were the same in the majority of the trials. However, the Hamming loss between two participants ranged from 12.5% and 47.2% . This suggests that the pairs exhibited error-correction capability in a large percentage of trials.p = 0.646). Given its high performance, of course the ResNet had minimum Hamming loss with the participants with the highest accuracy . Yet, even when paired with them, the pair still exhibited error-correction capability in more than 12% of the trials.When looking at the Hamming loss between the ResNet and each participant, we found similar results. In this case, the Hamming loss ranged between 12.2% and 48.3% . A Wilcoxon signed-rank test showed no differences between the Hamming loss of humans and ResNet  between corresponding nodes. Then, utilising a graph layout algorithm one can flattened the graph to obtain an intuition of similarities and differences in decision-making behaviour. This diagram as well as the previous analyses show how the ResNet behaves differently from virtually all other participants.m) using the five different methods for making group decisions analysed in this study . Since ResNet-assisted groups are based on standard majority, the smaller the group, the bigger the influence of the ResNet on the group decision. Indeed, the biggest improvement brought by ResNet-assisted groups over traditional groups occurs for m = 2, where the addition of the ResNet allows us to turn most of the ties generated by the two humans into correct decisions. This results in a boost in accuracy of more than 10% over traditional pairs. For bigger group sizes, the difference in average performance between ResNet-assisted and traditional groups gradually diminishes. We should expect the performance of ResNet-assisted groups to eventually converge to that of traditional groups. Yet, even for the larger group sizes considered in our study, ResNet-assisted groups were still better that traditional groups by 2\u20133%.Of course, one may expect methods which use some form of confidence estimation on a decision-by-decision basis to break ties, such as the BCI-assisted groups , but less accurate than BCI-and-ResNet-assisted groups. This indicates that, contrary to what is suggested in the literature for human groups [Interestingly, when using groups of BCI-and-ResNet-assisted humans . Conversely, BCI-assisted groups are significantly better than ResNet-assisted groups for large group sizes (m = 8\u20139). The two methods were on par for groups of size 7. For all group sizes m, BCI-and-ResNet-assisted groups  vs (e)\u201d column in As expected, BCI-assisted groups are significantly more accurate than equally-sized traditional groups based on standard majority for all group sizes\u2014see \u201c(a) vs (b)\u201d column in m = 3 \u2212 10, while the opposite happened for accuracy. On the other hand, the sensitivity of groups of different sizes . The median BCI confidence values for incorrect and correct trials are 0.747 and 0.850, respectively. The ResNet is also able to assess its degree of confidence via p = 0.191).p = 2.9 \u00d7 10\u22127 and p = 2.0 \u00d7 10\u22123, respectively). Once again, the difference in standard deviations between correct and incorrect confidence values is bigger for the ResNet.From Taken together, these statistical differences suggest that both the BCI and the ResNet are able to assess the decision confidence, albeit not perfectly. Of course, this is not entirely unexpected. The BCI bases its confidence estimates on the brain signals of the users, hence facing two major challenges that affect its certainty. Firstly, EEG signals are generally affected by a high level of noise caused by various sources . Secondly, humans participants may be overconfident or underconfident, two behaviours which might in turn also impact their brain patterns, thereby making it potentially difficult for our algorithms to reliably infer the probability of a decision being correct. The ResNet decisions and confidence are also affected by noise from different sources .Irrespective of the reasons for the differences in confidence distributions between ResNet and BCI-assisted humans reported in A cyborg is typically defined as the combination of a human and a machine, which is either invasively embedded inside the human or replaces/extends a body part, with the aim of restoring some function or augmenting human capabilities. These types of cyborgs were once only the domain of science fiction, but are nowadays becoming a reality thanks to the advancements in neural engineering and prosthetics. However, we believe that the terms cyborg and cyborg group are also appropriate to describe non-invasive combinations of one or more humans and one or more machines, such as the systems described in this paper to augment performance in face recognition.shared-control systems, where the artificial component of the HMI generally takes care of all low-level decisions [Of course, such systems can also be considered as forms of Human-Machine Interaction (HMI), consisting of a machine analysing information, and humans using this analysis to make informed decisions. In this area fall hybrid BCIs  that allecisions . For exaecisions  or contrecisions  with a hecisions . HoweverA more recent and promising avenue of research has been looking at Human-Machine Cooperation (HMC), which further extend HMI to allow the human and the machine to cooperate on a more equal footing and at a higher (cognitive) level . This noThe cyborgs and cyborg groups proposed in this study represent operational solutions to the problem of how HMC could be implemented in an uncentralised and self-organising manner in the domain of decision making. The simplest solution would be creating groups where humans and machines have an equal weight in the decision making (majority-based groups). In our face-recognition task, the ResNet achieved significantly better performance than the average participant, hence allowing ResNet-assisted groups to significantly improve performance over traditional groups of humans. However, this approach is situation-independent, i.e., it is not able to discriminate in which cases the machine or the human should be trusted more.Cyborg group performance could be significantly boosted by assessing the decision confidence of team members (computers or humans) in each decision and weighing their decisions accordingly. We have shown that machine-vision algorithms could be designed that also estimate their degree of confidence, while BCIs could be used to decode the decision confidence of humans from their brain activity. These BCI-and-ResNet-assisted groups fully implement the HMC strategy, allowing to decide the contribution to the group decision of each team member (human or machine) on a decision-by-decision basis, depending on the degree of confidence of each actor. Moreover, contrary to what has been suggested in the literature for human groups , cyborg The cyborgs proposed in this study also take into account ethical and liability issues concerning automated decision making. We have shown that the confidence values provided by the BCI for humans are higher than the confidence of the ResNet, leading to humans counting often more than the ResNet in uncertain decisions. When cyborg groups are used at an operational level, it is ethically simpler  to let humans decide in cases where there is no clear majority of opinions between humans and ResNet. However, when humans in a group cannot agree on a decision, it is more reasonable to trust fully-automated systems than flipping a coin to break ties.Overall, all strategies explored in this study on how to integrate humans and machines have shown significant advantages in face-recognition performance over traditional groups of only humans. When fully-automated machine-vision algorithms are not reliable enough, BCIs could be used to significantly boost the performance of groups of human operators. In other situations, pairing machines and BCI-assisted humans may provide the best performance. Future work will verify these findings with other tasks where automated algorithms could be developed. Furthermore, ensemble of ResNets or similar artificial agents could be created and their performance compared with that of groups of humans or humans and machines, to further verify the need of humans in the loop to achieve maximum accuracy."}
{"text": "Data on the relationship between the triglyceride glucose (TyG) index and coronary artery calcification (CAC) progression is limited. This longitudinal study evaluated the association of TyG index with CAC progression in asymptomatic adults.We enrolled 12,326 asymptomatic Korean adults who had at least two CAC evaluations. The TyG index was determined using ln (fasting triglycerides [mg/dL]\u2009\u00d7\u2009fasting glucose [mg/dL]/2). CAC progression was defined as a difference\u2009\u2265\u20092.5 between the square roots (\u221a) of the baseline and follow-up coronary artery calcium score (CACS) (\u0394\u221atransformed CACS). Annualized \u0394\u221atransformed CACS was defined as \u0394\u221atransformed CACS divided by the inter-scan period.During a mean 3.3\u00a0years, the overall incidence of CAC progression was 30.6%. The incidence of CAC progression  and annualized \u0394\u221atransformed CACS  were markedly elevated with increasing TyG index tertiles. Multivariate linear regression analysis showed that TyG index was associated with annualized \u0394\u221atransformed CACS . In multivariate logistic regression analysis, the TyG index was significantly associated with CAC progression in baseline CACS\u2009\u2264\u2009100.The TyG index is an independent predictor of CAC progression, especially in adults without heavy baseline CAC. Recent epidemiologic study revealed that the number of people who died from cardiovascular (CV) disease in 2013 was more than 17.3 million, representing an increase from 1990 of 40.8% .The triglyceride glucose (TyG) index has been suggested as a reliable surrogate marker of insulin resistance (IR), which is a substantial risk factor for ASCVD \u20139. PreviData from the Korea Initiatives on Coronary Artery Calcification (KOICA) registry were analysed in the present study. Briefly, the KOICA is a retrospective, multicentre, and observational registry with single ethnicity in the setting of self-referral for asymptomatic subjects who underwent general health examination at six healthcare centres in South Korea . Overall2). Blood pressure of the right arm was measured using an automatic manometer after resting for at least more than 5\u00a0min. All blood samples including total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), creatinine, glucose, and glycated haemoglobin A1C (HbA1C) levels were obtained after at least 8\u00a0h of fasting and analysed. All methods were performed in accordance with the relevant guidelines and regulations.Information on history of hypertension, diabetes, hyperlipidaemia, and smoking status for each subject was systematically collected. Weight, height, and blood pressure were measured during the healthcare centre visits. Weight and height were measured during the subjects wore light clothing without shoes. The body mass index was calculated as weight (kg)/height  and SAS version 9.1.3 . A P-value\u2009<\u20090.05 was considered significant in all analyses.Continuous variables are expressed as the mean\u2009\u00b1\u2009standard deviation. Categorical variables are presented as absolute values and proportions. After checking the distribution status of variables, the one-way analysis of variance test was used to analyse continuous variables and the \u03c7The mean age of the 12,326 participants  was 51.7\u2009\u00b1\u20098.5\u00a0years. The prevalence of hypertension, diabetes, hyperlipidaemia, and current smoking was 33.5%, 13.8%, 28.0%, and 28.5%, respectively. The ranges of the TyG index in stratified groups of I (lowest), II, and III (highest) were 6.77\u20138.40, 8.41\u20138.91, and 8.92\u201311.62, respectively. Systolic and diastolic blood pressure; body mass index (BMI); the levels of total cholesterol, triglyceride, LDL-C, glucose, HbA1C, and creatinine; and the prevalence of male sex, hypertension, diabetes, hyperlipidaemia, and current smoking significantly increased with increasing TyG index tertiles. In contrast, the mean age and HDL-C levels significantly decreased with increasing tertiles . Annualised \u0394\u221atransformed CACS was elevated with increasing TyG index tertiles in categorical CACSs of 0 , 1\u201310 , and 11\u2013100 , not in CACS\u2009>\u2009100   Table\u00a0. The resWith regards to the relation of TyG index with CAC progression according to the baseline CACS, the TyG index (per 1-unit increase) was associated with an increased risk of CAC progression in baseline CACSs of 0, 1\u201310, and 11\u2013100 after adjusting for age, male sex, BMI, hypertension, diabetes, hyperlipidaemia, current smoking, and serum creatinine level. However, this association of the TyG index and CAC progression was not observed in the condition with baseline CACS\u2009>\u2009100 Table\u00a0. After aThe present study aimed to evaluate longitudinal change of CAC related to TyG index after considering baseline CAC status in asymptomatic adults. A number of studies reported that the TyG index was related to the risk of developing traditional CV disease and adverse clinical events. Recent cross-sectional data from Brazilian Cardioprotective Nutritional Program Trial showed the positive association of TyG index with metabolic and behavioral risk factors . In the The homeostatic model assessment of IR (HOMA-IR) has been traditionally used to measure IR , 29. HowAlthough several cross-sectional studies previously reported a significant relationship between the TyG index and CAC prevalence, longitudinal data have been limited in clinical practice. Recently, Park et al. observed that an elevated TyG index was related to an increased risk of CAC progression in 1175 subjects ; howeverIn the current study, CAC progression was observed in 13.0% of participants without CAC at baseline. Importantly, we found that a high TyG index has a strong association with an increased risk of CAC progression in this population after adjusting for confounding clinical factors. This relation of the TyG index with CAC progression was consistent in the baseline conditions of CACS of 1\u201310 and 11\u2013100. However, there was no significant impact of the TyG index on CAC progression in participants with baseline CACS\u2009>\u2009100. This result might be affected by the relatively small sample size of participants with CACS\u2009>\u2009100, which was about 10.6% in our study. Thus, the significance of the TyG index for predicting CAC progression is still uncertain in individuals with heavy calcification at baseline. Further prospective studies with a large sample size are necessary to confirm this issue.The mechanism underlying the relationship of the TyG index with atherosclerosis is unclear. However, previous studies revealed the pivotal role of IR in atherosclerosis progression via promoting apoptosis of macrophages, endothelial cells, and vascular smooth muscle cells \u201338. ReceThere are some limitations to the present study. First, the KOICA registry was based on a relatively healthy population who underwent health check-ups in healthcare centres. The present study only included subjects who experienced at least two CAC scan examinations with available data on the TyG index and diabetic status from the KOICA registry. Thus, this study could not represent the characteristics of overall participants of KOICA registry and a potential selection bias might be present. Second, this was a retrospective study, which may be affected by unidentified confounders. Third, we only evaluated the association between the baseline TyG index and CAC progression; however, the mean change of the TyG index was very small: \u2212\u20090.02\u2009\u00b1\u20090.48. Longitudinal consecutive changes of the TyG index during follow-up could not be confirmed. Fourth, the homeostatic model assessment of IR was not analysed because insulin levels were not measured in the KOICA registry. Fifth, some important data including physical activity, alcohol consumption, family history of disease were unavailable. Sixth, we could not control the effect of medications for hypertension, diabetes, and hyperlipidaemia on CAC progression because of the observational study design. Finally, the present study included only the Korean population. However, this study is unique in that we identified the predictive value of the TyG index for CAC progression according to the baseline CAC status in a large sample of asymptomatic adults.The TyG index is strongly associated with CAC progression after adjusting for confounding clinical variables, especially in adults without heavy CAC at baseline.Additional file 1: Table S1. Association between clinical variables and CAC progression.Additional file 2: Table S2. TyG index (per 1-unit increase) and the risk of CAC progression according to baseline CACS 100."}
{"text": "P\u2009<\u20090.001) and 1\u2009\u2212\u2009100 , but not in those with baseline CACS\u2009>\u2009100 . After adjusting for traditional risk factors, the AIP was significantly associated with CAC progression in those with baseline CACS\u2009\u2264\u2009100. The AIP has value for predicting CAC progression in asymptomatic adults without heavy baseline CAC.This study aimed to evaluate the association between the atherogenic index of plasma (AIP), which has been suggested as a novel marker for atherosclerosis, and coronary artery calcification (CAC) progression according to the baseline coronary artery calcium score (CACS). We included 12,326 asymptomatic Korean adults who underwent at least two CAC evaluations from December 2012 to August 2016. Participants were stratified into four groups according to AIP quartiles, which were determined by the log of (triglyceride/high-density lipoprotein cholesterol). Baseline CACSs were divided into three groups: 0, 1\u2009\u2212\u2009100, and\u2009>\u2009100. CAC progression was defined as a difference\u2009\u2265\u20092.5 between the square roots (\u221a) of the baseline and follow-up CACSs (\u0394\u221atransformed CACS). Annualized \u0394\u221atransformed CACS was defined as \u0394\u221atransformed CACS divided by the inter-scan period. During a mean 3.3-year follow-up period, the overall incidence of CAC progression was 30.6%. The incidences of CAC progression and annualized \u0394\u221atransformed CACS were markedly elevated with increasing AIP quartile in participants with baseline CACSs of 0 and 1\u2009\u2212\u2009100, but not in those with a baseline CACS\u2009>\u2009100. The AIP level was associated with the annualized \u0394\u221atransformed CACS in participants with baseline CACSs of 0 (\u03b2\u2009=\u20090.016;  Furthermore, CAC progression is a powerful predictor of mortality beyond traditional CV risk factors4. Recently, the Heinz Nixdorf Recall (HNR) study suggested that CAC progression may only offer additional prognostic benefit in the asymptomatic adult population, and that what counts is the baseline coronary artery calcium score (CACS) and risk factor assessment5. In particular, additional CAC evaluation was not recommended in cases of heavy baseline CAC, although a high CV risk was present in the HNR study5. This result suggests that early detection of the presence and progression of subclinical atherosclerosis is important in conditions with low to intermediate CV risk burden.Coronary artery calcification (CAC) is associated with atherosclerotic burden and adverse cardiovascular (CV) clinical outcomes8. Notably, evidence suggests that the AIP may be more closely related to CV risk than are other atherogenic indices or individual lipoprotein cholesterol concentrations alone11. However, there is a paucity of data on the association between the AIP and CAC progression according to the baseline CACS. Thus, the present study aimed to evaluate the association between the AIP and CAC progression, focusing on the baseline CACS.The atherogenic lipoprotein profile of plasma is a substantial risk factor for atherosclerosis. The atherogenic index of plasma (AIP) has been considered a marker of plasma atherogenicity based on its strong and positive association with lipoprotein particle size, cholesterol esterification rates, and remnant lipoproteinemia13. Briefly, the present study included 12,326 participants who underwent at least two CAC scan examinations, with available data on the AIP and diabetic status, from December 2012 to August 2016. All data were obtained during visits to each healthcare center. Self-reported medical questionnaires were used to obtain information on the participant\u2019s medical history. Blood pressure of the right arm was measured using an automatic manometer, after resting for at least 5\u00a0min. Weight and height were measured with the participants wearing light clothing without shoes. The body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. All blood samples were obtained after at least 8\u00a0h of fasting and analyzed for total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), creatinine, glucose, and glycated hemoglobin A1C (HbA1C) levels. Hypertension was defined as systolic blood pressure (BP)\u2009\u2265\u2009140\u00a0mmHg or diastolic BP\u2009\u2265\u200990\u00a0mmHg, a previous diagnosis of hypertension, or use of anti-hypertensive medication. Diabetes was defined as either a fasting glucose level\u2009\u2265\u2009126\u00a0mg/dL, HbA1C level\u2009\u2265\u20096.5%, a referral diagnosis of diabetes, or use of anti-diabetic treatment. Hyperlipidemia was defined as a total cholesterol level\u2009\u2265\u2009240\u00a0mg/dL or anti-hyperlipidemic treatment. Obesity was defined as BMI\u2009\u2265\u200925.0\u00a0kg/m2 using cut-offs for the Asian population. Current smoking history was considered present if participants currently smoked or smoked until 1\u00a0month before the study. Hypertension, diabetes, hyperlipidemia, obesity, and current smoking status were considered as traditional risk factors. The AIP was defined as the base 10 logarithm of the ratio of the concentration of triglycerides to HDL-C6. All participants were categorized into four-groups based on AIP quartiles.Data from the Korea Initiatives on Coronary Artery Calcification (KOICA) registry were analyzed in this study. The KOICA registry is a retrospective, observational, and multicenter registry of self-referred asymptomatic adults who underwent a general health examination at six healthcare centers in South Korea14. CAC progression was defined using the SQRT method; specifically, as a difference\u2009\u2265\u20092.5 between the square roots (\u221a) of the baseline and follow-up CACSs (\u0394\u221atransformed CACS), with consideration of the inter-scan variability15. Annualized \u0394\u221atransformed CACS was defined as \u0394\u221atransformed CACS divided by the inter-scan period. All computed tomography (CT) scans used to assess CAC were obtained from a\u2009>\u200916-slice multi-detector CT scanner . Standard prospective or retrospective methods were used in all centers. All methods were performed in accordance with the relevant guidelines and regulations. The appropriate institutional review board committees of Severance Cardiovascular Hospital approved the protocol of the present study.CACS was measured using the scoring system described by Agatston et al.P value\u2009<\u20090.05 was considered significant in all analyses.Continuous variables are expressed as the mean\u2009\u00b1\u2009standard deviation. Categorical variables are expressed as absolute values and percentage. The one-way analysis of variance was used to analyze continuous variables. The \u03c72 or Fisher\u2019s exact test was used to analyze categorical variables, as appropriate. Univariate linear regression analysis was used to assess the association of clinical variables with the annualized \u0394\u221atransformed CACS in each categorical CACS group. Multiple logistic regression models were used to identify the independent predictive value of the AIP for CAC progression beyond that for traditional risk factors according to the baseline CACS status, using the forced entry method. Relative risk was estimated by a log-binomial regression analysis for generalized linear models. All statistical analyses were performed using the Statistical Package for the Social Sciences version 19  and SAS version 9.1.3 . A The protocol of the present study was approved by the institutional review board of each institution, and written informed consent was obtained from each participant.Clinical characteristics of the 12,326 participants  are presented in Table P\u2009<\u20090.001), \u0394\u221atransformed CACS , and annualized \u0394\u221atransformed CACS  were elevated with increasing AIP quartile  and 1\u2013100 , but not in those with baseline CACS\u2009>\u2009100 . The annualized \u0394\u221atransformed CACS was significantly different among AIP quartiles in those with baseline CACSs of 0  and 1\u2013100 , but not in those with baseline CACS\u2009>\u2009100   and 1\u2013100 . However, no significant association between the AIP and annualized \u0394\u221atransformed CACS was observed in those with baseline CACS\u2009>\u2009100  (Table The AIP (per 0.1-unit increase) was associated with the annualized \u0394\u221atransformed CACS in those with baseline CACSs of 0  trial found only a modest association between CAC progression and CV outcomes, although additional prognostic information was not provided in comparison with that for the latest single CAC value, during the mean 3.5-year period16. Lehmann et al. recently reported that CAC progression may only add prognostic benefit for participants without CV disease, based on the HNR study data , during a mean 5.1-year follow-up5. This previous study emphasized the significance of the most recent CAC value and risk factor assessment. Additional CAC examination is not required in cases of heavy CAC at baseline, which implies the presence of a high CV risk. Considering that atherosclerosis-related adverse events commonly occur, even in populations with low CV risk burden19, these results strongly suggest that early detection of the presence and progression of subclinical atherosclerosis is an important issue in clinical practice.Budoff et al. previously reported that CAC progression had additive value in predicting all-cause mortality, beyond that for the baseline score and CV risk factors, in 4,609 asymptomatic participants who underwent repeat screening during a mean 3.1-year period21. In addition, a number of cross-sectional studies demonstrated that the AIP had predictive value for coronary artery disease in various clinical conditions24. However, earlier studies did not address the relationship between the AIP and CAC progression, especially in terms of the baseline CACS. In the present study, we considered a CACS\u2009>\u2009100 as indicating heavy CAC status based on the following facts: (1) Nasir et al.25 reported that the frequency of CACS\u2009>\u2009100 in the Asian population is significantly lower than that in western populations; and (2) the overall proportion of baseline CACS\u2009>\u2009100 was 10.6% in our participants. The presence of CAC at baseline and CAC progression during follow-up were more frequently observed with increasing AIP quartile. However, the AIP was associated with the risk of CAC progression, over that for traditional CV risk factors, in participants without heavy CAC at baseline. This finding suggests that the AIP is a useful marker for the stratification of CV risk, in addition to the baseline CAC status, in conditions with low to intermediate CV risk.Previous studies revealed that the AIP is significantly related to diverse metabolic disorders, such as fatty liver disease, hypertension, and diabetes26. However, a recent CONFIRM  sub-study found that further prognostic benefit was not offered by coronary computed tomographic angiography (CCTA) findings when added to traditional risk factors and CACS in 1,226 asymptomatic participants, during a mean 5.9\u2009\u00b1\u20091.2\u00a0years of follow-up27. This finding suggests that CCTA may have a limited role in future CV risk stratification in the asymptomatic population, until further proven.Because the present study only evaluated the change in CACS in the asymptomatic adult population, data on changes in the regional distribution or pattern of CAC were unavailable. In addition, the effect of medications such as statins, which could influence the changes in plaque sub-types, was not consideredWe should acknowledge some limitations of the present study. First, this study included participants from the KOICA registry who underwent at least two CAC scans with available data on the AIP and diabetic status. Thus, a potential selection bias might be present. Second, we only identified the association between the baseline AIP level and CAC progression, focusing on the baseline CACS; consecutive changes in AIP during the follow-up period were not confirmed. However, the mean change in the AIP between the initial and follow-up CAC scan was very small:\u2009\u2212\u20090.02\u2009\u00b1\u20090.23. Third, the use of medications for hypertension, diabetes, and hyperlipidemia was not controlled during the follow-up period because this study had an observational design. Fourth, although we evaluated the predictive value of the AIP for CAC progression after adjusting for sex, the predominance of male sex in this cohort registry could have impacted the results. Thus, it might be hard to apply the results of present study to the female population. Fifth, although this study was performed in an asymptomatic and relatively healthy population, there was a paucity of data on environmental risk factors, such as physical activity, exercise, and diet, which could influence CAC progression. Sixth, different CT scanners were used among the participating centers because the KOICA registry was a retrospective, observational, and multicenter registry. However, all participants were examined using the identical CT scanner at both the initial and follow-up image acquisitions. Finally, the generalizability of our results may be limited considering that all participants were Korean. Despite these limitations, we firstly identified the predictive value of the AIP for CAC progression according to the baseline CACS in a large sample of the asymptomatic adult population.In summary, the present study demonstrates that the AIP is independently associated with CAC progression, especially in the absence of heavy CAC at baseline. This result suggests that the AIP has the potential to predict early coronary atherosclerotic changes and to stratify CV risk in asymptomatic adults with low to intermediate CV risk. Further large-scale prospective studies are necessary to confirm the significance of the AIP in primary prevention.Supplementary Information 1."}
{"text": "However, the prognostic value of TyG index in patients with type 2 diabetes mellitus  was recorded. The TyG index was calculated as the ln [fasting triglycerides (mg/dL)\u2009\u00d7\u2009fasting plasma glucose (mg/dL)/2].A total of 1932 consecutive patients with Tp\u2009<\u20090.001], non-fatal MI , cardiac rehospitalization , revascularization  and composite MACCEs . The area under ROC curve of the TyG index for predicting the occurrence of MACCEs was 0.604 , with the cut-off value of 9.30. The addition of TyG index to a baseline risk model had an incremental effect on the predictive value for MACCEs .Competing risk regression revealed that the TyG index was positively associated with CV death [2.71(1.92 to 3.83), 2DM and AMI.The TyG index was significantly associated with MACCEs, suggesting that the TyG index may be a valid marker for risk stratification and prognosis in patients with TTrial registration Retrospectively registered. Finally, 1932 patients were included in this analysis. The patients were divided into 2 groups according to the occurrence of MACCEs during the follow-up: the MACCEs group (n\u2009=\u2009735) and the No-MACCEs group (n\u2009=\u20091197). In addition, the patients were also divided into tertiles according to their TyG index levels . All patients were followed up till October 31, 2020 with a median follow up of 26.8  months.Study subjects were identified from the Cardiovascular Center of Beijing Friendship Hospital Database (CBD) Bank. A total of 5169 consecutive patients were diagnosed with AMI and underwent coronary angiography from January 2013 to August 2020. Of the 5169 patients, 3237 were excluded according to the exclusion criteria, which were (1) without TThe data collection process was approved by the Institutional Review Board of Beijing Friendship Hospital affiliated to Capital Medical University and was in accordance with the Declaration of Helsinki.0.185/(TGs0.2\u2009\u00d7\u2009body mass index(BMI)1.338)  . The SinI)1.338) , with fa2DM include: (1) previously diagnosed T2DM under treatment of antidiabetic medication; (2) the typical symptoms of DM with a FPG\u2009\u2265\u20097.0\u00a0mmol/L, and/or random blood glucose (RBG)\u2009\u2265\u200911.1\u00a0mmol/L, and/or 2-h plasma glucose level after oral glucose tolerance test (OGTT)\u2009\u2265\u200911.1\u00a0mmol/L . Figure\u00a05.1%; CV p\u2009<\u20090.001)  of the TyG index for predicting the occurrence of MACCEs was 0.604  Fig.\u00a0. The cutp\u2009<\u20090.001). In addition, the C-index of the baseline risk model  changed after addition of the TyG-index .Table 2.Subgroup analysis was performed according to age, sex, BMI, smoker, HT, eGFR, LVEF and AMI type Fig.\u00a0. We foun2DM. Our main findings include: (1) the incidences of MACCEs significantly increased with the increase of TyG index, and (2) the TyG index was an independent predictor of MACCEs The AUC of the TyG index for predicting the occurrence of MACCEs was 0.604 with a cut-off value of 9.30, and (4) The addition of TyG index to a baseline risk model had an incremental effect on the predictive value for MACCEs, and (5) the predictive effect of TyG index on MACCEs is ineffective in patients with eGFR\u2009<\u200960\u00a0ml/min/1.73\u00a0m2. According to this study, we confirmed that the TyG index was positively associated with increased MACCEs. Most importantly, this study suggested that a simple method of estimating IR may optimize the risk stratification of recurrent cardiovascular risk in AMI patients with T2DM.To the best of our knowledge, this is the first study to explore the association between the TyG index and MACCEs in AMI patients with TIR is defined as a decrease in the efficiency of insulin in promoting glucose uptake and utilization, which is an indicator of abnormal metabolism. IR promotes the progression of CVDs by inducing glucose metabolism imbalance, altering systemic lipid metabolism, and causing endothelial dysfunction . Severalp\u2009=\u20090 003] , and the addition of TyG index to a baseline risk model had an incremental effect on the predictive value for adverse prognosis   . Consideas 9.323 . A study<\u20090.001] . However2DM for the first time. To better understand the predictive power of TyG index for different CVEs, we analyzed the correlation between TyG index and each type of MACCEs , which other studies have not tried. The conclusions drawn by this research have important guiding role for clinicians to predict the occurrence of future clinical events in patients with AMI and T2DM. In addition, we also found that adding TyG index to the baseline risk model had a significantly incremental effect on the predictive value for MACCEs, which is consistent with the conclusions of Zhao et al. [2. For this result, the mechanism is still unclear. There are relatively few studies on TyG index and kidney disease. Zhao et al. [2). Maybe we need to do some work on that.In this study, we investigated the prognostic value of the TyG index in patients with AMI combined with To et al. . Anothero et al.  found tho et al.  showed tOur study had several limitations. First, this was a single-center study although including a large sample size; thus, generalization of the findings should be cautious. Second, laboratory parameters were only measured once after hospital admission, which could cause potential bias due to measurement error. Third, conventional laboratory testing methods for IR, such as HOMA-IR, has not been tested, the relationship between TyG index and IR cannot be verified directly in this study. In addition, prospective cohort studies are required to confirm our findings.2DM. In addition, adding the TyG index to a baseline risk model had an incremental effect on the predictive value for MACCEs.In conclusion, the current study firstly demonstrated that elevated TyG index level was a strong independent predictor of MACCEs in patients with AMI and T"}
{"text": "Mammalian heart valves are soft tissue assemblies with multi-scale material properties. This is because they are constructs comprising both muscle and non-contractile extracellular matrix proteins (such as collagens and proteoglycans) and transition regions where one form of tissue structure becomes another, significantly different form. The leaflets of the mitral and tricuspid valves are connected to chordae tendinae which, in turn, bind through papillary muscles to the cardiac wall of the ventricle. The transition regions between these tissue subsets are complex and diffuse. Their material composition and mechanical properties have not been previously described with both micro and nanoscopic data recorded simultaneously, as reported here. Annotating the mechanical characteristics of these tissue transitions will be of great value in developing novel implants, improving the state of the surgical simulators and advancing robot-assisted surgery. We present here developments in multi-scale methodology that produce data that can relate mechanical properties to molecular structure using scanning X-ray diffraction. We correlate these data to corresponding tissue level (macro and microscopic) stress and strain, with particular emphasis on the transition regions and present analyses to indicate points of possible failure in these tissues. It is estimated that \u223c11.7 million people in the United State of America suffer from heart disease . A majorHuman heart valves act as gatekeepers to help ensure unidirectional flow of blood under physiological conditions. Because of their different placements, they control blood flow in the pulmonary and systemic circuits and between the atria and ventricles. The four main valves experience significantly different blood pressures and rates of activation. Blood flow/pressure activates the valves to allow the flow of blood away from a chamber of the heart and they close to prevent blood flow in the wrong direction. The aortic valve (AV) allows the outflow of blood from the left ventricle to the aorta and the pulmonary valve (PV) controls the outflow of blood from the right ventricle to the pulmonary artery . These vAs is clear from visual inspection, the MV and TV have two distinct tissue transitions, where one specialized tissue organizational domain becomes another with a different organization, even though they are principally made of the same materials; namely (i) LL to CT (one type of fibrillar collagen and proteoglycan (PG) arrangement to another; see One may expect, a priori, some manner of muscle-to-tendon transition in cardiac valves, analogous to that found in skeletal tissues. So, the mechanism of injury to the latter may be beneficial for determining similar responses in the former. Previous studies have demonstrated that injury to tissues that possess muscle-to-tendon transitions originates in those transition regions ,11,12. RImportantly, these data are needed to develop high definition computational models of the heart that are intended to closely reflect physiological function. With the development of high-speed computing and modeling techniques, cardiac flow simulations have made it possible to establish potential cardiac injury mechanisms in simulations. These simulations can aid in predicting cardiac damage, particularly chordal tear and valvular defects ,18,19,20A major hurdle in improving the quality and reliability of simulations focused on the function and injury in heart valves is the lack of relatively high-resolution data  concerning tissue composition, tissue boundaries, and elastic properties of heart valves. Several modeling efforts have been focused on developing the elastic contributions of the various elements of a tissue assembly ,29,30,31We present here expanded methods and analyses to establish material and mechanical properties of cardiac tissues, mitral and tricuspid valve transition regions. These methods are an advancement of those described in Madhurapantula et al. (2017)  that werXRD scanning was performed along the samples dissected from pig valves. Diffraction patterns were analyzed to identify and measure the collagen fifth-order meridional and muscle  equatorial peaks. The relative intensity of these peaks was used to calculate the relative percentage composition of muscle and collagen along the transition regions , as per MV and TV have clear differences in mechanical properties, evident also from consideration that one is a bi-part and the other a tri-part valve. However, the material composition from the respective muscle to collagenous tissue transitions are similar. As noted in the study of skeletal muscle to tendon molecular composition transition is not a \u2018step-change\u2019, but is diffuse or gradual over a section of the fibrous tissue. XRD data shows a near-linear decrease in collagen percentage is observed in the PM to CT transition, going into the belly of CT (no muscle detected), the length of this transition spanning a few millimeters.The transition between CT\u2013LL, as determined by XRD, appears to be somewhat more homogeneous than the PM\u2013CT transition. This is due to fact that the LL and CT are both primarily constituted of fibrillar collagen , unlike The AV is distinctly different from the MV and TV, consisting of three cusps attaching to the wall of the aorta see . ComposiMV and TV samples were cut and separated, halfway along the CT (\u2018bisected\u2019) to capture stress vs. strain properties of these segments. XRD scans along these \u2018bisected\u2019 samples were used to determine changes in the molecular packing structures of muscle and collagen along the tissues .Molecular strain was calculated as the change in the length of the collagen D-period from that point recorded at \u2018Resting\u2019  state as described in Madhurapantula et al. (2017) . Ten datThe PM region is most compliant and the greatest strain can be observed in this element in the PM\u2013CT transition in both MV and TV. The molecular strain is highest at around the PM\u2013CT transition, where the corresponding composition scan shows a steep increase in collagen content in the tissue see . This stA similar trend of localization of molecular strain is observed in the CT\u2013LL transition of the MV and TV. Strain is localized at the point in the transition where there appears to be a muscle-like diffraction series. This can be observed using the corresponding composition scans.2D XRD scans were performed on \u2018bisected\u2019 CT\u2013LL samples from MV and TV. Local collagen fiber orientations are reported as aligned ellipses with the direction of the major axis pointed along the fiber direction . See MusIndividual sample elements, i.e., PM, CT and LL, were stretched continuously until breakage. The stress vs. strain was calculated and is reported as below. The CT assumes most stress and the least amount of strain. This is in line with the functional aspects of the highly tensile tendons. PM assumes most strain for the least stress, with LL lying between the two other elements. The PM to CT mechanical differences are relatively comparable to data from skeletal muscle\u2013junction tissues examined previously Madhurapantula et al. (2017) , althougWhile application of stretch on individual tissue elements is important to understand material and mechanical properties, transitions may be understood by performing similar stretch experiments on \u2018bisected\u2019 samples that leaves these regions (and surrounding structures) intact. As observed in As a crude test of valve material vulnerability in the transition regions, sample failure pulls were performed for the MV and TV. Since the CT possesses the ultimate strength of the assembly, it was bisected and the PM\u2013CT and CT\u2013LL assembles tested. Ultimate strength (stress before the sample is permanently deformed) can be determined from As is evident from the data presented, the PM\u2013CT and the CT\u2013LL transitions present different stress\u2013strain properties in comparison to the \u2018pure\u2019 LL, CT and PM sections of the sample . PM is tThe ultimate strength (point on the stress\u2013strain curve marked by permanent deformation) of each region is markedly different . This maFor both the TV and MV, the ultimate stresses within the transition regions (PM\u2013CT and CT\u2013LL) are not much greater than the PM or LL regions  even witCumulatively, this indicates a mechanical vulnerability to failure relative to those more compliant but thick (PM) or compliant due to fibrillar re-arrangement (LL) or materially durable due to dense construction of relativity non-compliant but strong material (CT and LL). Additional materials and methods are located within the Whole, snap-frozen porcine hearts were procured from Carolina Biological Supply . The mitral (MV), tricuspid (TV) and the aortic (AV) valves were carefully dissected en bloc to preserve underlying structures . Valves were dissected with enough clearance to ensure that the samples of interest were not damaged during dissection. The dissected valves were then stored at \u221220 \u00b0C. Samples were thawed overnight at 4 \u00b0C before experiments.Clear sections of these valves that consisted of a portion of the leaflet with a singular connection to a CT leading into a PM were identified and dissected. Any branches of the CT were severed, to ensure that there was one main connection between the three tissue elements. The samples were dissected under 1X phosphate buffered saline (PBS) to keep them hydrated during the course of dissection and were stored at room temperature under a paper towel soaked in PBS before experiments. Samples were not stored in PBS buffer longer than 6 h.Samples were evaluated in three modes: (i) full valve samples (LL to CT to PM); (ii) samples \u2018bisected\u2019 by cutting the full valve assembly at the middle of the CT, to evaluate the properties of LL to CT and CT to PM transitions, and; (iii) \u2018trisected\u2019 samples, i.e., valve elements separated to evaluate the properties of the LL, CT and PM individually.It was determined at an early stage of investigation that a more direct comparison of structure and physiological roles of the MV and TV would be the study priority. Due to the anatomical placement of the aortic and pulmonary valves, dissecting both valves from the same heart may compromise one or both of these valves. Moreover, MV and TV present higher complexity in valve architecture than the pulmonary and aortic valves. The pulmonary valve was excluded due to its relatively low-pressure function and because it is rarely reported to fail. Given these priorities, in a number of instances it was viable to retrieve the aortic valve, hence allowing some limited study of these samples also, reported in the A strain apparatus was constructed based on that previously described in Madhurapantula et al. (2017) . BrieflySamples were mounted between the top and the bottom sample brackets. The stage on the actuator is moved by the stepper motor. An Arduino based program was used to issue commands to stretch the sample. The Arduino also mediates the collection of displacement (mm) vs. load (g) information into text files, which were used for analyses presented here .See Molecular strain, is defined after Madhurapantula et al. (2017) : for colFor muscle, it is the increase in sarcomere length which is directly related to lengthening of the muscle fibers due to myosin and actin fiber sliding in response to an application of stretch . Muscle 2D scans were performed on bisected samples to determine changes in alignment of collagen fibers in the PM\u2013CT and the CT\u2013LL junctions with application of stretch. The initial sample measurements were noted and stretch was applied to the samples at increments of 2% engineering strain. The sample was allowed to relax for 2 min before 10 XRD patterns were collected along the central sections of the LL\u2013CT and CT-PM samples.\u2018Bisected\u2019 valve samples  were loaded on to the strain rig described herein. Visual markers (\u2018dots\u2019) were placed on the sample using a marking pen (Sharpie\u2122). These dots were used as fiducial markers. These markers were placed so there was at least one each in the PM or LL and a pair of dots on either side of the transition region . The eloThe video was then used to generate still frames at 5 fps. These frames were imported into ImageJ  for analAlthough it is often reported that CT is the point of valve failure, our data indicate that the definition of which portion of the valve is CT is highly relevant to the material outcomes of the tissue. Although the CT tissue is present through transition regions from the PM to CT and from the CT to LL, its durability through these material regions is different from that of the CT main section. The significance of tissue transition regions have often been overlooked, when studying mechanical behaviors of soft tissues. Emphasis is usually laid on understanding the individual tissue components. However, as is evident from earlier surgical observations ,43 and dThe methods and data presented here allow attribution of material properties to these regions and their behavior under strain. XRD scans enable determination of high-resolution tissue composition in transition regions in their native state (ex vivo) without introducing possible artifacts due to sample preparation  or in downstream data preparation where 2D sample image sections need to be reconstructed into a 3D volume before detailed analysis is performed. The molecular strain analyses in the context of engineering strain, in relation to the tissue composition, provide insights into changes in the molecular packing of materials in samples that lead to strain distribution in these regions in relation to its change in material composition as it transitions from one tissue into another. No other technique, to our knowledge, allows similar static and dynamic analyses of the simultaneously multi-scale properties  collected from a native untreated sample while it is stretched.Studies have shown that virtual reality-based technique used to train surgeons are highly beneficial ,47. The"}
{"text": "High strain rate biaxial forging (HSRBF) was performed on AZ31 magnesium alloy to an accumulated strain of \u03a3\u0394\u03b5 = 1.32, the related microstructure, texture and mechanical properties were investigated. It was found that the microstructure evolution can be divided into two steps during HSRBF. In the early forging processes, the refinement of the grain is obvious, the size of ~10 \u03bcm can be achieved; this can be attributed to the unique mechanisms including the formation of high density twins ({10 Magnesium alloys are potential materials in airplanes and automobiles, they have good machinability, excellent damping capacity and favorable recycling capability, besides which, the advantage of light weight can save energy and reduce emissions ,2. HowevIt is well known that, different strain paths during forging lead to different metal plastic flows, which consequently results in different shapes and properties . For exaI(0002):I(10:I(10 = 1:0.83:2.21) were comparable to those of a completely random Mg powder (I(0002):I(10:I(10 = 1:0.85:2.44).A commercial AZ31 magnesium alloy with the chemical composition of Mg-3%A1-1%Zn-0.3%Mn was selected in the present study. The as-cast billets were homogenized at 400 \u00b0C for 12 h followed by water quenching. The homogenized material was characteristic of coarse grains with grain size of ~400 \u03bcm, and few twins were detected . X-ray dRectangular samples for HSRBF were machined from the homogenized ingot, and the height, width and length for the sample was 40, 35 and 35 mm, respectively. The samples were heated in a muffle furnace before forging, and the hold temperature was 350 \u00b0C for 5 min. An air hammer was used to conduct biaxial forging along two orthogonal directions in turn as illustrated in \u03bb), and HSRBF was carried out to an accumulated strain of \u03a3\u0394\u03b5 = 1.32, i.e., 6 passes of forging. The billets stretched along the rolling direction (RD) after HSRBF, and no obvious crack was observed.A pass strain of \u0394\u03b5 = 0.22 was obtained by accurate controlling of the pass reduction  was selected to make different microstructure characterizations. Optic microstructures were observed using an optical microscope  after etched with a solution of 1 g oxalic acid, 1 mL nitric acid and 98 mL water. Electron back-scatter diffraction (EBSD) observation was conducted on a scanning electron microscope  at 20 kV, and 1.5 mm was selected as a step size in the measure of the orientation imaging. The Schultz reflection method was carried out on X-ray diffraction  to analysis texture. Dog-bone like tensile specimens were machined with a gauge length of 10 mm, and abrasive papers were utilized to polish the surfaces of tensile specimens. A tensile test was conducted under a constant tensile rate of 0.5 mm/min at room temperature, and the tensile direction was parallel to RD. In order to make sure of the repeatability, three experiments for each condition were conducted. The fracture characters of the tensile samples were observed on SEM.Shown in 1,a2,b1,b2) was similar to that observed by OM, in the twinned regions and at grain boundaries some DRX grains were detected, and the fraction of DRX increased significantly with the increase of accumulated strain. Moreover there are two peaks around 38\u00b0and 86\u00b0 in the misorientation distribution map , indicating that {10Shown in tion map a3,b3, inu et al.  and Li eu et al.  have repThe IPF maps and grain size distribution maps of AZ31 alloys HSRBFed with accumulated strain of \u03a3\u0394\u03b5 = 0.88 and 1.32 are shown in As mentioned above, the microstructure evolution of the AZ31 alloy during HSRBF can lead to the refinement and growth of the grain, which was different from alloys forged at low strain rates as reported in references ,21,22,23w strain ,26. As sw strain ,21,22,23w strain .1,b1) also confirms the strong basal texture as the alloys HSRBFed to the accumulated strain of \u03a3\u0394\u03b5 = 0.22 and 0.44. With further increase of accumulated strain, DRX developed fast at twins and the fraction of the DRX grain increased. It was reported that during the hot deformation of magnesium alloys TDRX was of great importance in texture weakening [During uniaxial compression, magnesium tends to form a basal texture with the c-axis parallel to the loading direction, and the (0002) pole rotates towards the compression direction owing to the development of the {10 strains . It is oeakening ; therefoeakening  have reveakening . TherefoIt is acknowledged that a magnesium alloy with tiled texture displays excellent formability; therefore, basal pole inclining is widely used in tailing the texture of a magnesium alloy. Shear deformation is one of the effective methods to achieve tilted-basal texture, which is commonly seen in processes such as equal channel angular pressing (ECAP), differential speed rolling (DSR) and friction stir processing (FSP), and it is reported that the inclinations were 20\u201345\u00b0, 5\u201315\u00b0 and up to 55\u00b0 in ECAP, DSR and FSP alloys, respectively . It was s, ultimate tensile strength (UTS) \u03c3b and elongation \u03b4. The alloy in its initial state exhibits poor mechanical properties. However, it is obvious from The microstructure, texture and mechanical properties of the AZ31 alloy during high strain rate biaxial forging (HSRBF) were investigated. The main conclusions were listed as below.The microstructure evolution during HSRBF can be divided into two steps, i.e., grain refinement in the early stage and grain growth with further increase of accumulated strain. A homogeneous fine DRX structure with the average grain size of 9.2 \u03bcm was obtained as accumulated strain was 0.88, implying that DRX developed at lower strain during HSRBF.The formation of high density twins, and subsequently twining induced DRX, leads to the grain refinement, and {10The main texture in the HSRBFed alloys is a typical hot-rolled texture, and the intensity of the texture decreases with increasing of the accumulated strain. Moreover, the basal pole rotates towards the direction of forging direction (FD) after each pass, and a basal texture with a basal pole inclining at 15\u201320\u00b0 form the rolling direction (RD) formed in the full recrystallized HSRBFed alloy.The strength and elongation increase with the strain as the accumulated strain is lower than 0.88, but decrease slightly with further forging, and an excellent combination of mechanical property with UTS of 331.2 MPa and elongation of 25.1% was achieved at \u03a3\u0394\u03b5 = 0.88, which resulted from the combined effects of grain refinement and weakened basal texture. Therefore, HSRMF is an efficient way to produce strong and ductile wrought AZ31 alloy."}
{"text": "Passive acoustic monitoring systems allow for non-invasive monitoring of underwater species and anthropogenic noise. One of these systems has been developed keeping in mind the need to create a user-friendly tool to obtain the ambient noise indicators, while at the same time providing a powerful tool for marine scientists and biologists to progress in studying the effect of human activities on species and ecosystems. The device is based on a low-power processor with ad-hoc electronics, ensuring that the system has efficient energy management, and that the storage capacity is large enough to allow deployments for long periods. An application is presented using data from an acoustic campaign done in 2018 at El Gorguel . The results show a good agreement between theoretical maps created using AIS data and the ambient noise level indicators measured in the frequency bands of 63 Hz and 125 Hz specified in the directive 11 of the EU Marine Strategy Framework Directive. Using a 2D representation, these ambient noise indicators have enabled repetitive events and daily variations in boat traffic to be identified. The ship noise registered can also be used to track ships by using the acoustic signatures of the engine propellers\u2019 noise. Assessment of good environmental status (GES) in European seas is at the heart of the Monitoring Guidance for Underwater Noise in European Seas: Monitoring Guidance Specifications . It is eIt is not easy to monitor Directive 11 on environmental noise in the various marine areas. Indeed, it is essential not only to monitor underwater noise in specific locations, but also to use models that enable noise maps to be estimated. To create these models, many variables need to be known: water column salinity and temperature, marine traffic location, vessel type, speed, etc. The simulations carried out by the models must be checked and corrected using the experimental data obtained with PAM acoustic recorders, and complemented with the information regarding the sources of noise (mostly ships) that it is possible to obtain by analyzing real data.This manuscript is an extended version of the work published in . In thisWorking with an autonomous PAM recorder such as the SAMARUC involves several steps: first, the device is deployed for several months in a desired location where it registers acoustic information about the species and anthropogenic noises; after that, the system is recovered, the data extracted from device\u2019s memory and saved as wav files in a local server. Finally, the data is analyzed offline by means of specific software (SAMARUC software is registered by UPV ). The SAThe system\u2019s main component is a general-purpose, ultra-low power consumption digital signal processor (DSP) from Texas Instruments, the TMS320C5535. The selection of the DSP is made as a trade-off among price, low-power consumption, flexibility, and reliability. An adequate selection of the DSP and peripherals allows the time between service intervals to be increased and the battery life extended.The AIC3204 has been set up to perform a BIQUAD filter to establish 192 kHz as a sampling frequency, and it is able to add a gain in order to amplify the signal if it is necessary to increase the sensitivity of the selected hydrophone depending on the marine strategy deployment category. In any case, a system sensitivity (hydrophone + AIC3204 gain) of between \u2212165 and \u2212185 dB re 1 V/uPa is recommended [Data transmission is set up in DMA Ping-Pong mode, so the system records acoustic data without sample loss while storing that data in the microSD cards. The Ping-Pong settings allow a buffer (PING_PONG buffer) to be used, which is internally divided into two subbuffers , allowing the status of each subbuffer to be checked for the tasks to be carried out in parallel. Thanks to the integration of the two DMAs, simultaneous transmission between memory and peripherals is available. A dedicated electronic board was designed whose functions include, among others, adding a second microSD card to double the available storage capacity. This electronic design  and gain. All this information is loaded on powering up the device. The Block-File allocation table stores information about the block start and block end of each of the files, as well as the time stamp of the first sample. Finally, the data section stores the samples of the recording in the different blocks of the uSD card. The system is put into sleep mode during the OFF cycle to preserve battery life.A pressure case was built from a tube of 6082 aluminum anodized with NITUFF, measuring 12 \u00d7 70 cm (diameter \u00d7 length). This material was chosen according to relevant factors such as its durability (the anodized NITUFF is used in military marine applications) and light weight. Pressure tests were performed to guarantee that the housing can withstand a pressure of 50 atmospheres, corresponding to a depth of 500 m.Moreover, to maximize the energy density in the cylindrical housing, a hexagonal layout of D-cell batteries was created. The battery block to supply power to the electronic components is composed of 7 \u00d7 4 D-type batteries  on the south-east coast of the Mediterranean Sea . This location allows the existing marine traffic in the Port of Cartagena to be monitored.The seabed in this area is not uniform and consists basically of mud. As described above, the system is prepared for 500 m depth deployments, but in this case, a depth of 60 m was chosen given the bathymetric profile of the exploration area. The device was placed 10 m from the seabed (reducing its acoustic influence) at a depth of 50 m using a 70 kg anchoring weight. The minimum distance between the device and the vessels\u2019 permitted transit area was approximately 150 m  was choUsing the temperature, salinity, and depth/pressure data from the Argo profiler available on the EMODNET website , the souOne of the most important uncertainties in noise modelling is the source level assigned to each vessel, considered to be a noise emitter in the marine zone under study. Ships as an underwater acoustic source are a widely studied problem that remains open due to the complexity of the sound field emitted by each part of the ship at a given frequency. Several kinds of experimental models have been developed in recent decades, from the Ross model  that preSince the frequencies studied in this work are 63 Hz and 125 Hz, we chose the RANDI model to parameterize the source level of each ship considered in the computed AIS data. This method is validated for frequencies between 28.4 Hz\u2013191.6 Hz. The mean value of the Sound Pressure Level (SPL) over the location was calculated considering the AIS data from vessels that have non-zero speed over ground.v), closest point of approach (CPA), and draft can be estimated using a PAM system equipped with a single broadband hydrophone [Acoustic signatures of ship engine propeller noise can be used for noise characterization as well as for target tracking. Engine harmonics can be examined for full awareness of the effect of the different ships in the 1/3 octave ambient noise indicators. Additionally, vessel speed 2\u03c0fnt was obtained in Equation f(t)The results obtained considering ray theory approximation are summarized in the sound maps of daily SPL dB re 1 uPa at a depth of 50 m. Daily sound maps of Going back to real data obtained by SAMARUC, we calculated the average one-third-octave band  for an integration interval of one hour at the same timestamp times of the modelling calculations. As The results show a good agreement between experimental and theoretical calculations, verifying not only that the device worked properly and the numerical simulations were valid, but also that the calibration of the system performed well at IEO-UPV\u2019s facilities, an aspect not easy to achieve due to the low frequency range considered.In addition to the aforementioned results, the computed 1/3 octave indicators can be conveniently structured in hours and days, and represented as a 2D color map to provide an acoustic panorama and identify seasonal events . This reLet us now select one of the recordings containing a ship passing within the range of the SAMARUC system. The spectrogram for these real signals is shown in the left panel of v, and The U-shaped structure and Doppler shift can be used to estimate the values for quations  and  as an example, we have illustrated how the theoretical sound maps can be created using marine traffic information, and how the real data can be used to experimentally validate the estimated sound pressure levels associated with continuous noise coming from anthropogenic activities. The system designed efficiently managed the power consumption and thus it is appropriate for long deployments. This fact allowed 2D ambient noise maps to be created, which could be used to identify seasonal events and to characterize ships by means of their acoustic signatures."}
{"text": "Based on a three-stage stackelberg dynamic game analysis, this paper constructs a product quality control strategy model for three types of distribution channels  in a three-echelon supply chain, which is composed of one manufacturer, one retailer and the final customer. This paper studies how to design a distribution channel strategy and provides a product quality control strategy. Furthermore, this paper analyzes three types of distribution channels strategy in the context of how they influence a manufacturer\u2019s product quality decision and quality prevention strategy, a retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision. We compare the manufacturer\u2019s product quality level, quality prevention effort level, wholesale price, direct sale price and the retailer\u2019s quality inspection effort level, retail price in three types of distribution channels and determine the manufacturer\u2019s and retailer\u2019s expected profits function and the final customer\u2019s consumer surplus. In addition, we introduce the distribution channels demand elasticity ratio to analyze the influence of determining the product quality control strategy. Most importantly, we conduct a numerical sample analysis that will prove the model\u2019s effectiveness and indicate a specific application in practice. In recent years, with the rising of network economy and e-commerce, in addition to the traditional retail channel, more and more customers or consumers choose to purchase products from the internet channel(direct channel), which have become an important way of products sale. With the changing in customer or consumer buying behavior, more and more companies are beginning to redesign or rebuild their distribution channel structure; what\u2019s more, the different types of distribution channels structure in supply chain how to influence the manufacturer\u2019s product quality decision and quality prevention strategy, the retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision; above all, how to influence the manufacturer\u2019s and retailer\u2019s expected profits function and the final customer\u2019s consumer surplus, and how to determine a product quality control strategy in order to eliminate \u201cchannel conflict\u201d and \u201cfree-riding behavior\u201d. All of these problems and difficulties have not been fully resolved, which are also important research directions for current researchers.In this paper, we will construct a distribution channel strategy model in a three echelon supply chain that is composed of one manufacturer, one retailer and a final customer based on a three-stage stackelberg dynamic game. Furthermore, we will introduce the distribution channel demand elasticity ratio and investigate how to craft a product quality control strategy in three types of distribution channels , which will eliminate the influence of \u201cchannel conflict\u201d and \u201cfree-riding behavior\u201d. Most important, we will determine the manufacturer\u2019s product quality level, quality prevention effort level, wholesale price, direct sale price, and the retailer\u2019s product quality inspection effort level and retail price, the manufacturer\u2019s and retailer\u2019s expected profits function, and the final customer\u2019s consumer surplus. Then, we will conduct a numerical sample analysis that will indicate a specific application in practice.This paper is chiefly related to three streams of literature. The first stream is the research on how to design a distribution channels structure strategy, the different types of distribution channels structure and how to influence the product quality decision in a supply chain. Yunchuan Liu 2011)  establis  establiThe second stream pertains to designing a product quality contract and establishing a quality incentive mechanism in a supply chain. Peng Ma et al. 2013)  created 13  creatThe third stream of related literature concerns research on product quality risk sharing and the quality strategy of distribution channels in a supply chain. Zhu Lilong et al. (2011)  exploredIn this paper, first of all, we will introduce the distribution channel demand elasticity ratio and investigating how to construct a product quality control strategy model and channel coordination in three types of distribution channels ; what\u2019s more, we consider the manufacturer\u2019s product quality decision and quality prevention strategy, the retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision in a three-echelon supply chain; above all, we also establish a product quality control strategy model in three types of distribution channels to eliminate the influence of \u201cchannel conflict\u201d and \u201cfree-riding behavior\u201d, which will improve the manufacturer\u2019s and retailer\u2019s expected profits and the final customer\u2019s consumer surplus.The remainder of our paper is organized as follows: in section 3, we describe the model and the basic assumption; in section 4, we consider the product quality strategy in the direct channel and determine the first-best contact parameters; in section 5, we investigate the product quality strategy in the retail channel and establish the manufacturer\u2019s and retailer\u2019s stackelberg \u201cleader-follower\u201d quality control model, and we compare the contract parameter differences with the direct channel. In section 6, we investigate the product quality strategy in the mixed channel that includes a retail channel and a direct channel scenario simultaneously, and in section 7 we present a numerical example analysis to verify our model results. Finally, we provide the research conclusions and direction for future research.qi is the manufacturer\u2019s product quality level; furthermore, i \u2208 {D,R,MC} denote the direct channel, the retail channel and the mixed channel respectively. The product quality cost function is k is the manufacturer\u2019s production technology elasticity); so, we assume Ci (0) = Ci\u2032(0) = 0, Ci\u2032(+\u221e) = +\u221e, i.e. Ci(qi) is the convex function of increasing marginal cost. \u03bbm is the manufacturer\u2019s product quality prevention effort level, and \u03bbm \u2208 uniform distribution, i.e. a is the final customer lower limit of distribution quantity, b is the final customer upper limit of distribution quantity, and the corresponding final customer\u2019s consumer surplus is (\u03b8qi\u2013 pi).The final customer\u2019s quality utility is \u03b2i is the product demand price elasticity.The final customer\u2019s product demand function will be In this paper, the manufacturer will determine the three types of distribution channels including a direct channel, a retail channel and a mixed channel. The three-stage stackelberg game is in the following order: in stage one, the manufacturer determines the product quality level in a different distribution channel and determines the product quality prevention effort level; in stage two, the manufacturer determines the wholesale price in a retail channel or the direct sale price in a direct channel; and in stage three, the retailer determines the product quality inspection effort level and the retail price.And then, the three types of distribution channels decision system is described as qD, the quality prevention effort level \u03bbm and the direct sale price pD.In the direct channel, the manufacturer sells its product to the final customer directly through an internet or online ordering system; then, the manufacturer determines the product quality level, the quality prevention effort level and the direct sale price. Therefore, the manufacturer\u2019s expected profits\u2019 function model is as follows.Proposition 1 In the direct channel, with the final customer\u2019s product demand price elasticity decreases, the manufacturer\u2019s product quality level and direct sale price will increase, and the quality prevention effort level will also increase. In this scenario, the manufacturer\u2019s expected profits\u2019 function is concave; i.e. an optimal product quality level exists that will to be maximum. Then, the final customer\u2019s consumer surplus will increase with the decreasing in the demand price elasticity.Proof. Based on the stackelberg game analysis, this paper will use the backward induction method to solve the equation. Thus, using the first-order and second-order optimal condition with respect to pD in formula (1) yields the following:\u03bbm, which yields the following:pD and \u03bbmD is the manufacturer\u2019s first-best sales price, and the quality effort level occurs with a direct channel.qD in Thereafter, we use the first-order and second-order optimal conditions with respect to Based on proposition 1, we conclude that, in the direct channel, the manufacturer\u2019s product quality level, the direct sale price and the quality prevention effort level will increase with a decrease in the final customer\u2019s product demand price elasticity. In addition, the manufacturer\u2019s expected profits function is concave, and In the retail channel, the manufacturer sells product to the retailer, which will determine a product quality inspection strategy and then sell the product to the final customer. The manufacturer determines the product quality level, the quality prevention effort level and the wholesale price, and the retailer determines the quality inspection level and the retail price. Therefore, the manufacturer\u2019s and retailer\u2019s stackelberg \u201cleader-follower\u201d control model is described as follows:Proposition 2 In the retail channel, with the final customer\u2019s product demand price elasticity decreases, the manufacturer\u2019s product quality level and wholesale price will increase, and the retailer\u2019s product retail price will also increase. In comparison with the direct channel scenario, the product quality level and the retail price will be much higher.Proof. In this paper, we still use the backward induction method to solve the model. Thus, we use the first-order optimal condition with respect to pR and \u03bbr in formula (16), which yields the following:w, and we obtain that\u03bbm, which yields the following:\u03b2D = \u03b5\u03b2R, where \u03b5 is the demand elasticity ratio in a different distribution channel condition and \u03b5 >1. The demand price elasticity for the direct channel will be greater than for the retail channel; i.e., the final customers are more sensitive to price in the direct channel. Samar K.M (2008) earlier had proved that \u03b7m = \u03b7r.We compare Eqs Based on proposition 2, we conclude that the manufacturer\u2019s product quality level in the retail channel will be much higher than that in the direct channel scenario; the retailer\u2019s retail price will be greater than the wholesale price, which will be also much higher than the manufacturer\u2019s sales price in the direct channel scenario.Corollary 2.1\u03b5 > 2).Proof. We compare the manufacturer\u2019s quality prevention effort level and the retailer\u2019s quality inspection effort level in the retail channel with that in the direct channel; then, we determine thatBased on corollary 2.1, we can infer that the manufacturer\u2019s quality prevention effort level in the retail channel will be greater than the retailer\u2019s quality inspection effort level, which will also be greater than the manufacturer\u2019s quality prevention effort level in the direct channel.Corollary 2.2Proof. We substitute formula (25), (26) and (27) into Eqs Based on corollary 2.2, we conclude that the manufacturer\u2019s expected profits in the retail channel will be greater than that in the direct channel scenario.Corollary 2.3.R*CS  > D*CS .Proof. The final customer\u2019s consumer surplus in the retail channel will be described as followsBased on corollary 2.3, we conclude that the final customer\u2019s consumer surplus in the retail channel will be greater than that in the direct channel.T is the transfer payment.In the mixed channel, the manufacturer may sell a product to the final customer directly through an online ordering system or sell wholesale to the retailer who will continue to sell the product to the final customer. Thereafter, the manufacturer will determine a transfer payment to the retailer to eliminate the channel conflict. Therefore, the manufacturer determines the product quality level, the quality prevention effort level, the wholesale price and the direct sale price, and the retailer determines the quality inspection level and the retail price. The manufacturer and the retailer\u2019s stackelberg \u201cleader-follower\u201d control model can be described as follows:Proposition 3 In the mixed channel, in comparison with a direct channel and a retail channel scenario, the manufacturer\u2019s product quality level will be greater than which in the direct channel and less than which in the retail channel, i.e. Proof. We still use the backward induction method to solve the model. Thus, we use the first-order optimal condition with respect to Rp and r\u03bb in formula (32), which yields the following:w and Dp in formula (37) and obtain thatBased on proposition 3, we conclude that, in the mixed channel, the manufacturer\u2019s product quality level will be greater than which in the direct channel and less than which in the retail channel. In addition, the wholesale price will decrease, the manufacturer\u2019s direct sale price will increase and the retailer\u2019s retail price will decrease.Corollary 3.1Proof. We compare the manufacturer\u2019s quality prevention effort level and the retailer\u2019s quality inspection effort level in the mixed channel with which in the direct channel and the retail channel scenario and obtain the following:Based on corollary 3.1, we conclude that, in the mixed channel, the manufacturer\u2019s quality prevention effort level will be greater than that in the retail channel and the direct channel, and the retailer\u2019s quality inspection effort level will be greater than that in the retail channel, which will effectively eliminate the \u201cfree-riding behavior.Corollary 3.2Proof. We substitute formula (42), (43) and (44) into Eqs Based on corollary 3.2, we infer that, in the mixed channel, the manufacturer\u2019s expected profits will be less than that in the retail channel but will be greater than the profits in the direct channel; additionally, the retailer\u2019s expected profits will be greater than that in the retail channel.Corollary 3.3Proof. The final customer\u2019s consumer surplus in a mixed channel will be described as followsWe find that Based on corollary 3.3, we can infer that, in the mixed channel, the final customer\u2019s consumer surplus will be greater than that in the retail channel and the direct channel.k = 2, \u03b7m = \u03b7r = 1, \u03b1 = 60, \u03b8 ~ U, T = 120, \u03b5 = {2.5, 3.0}, \u03b2R ~ . We use numerical computing by Matlab 7.0 and obtain the results, as shown in Tables In this paper, we assume a manufacturer that sells computer components through a retailer (retail channel) or internet online system (direct channel) or through a mixed channel to the final customer. The parameters are described as follows: Based on Based on Based on Tables In this paper, we construct a product quality control model of three types of distribution channel  in a three-echelon supply chain, which is comprised by one manufacturer, one retailer and the final customer, and then we discuss how to design a distribution channel strategy and craft a quality control strategy. Furthermore, our paper analyzes three types of distribution channel strategies regarding how to influence the manufacturer\u2019s product quality decision and quality prevention strategy, the retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision. We compare the product quality level in three types of distribution channels and solve the manufacturer\u2019s and retailer\u2019s expected profits functions and the final customer\u2019s consumer surplus. In addition, we introduce the distribution channel demand elasticity ratio to analyze the influence of determining the product quality control strategy.Our paper demonstrates that, in the direct channel, the manufacturer\u2019s product quality level, the quality prevention effort level and the direct sale price will increase, and the customer\u2019s consumer surplus will also increase with the decreasing in the products demand price elasticity. In addition, in the retail channel which is compared with the direct channel scenario, the manufacturer\u2019s product quality level, the wholesale price, the quality prevention effort level and expected profits will increase, and the retailer\u2019s retail price, the quality inspection effort level and the customer\u2019s consumer surplus will be much higher. In the mixed channel, the manufacturer will determine the transfer payment to eliminate channel conflict, the manufacturer\u2019s quality prevention effort level and the retailer\u2019s quality inspection effort level will increase, which will effectively eliminate the \u201cfree-riding behavior\u201d. In addition, the manufacturer\u2019s expected profits will be higher than that in the direct channel and less than that in the retail channel. The retailer\u2019s expected profits and the final consumer surplus will also increase, and our conclusions will be a strong complement to the research field. Most importantly, we conduct a numerical sample analysis that demonstrates the model\u2019s effectiveness and the conclusions\u2019 correctness and will also indicate a specific application in practice.In further research, we will assume that the manufacturer\u2019s quality prevention effort level and the retailer\u2019s quality inspection effort level have incomplete information regarding how to craft a product quality control strategy in three types of distribution channels; then, we will also attempt to construct a multi-stage, repeat and asymmetry information dynamic game model and analyze the distribution channel strategy regarding how to influence the manufacturer\u2019s and retailer\u2019s expected profits function, the final customer\u2019s consumer surplus and social welfare. 8 Jan 2020PONE-D-19-20228Supply Chain Product Quality Control Strategy in Three Types of Distribution ChannelsPLOS ONEDear Authors,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by 30.1.2020. When you are ready to submit your revision, log on to If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Dejan Dragan, PhDAcademic EditorPLOS ONEAdditional Editor Comments (if provided):Editor's initial comments to the paper:Supply Chain Product Quality Control Strategy in Three Types of Distribution ChannelsThe paper deploys a three-stage Stackelberg dynamic game analysis and constructs a product quality control strategy model for three types of distribution channels  in a three-echelon supply chain, which is composed of one manufacturer, one retailer and the final customer. Furthermore, the paper studies how to design a distribution channel strategy and provides a product quality control strategy. Here, three types of distribution channels strategy in the context of how they influence a manufacturer\u2019s product quality decision and prevention strategy, a retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision, are analyzed. The subject of this research is up-to-date and fundamentally interesting for scholars from the field of SCM and OR.The paper, in general, roughly satisfies all rigor requirements that are demanded from Plos One. The red clue remains more or less consistent all over the paper, while the derivations of equations seem to be adequate.However, the editor has detected some major issues based on his own perception, the review of the additional reviewer, and the Plos One criteria for the papers, which should be corrected prior to the further publishing process.Comments of the AE:1. It is not clear enough emphasized what the main contribution of the paper is, i.e., what has been done new, if compared with the work of the other researchers. Here, the borderline should be clearly highlighted, while the main contributions  should be more explicitly given in the introduction and other places, where necessary.2. Figure 1 should be improved in the sense of informative content meaning that the reader immediately understands the main point without even looking at the corresponding text.Comments regarding the Plos One criteria for the papers:http://journals.plos.org/plosone/s/submission-guidelines#loc-methods-software-databases-and-tools. On this basis, the AE warns the authors to reconsider these facts and give the rationale and/or explanation fortified with the facts, let be in the paper itself or in the answers to reviewers or cover letter.There exists a certain doubt whether the paper meets PLOS ONE criteria for papers that describe new methods or software for applications. Specifically, these reports must meet the criteria of utility, validation, and availability, which are described in detail at Comments of Reviewer #1Summary (see reviewer 1 comments.pdf):Additional comments are available in the file: reviewer 1 comments.pdf.Journal Requirements:When submitting your revision, we need you to address these additional requirements:http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: 1. The language of the article should be more concise and accurate.2. It is better to complement the differences between this study and the existing research literature.3. The conclusions of the study for the the objectives mentioned in the abstract as \u2018This paper studies how to design a distribution channel strategy and provides a product quality control strategy\u2019 are not very clear.4. In this article, we can not see how consumer utility and consumer behavior affect product market demand.5. In Mixed Channel, how does the product demand between the two channels affect each other?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentEditor - initial review.docxSubmitted filename: Click here for additional data file.AttachmentReviewer 1 Comments.pdfSubmitted filename: Click here for additional data file. 2 Feb 2020Responses to Editors and ReviewersDear Professor Dejan Dragan and Reviewers,Thank you very much for your suggestions and critical comments about our paper submitted to PLOS ONE (Manuscript ID: PONE-D-19-20228). The revised title is \u201cSupply Chain Product Quality Control Strategy in Three Types of Distribution Channels\u201d.We are also thankful to the reviewers for their critical reading and valuable comments on the manuscript. Those comments were very helpful for providing direction for our further studies. We have tried our best to revise our manuscript according to the comments. Attached, please find the revised version, which we would like to resubmit for your kind consideration. The main revised parts are marked in blue in the paper. The following is a detailed explanation how we have complied with the editor\u2019s and reviewers\u2019 suggestions.Responds to the editor\u2019s comments:Comment #1:The paper deploys a three-stage Stackelberg dynamic game analysis and constructs a product quality control strategy model for three types of distribution channels  in a three-echelon supply chain, which is composed of one manufacturer, one retailer and the final customer. Furthermore, the paper studies how to design a distribution channel strategy and provides a product quality control strategy. Here, three types of distribution channels strategy in the context of how they influence a manufacturer\u2019s product quality decision and prevention strategy, a retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision, are analyzed. The subject of this research is up-to-date and fundamentally interesting for scholars from the field of SCM and OR.The paper, in general, roughly satisfies all rigor requirements that are demanded from Plos One. The red clue remains more or less consistent all over the paper, while the derivations of equations seem to be adequate.Response: Thank you very much for the highly praises and the valuable suggestions. Our paper constructs a product quality control strategy model for three types of distribution channels  in a three-echelon supply chain which use a three-stage stackelberg dynamic game analysis, and then, analyzes three types of distribution channels strategy in the context of how they influence a manufacturer\u2019s product quality decision and quality prevention strategy, a retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision. Most importantly, we conduct a numerical sample analysis that will prove the model\u2019s effectiveness and indicate a specific application in practice.Comment #2: It is not clear enough emphasized what the main contribution of the paper is, i.e., what has been done new, if compared with the work of the other researchers. Here, the borderline should be clearly highlighted, while the main contributions  should be more explicitly given in the introduction and other places, where necessary.Response: Thank you very much for the valuable suggestions. We rewrite and emphasize the paper\u2019s main contributions in every paragraph in \u201c2 Related Literature\u201d, just like: (1) Our paper differs from the existing literature by introducing the distribution channel demand elasticity ratio and investigating how to construct a product quality control strategy model and channel coordination in three types of distribution channels  by providing a new perspective. (2) Our model contributes to the product quality control strategy research by constructing a distribution channel model in a three-echelon supply chain which is composed of one manufacturer, one retailer and the final customer based on a three-stage stackelberg dynamic game. Then, the model considers the manufacturer\u2019s product quality decision and quality prevention strategy, the retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision. (3) we also establish a product quality control strategy model in three types of distribution channels to eliminate the influence of \u201cchannel conflict\u201d and \u201cfree-riding behavior\u201d, which will improve the manufacturer\u2019s and retailer\u2019s expected profits and the final customer\u2019s consumer surplus.Comment #3: Figure 1 should be improved in the sense of informative content meaning that the reader immediately understands the main point without even looking at the corresponding text.Response: Thank you very much for the valuable suggestions. We redraw the Figure 1 Three types of distribution channels decision system which improve in the sense of informative content meaning that the reader immediately understands the main point of our paper.Figure 1 Three types of distribution channels decision systemResponds to the reviewer\u2019s comments:Comment #1:The language of the article should be more concise and accurate.Response: Thank you very much for the valuable suggestions. We made necessary revisions and language editing in the manuscript according to your suggestions, and the English has also been edited by Wiley English Language Editing Services.Comment #2:It is better to complement the differences between this study and the existing research literature.Response: Thank you very much for the valuable suggestions. We rewrite and emphasize the paper\u2019s main contributions in every paragraph in \u201c2 Related Literature\u201d, just like: (1) Our paper differs from the existing literature by introducing the distribution channel demand elasticity ratio and investigating how to construct a product quality control strategy model and channel coordination in three types of distribution channels  by providing a new perspective. (2) Our model contributes to the product quality control strategy research by constructing a distribution channel model in a three-echelon supply chain which is composed of one manufacturer, one retailer and the final customer based on a three-stage stackelberg dynamic game. Then, the model considers the manufacturer\u2019s product quality decision and quality prevention strategy, the retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision. (3) we also establish a product quality control strategy model in three types of distribution channels to eliminate the influence of \u201cchannel conflict\u201d and \u201cfree-riding behavior\u201d, which will improve the manufacturer\u2019s and retailer\u2019s expected profits and the final customer\u2019s consumer surplus.Comment #3: The conclusions of the study for the objectives mentioned in the abstract as \u2018This paper studies how to design a distribution channel strategy and provides a product quality control strategy\u2019 are not very clear.Response: Thank you very much for the valuable suggestions. With the changing in customer or consumer buying behavior, more and more companies are beginning to redesign or rebuild their distribution channel structure, Such as HP, Nike, Lenovo, in addition to focus on the traditional retail channel, have also opened up an internet channel(direct channel); Dell, MI has been focused on internet channel in the past, and now also began selling products in traditional retail channel; and Apple, Haier sell their products in the traditional retail channel and internet channel in the same time, which used a mixed channel structure. In our paper, we will construct a distribution channel strategy model in a three echelon supply chain that is composed of one manufacturer, one retailer and a final customer based on a three-stage stackelberg dynamic game. Furthermore, we will introduce the distribution channel demand elasticity ratio and investigate how to craft a product quality control strategy in three types of distribution channels , Most important, we will determine the manufacturer\u2019s product quality level, quality prevention effort level, wholesale price, direct sale price, and the retailer\u2019s product quality inspection effort level and retail price, the manufacturer\u2019s and retailer\u2019s expected profits function, and the final customer\u2019s consumer surplus.Comment #4: In this article, we can not see how consumer utility and consumer behavior affect product market demand.Response: Thank you very much for the valuable suggestions. In \u201c3 The Model and Assumption\u201d, we describe that the final customer\u2019s quality utility is, and denotes the type of final customer, is the manufacturer\u2019s product quality level; furthermore, denote the direct channel, the retail channel and the mixed channel respectively; then, we assume \uff5e uniform distribution, and the corresponding final customer\u2019s consumer surplus is. Therefore, in direct channel, we can describe the final customer\u2019s consumer surplus as = = (14)In retail channel, we can describe the final customer\u2019s consumer surplus as = = (30)In mixed channel, we can describe the final customer\u2019s consumer surplus as = + = (52)Comment #5: In Mixed Channel, how does the product demand between the two channels affect each other?Response: Thank you very much for the valuable suggestions. In our paper, we describe the mixed channel that the manufacturer sells their products in the traditional retail channel by the retailer, in the same time, the manufacturer sells their products to the final customer in direct channel (internet channel). The final customer\u2019s product demand function will be ; denotes the market maximum demand, and is the product demand price elasticity. Therefore, In mixed channel, the manufacturer and the retailer\u2019s stackelberg \u201cleader-follower\u201d control model can be described as follows:  = + - - (31) s.t.  = (32)The product demand in retail channel as The product demand in direct channel as  is the manufacturer\u2019s product quality level in mixed channel, is the product demand price elasticity in retail channel, is the product demand price elasticity in direct channel, is the manufacturer\u2019s wholesale price, is the retailer\u2019s retail price, is the direct sale price in a direct channel, the manufacturer\u2019s product quality cost function in direct channel is .We have tried our best to improve the manuscript and made some substantial changes and necessary deletions according to the editors\u2019 and reviewers\u2019 comments. We earnestly appreciate the editors\u2019 and reviewers\u2019 professional work and hope that the corrections will make our manuscript suitable for publication in PLOS ONE. We are looking forward to receiving comments from reviewers in the future.Once again, thank you very much for your valuable comments and suggestions.Best wishes.Yours sincerely,Lilong Zhu (Correspondence)zhulilong2008@126.comE-mail: Tel: + 86 13853193366, Fax: + 86 531 8618 2769School of Management, Shandong University, Ji\u2019nan Shandong, 250100, ChinaCollege of Business, Shandong Normal University, Ji\u2019nan 250014, Shandong, ChinaAttachmentResponse to Reviewers.docSubmitted filename: Click here for additional data file. 16 Mar 2020PONE-D-19-20228R1Supply Chain Product Quality Control Strategy in Three Types of Distribution ChannelsPLOS ONEDear Authors,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.==============================Please see below==============================https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by 29.3.2020. When you are ready to submit your revision, log on to If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Dejan Dragan, PhDAcademic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #2: (No Response)Reviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: YesReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: YesReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #2: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #2: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #2: Really, the paper has been greatly improved as all comments provided by all other reviewers in the first round.Reviewer #3: 1. It is a very nice thing that the new literature review part is added. But it seems to be to be too detailed. It will be better that the author just mentioned 2~3 related papers and on top of that, provide their own contributions. Moreover, those contributions should be more precise, instead of \"..by providing a newperspective.\", the author need to be more specific about what exactly the new perspective is.2. The model and assumption need to be more concise and concrete. For example, when the author mentioned the customer type follow a U, it needs more clarify about the value means and the types they are referring to. It will be better if the author check the model and assumption part one more time and provide additional explanation if necessary**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  25 Mar 2020Responses to Editors and ReviewersDear Professor Dejan Dragan and Reviewers,Thank you very much for your suggestions and valuable comments about our paper submitted to PLOS ONE (Manuscript ID: PONE-D-19-20228R1). The manuscript\u2019s title is \u201cSupply Chain Product Quality Control Strategy in Three Types of Distribution Channels\u201d.We are also thankful to the reviewers for their critical reading and valuable comments on the manuscript. Those comments were very helpful for providing direction for our further studies. We have tried our best to revise our manuscript according to the comments. Attached, please find the revised version, which we would like to resubmit for your kind consideration. The main revised parts are marked in red in the paper. The following is a detailed explanation how we have complied with the editor\u2019s and reviewers\u2019 suggestions.Responds to the editor\u2019s comments:Comment:Please include the following items when submitting your revised manuscript:\u2022 A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.\u2022 A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.\u2022 An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.Response: Thank you very much for the highly praises and the valuable suggestions for this manuscript. According to the editor\u2019s and reviewers\u2019 suggestions, we have completed all the revision which each point raised by the academic editor and reviewers, the file is labeled 'Response to Reviewers'. We have completed a marked-up copy of our manuscript that highlights changes made to the original version, the file is labeled 'Revised Manuscript with Track Changes'. We also have completed an unmarked version of our revised paper without tracked changes, the file is labeled 'Manuscript'.Comment #2: Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.Response: Yes, I would like to make the peer review history publicly available.Responds to the reviewer\u2019s comments:Comment #1:If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #2: (No Response)Reviewer #3: All comments have been addressedResponse: Thank you very much for the valuable suggestions. We have addressed and completed all the revised comments in the new 'Manuscript'.Comment #2:Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #2: YesReviewer #3: YesResponse: Thank you very much for the valuable suggestions. In this manuscript, all the data support the conclusions.Comment #3:Has the statistical analysis been performed appropriately and rigorously?Reviewer #2: YesReviewer #3: YesResponse: Thank you very much for the valuable suggestions.Comment #4:Have the authors made all data underlying the findings in their manuscript fully available?Reviewer #2: YesReviewer #3: YesResponse: Thank you very much for the valuable suggestions.Comment #5:Is the manuscript presented in an intelligible fashion and written in standard English?Reviewer #2: YesReviewer #3: YesResponse: Thank you very much for the valuable suggestions. We made necessary revisions and language editing in the manuscript according to your suggestions, and the English has also been edited by Wiley English Language Editing Services.Comment #6:Review Comments to the Author.Reviewer #2: Really, the paper has been greatly improved as all comments provided by all other reviewers in the first round.Reviewer #3: 1. It is a very nice thing that the new literature review part is added. But it seems to be to be too detailed. It will be better that the author just mentioned 2~3 related papers and on top of that, provide their own contributions. Moreover, those contributions should be more precise, instead of \". by providing a new perspective, the author need to be more specific about what exactly the new perspective is.2. The model and assumption need to be more concise and concrete. For example, when the author mentioned the customer type follow a U, it needs more clarify about the value means and the types they are referring to. It will be better if the author check the model and assumption part one more time and provide additional explanation if necessary.Response to Reviewer #2: Thank you very much for the valuable suggestions. We have revised and completed all comments provided by the editor and reviewers in the first round.Response to Reviewer #3: Thank you very much for the valuable suggestions.1. We have revised the literature review part, which is chiefly related to three streams of literature, it seems not to be too detailed, which is mentioned the related papers and on top of that, provide their own contributions. Moreover, those contributions should be more precise as line 150-160.In this paper, first of all, we will introduce the distribution channel demand elasticity ratio and investigating how to construct a product quality control strategy model and channel coordination in three types of distribution channels ; what\u2019s more, we consider the manufacturer\u2019s product quality decision and quality prevention strategy, the retailer\u2019s product pricing decision and quality inspection strategy, and the final customer\u2019s product demand decision in a three-echelon supply chain; above all, we also establish a product quality control strategy model in three types of distribution channels to eliminate the influence of \u201cchannel conflict\u201d and \u201cfree-riding behavior\u201d, which will improve the manufacturer\u2019s and retailer\u2019s expected profits and the final customer\u2019s consumer surplus.2. We have revised model and assumption more concise and concrete in line 174-l96. For example, The final customer\u2019s quality utility is , and denotes the type of final customer; then, we assume \u019f~U uniform distribution, i.e. a is the final customer lower limit of distribution quantity, b is the final customer upper limit of distribution quantity, and the corresponding final customer\u2019s consumer surplus is .We have checked the model and assumption part one more time, and then, provided additional explanation more clearly and accurately.We have tried our best to improve the manuscript and made some substantial changes and necessary deletions according to the editors\u2019 and reviewers\u2019 comments. We earnestly appreciate the editors\u2019 and reviewers\u2019 professional work and hope that the corrections will make our manuscript suitable for publication in PLOS ONE. We are looking forward to receiving comments from reviewers in the future.Once again, thank you very much for your valuable comments and suggestions.Best wishes.Sincerely yours,Lilong ZhuAttachmentResponse to Reviewers.docSubmitted filename: Click here for additional data file. 31 Mar 2020Supply Chain Product Quality Control Strategy in Three Types of Distribution ChannelsPONE-D-19-20228R2Dear authors,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the \"Update My Information\" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Dejan Dragan, PhDAcademic EditorPLOS ONEAdditional Editor Comments :The improvements have been noticeable so that the paper deserves to be published in the Plos One.Reviewers' comments: 6 Apr 2020PONE-D-19-20228R2 Supply Chain Product Quality Control Strategy in Three Types of Distribution Channels Dear Dr. Zhu:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Dejan Dragan Academic EditorPLOS ONE"}
{"text": "During mechanical recanalization of large vessel occlusions (LVO), the use of proximal flow arrest with balloon guide catheters (BGC) was shown to be associated with better angiographic and even clinical outcome. The aim of the study was to analyze the impact of BGC use on microstructural alterations in the salvaged penumbra.All patients who underwent mechanical recanalization of LVO of the anterior circulation were reviewed within a prospective stroke registry of a single comprehensive stroke center. Fifty-two patients received an admission CT perfusion together with post-interventional diffusion tensor imaging. Technical details such as BGC usage were correlated with microstructural integrity changes of the salvaged gray matter through the mean diffusivity (MD) index. Moderation analysis was performed to test the interaction of BGC on the correlation between angiographic and clinical outcomes.p = 0.04). The importance of complete reperfusion for good clinical outcome is predominantly based on patients treated with BGC .For all patients with complete reperfusion, microstructural integrity changes with lowered MD index were found within the salvaged penumbra for cases of non-BGC usage (mean \u2212 0.02) compared to cases with BGC usage  contains supplementary material, which is available to authorized users. Endovascular mechanical thrombectomy (MT) is used as the current standard procedure for large intracranial vessel occlusions (LVO) of the anterior circulation, based on the major five randomized trials, in which so-called stent retrievers were an integral part of endovascular therapy (EVT) \u20135. RecenEspecially the use of balloon guide catheters (BGC) has been established within the standard procedure in many neuro-interventional institutions by now, as it became evident that improved angiographic and clinical results can be achieved by using this technique \u201313. ThisThe aim of acute stroke treatment is to save the so-called penumbra. \u201cPenumbra\u201d means the potentially salvageable brain tissue that is at risk of definite infarction if no successful reperfusion therapy will be performed immediately.The penumbra presents the range of cerebral perfusion between temporary loss of electrical activity and irreversible neuronal depolarization and plays an essential role as the degree of \u201ctissue at risk\u201d can be assessed , 16. ThiPrevious analyses have suggested that microstructural alterations also occur in the salvaged penumbra, which does not undergo final infarction \u201321. MicrWe hypothesize that the processes that take place in the penumbra until successful reperfusion are influenced by technical specifications of the MT technique such as usage of BGC.Therefore, the primary aim of the present study was to analyze the impact of applied procedural techniques on penumbral processes. A specific focus was laid on the use of BGC, and the microstructural changes within the salvaged penumbra in the acute stroke phase after mechanical recanalization of an acute intracranial vessel occlusion were assessed. Additionally, the interaction of the techniques on the correlation between angiographic and clinical outcomes should be tested.The study cohort is based on a prospectively collected stroke registry of a single comprehensive stroke center. All patients treated with MT between April 2016 and December 2018 were included. The database query revealed four hundred thirty-nine patients that had undergone EVT due to LVO of the anterior circulation .n = 192). Exclusion criteria were incomplete or insufficient MRI acquisition (n = 20), space-consuming malignant infarction, or intracranial hemorrhage (n = 7). From the residual 165 patients, 52 met the further required inclusion criteria: (a) in-house-acquired admission CT perfusion imaging that was essential to determine the penumbral tissue immediately before EVT; (b) clearly identifiable angiographic technique  after MT, including diffusion tensor imaging (DTI) and structural T1-weighted imaging  score at the time of admission and at the time of MRI acquisition. The modified Rankin Scale (mRS) score was used to measure disability at day 90 after stroke onset, either on a routinely scheduled clinical visit or by a structured telephone interview.This study was approved by the local ethics committee and the need for patient consent was waived.For all study patients, the procedure of MT was classified by two experienced neuro-interventionalists  in consensus regarding the technical approach. The following categories of intervention techniques were differentiated  is composed of all patients that were treated with proximal flow arrest in a setting of BGC usage (PROTECT or PROTECTPLUS):n = 11) [PROTECT: PRoximal balloon Occlusion TogEther with direCt Thrombus aspiration during stent retriever thrombectomy (n = 11) : BGC + aPLUS (n = 18) [PROTECT(n = 18) : BGC + aThe BGC group (n = 23) is composed of all patients treated without BGC usage:SOLUMBRA : GuidingThe non-BGC group ) were excluded for direct comparisons of BGC vs. non-BGC. A possible bias within this group arises from the high conversion rates of this approach (secondary stent retriever application). Consequently, procedural and outcome results of the cases that used only the ADAPT technique are artificially good.Due to the exclusion criteria of mixed techniques , patients treated with only ADAPT  was necessary.The modified thrombolysis in cerebral infarction (mTICI) score  was deteSalvaged penumbral tissue was identified for each patient. Final infarct assessed with an MRI acquisition including DTI in the acute post-stroke phase was subtracted from the tissue at risk (penumbra) of co-aligned admission CT perfusion imaging. Hereby, salvaged penumbra was identified in the acute post-stroke phase and its microstructural integrity was analyzed see Fig. .Fig. 1ExTo identify the tissue at risk (penumbra) in the acute stage immediately before mechanical recanalization, CT perfusion imaging was acquired and post-processed by the fully automated software RAPID (iSchemaView Inc.). Perfusion maps were verified by an at least 3-year experienced neuroradiologist (M.B.)  with suI \u2212 MDH) / (MDI + MDH). A negative MD index means lower MD values within the penumbral gray matter compared with the non-affected side. In a previous work, this MD index was effectively used for characterizing microstructural integrity within the salvaged penumbra [By co-registering of admission CT perfusion imaging and MRI acquisition in the acute post-stroke phase, salvaged penumbra was calculated for each patient by a subtraction of tissue at risk and final infarction. After processing of DTI images using FSL\u2019s FDT toolbox, MD maps were created. To find gray matter alterations within the salvaged penumbra, MD values of the penumbra on the infarcted side (I) were assessed and compared with MD values of the corresponding voxels on the contralateral, non-affected side (H). MD index was calculated by using the following formula: MD index =  and good clinical outcome (mRS \u2264 2 after 90 days) under consideration of the covariates age, sex, and reperfusion time. Moderation analysis was performed to check the interaction of the subgroups of BGC/non-BGC on the association between complete reperfusion and good clinical outcome.In total, 52 patients met the inclusion criteria as described above. An overview of demographic, clinical, and interventional data of patients and a comparison between the BGC and non-BGC groups can be found in Table p = 0.016).For completely reperfused patients, there is a significantly lower incidence of peripheral emboli in the BGC group (50%) in comparison with the non-BGC group .For directly comparing penumbral integrity of the different techniques, the precondition of mTICI 3 should be met. MD index of the BGC group shows significantly higher values (mean \u00b1 SD: 0.009 \u00b1 0.035) than MD index of the non-BGC group . To exclude an influence of the volume of infarction on the impact of BGC on MD index as mentioned above, volume distributions for the technique groups in dependency of angiographic success can be found in Fig. p = 0.5) groups.Values of MD index are inversely correlated to the volume of infarction (Pearson\u2019s p = 0.017). The covariates age, sex, and reperfusion time showed no significant influence on the analysis.Complete reperfusion success (mTICI 3) is significantly associated with good clinical outcome (mRS \u2264 2 after 90 days) in a logistic regression analysis . For the non-BGC group, the conditional effect was lowered and lost its significance on the 5% level . That implies that the importance of mTICI 3 reperfusion for good clinical outcome is predominantly based on patients treated with BGC.In a second step, the impact of the subgroups BGC/non-BGC on this association was tested in a moderation analysis under consideration of the abovementioned covariates. The conditional effect of complete reperfusion success on good clinical outcome remained significant for the BGC group  by BGC usage. The main new finding is the preserved microstructural integrity (higher MD index) when using BGC that is known to impact the clinical course of patients . To avoiAs expected, there is an interaction of MD index of the salvaged penumbra with the volume of infarction. The more the ischemic demarcation is visible, the higher the integrity loss of salvaged penumbra can be measured. This coherence makes it necessary to check the distribution of ischemic volume for the technical subgroups (BGC/non-BGC) that in the end showed no volume differences. That confirms the supposed hypothesis that BGC usage saves the microstructural integrity of the salvaged penumbral tissue for the case of complete reperfusion, independently from the extent of final infarction. However, this point does not apply to the same extent to incomplete reperfusions. The remaining vessel occlusions impact penumbral tissue integrity so much (broad range of infarction volume for both groups) that no clear microstructural benefit is detectable for the cases of BGC usage, but only a trend to higher MD indices.It can be supposed that clot fragmentation and micro-embolization into the occlusion-dependent brain tissue during MT would affect microstructural, ischemic-like damage within the salvaged penumbra. This damage can be measured by MD index that gives information about the tissue integrity of the salvaged gray matter, compared with the contralateral non-affected hemisphere. By assessing MD values using DTI, changes of the molecular diffusion rate can be detected that give information about subtle integrity alterations . For theA previous study showed that MD index for gray matter is a very accurate parameter for characterizing penumbral integrity . CertainFinally, it cannot be proven that the microstructural damage in the salvaged penumbra, that was shown for the non-BGC group, did not exist before recanalization. However, the mTICI 3/BGC group showed no microstructural damage, which implies that this developed secondarily, possibly induced by clot fragmentation and micro-embolization. A bias in performing the groups of BGC/non-BGC cannot be excluded, but it is rather unlikely in the study\u2019s cohort: since the implementation of BGC in this single comprehensive stroke center, nearly every endovascular treatment was made by the use of BGC, and the non-BGC group consists of patients mostly treated in 2016\u20132017, before BGC was routinely applied. The exclusion of ADAPT cases reduces the generalizability of the findings as these are only applicable for the case of stent retriever application. A further limitation is the moderate sample size within the groups that makes a fine matched analysis too powerless. Especially in the beginning phase of the study, not all patients who were mechanically recanalized were examined with MRI, arising from internal organization problems . Patients with disability for an MRI examination could also not be included. These patients often present extended ischemia or space-consuming hemorrhage that makes a DTI analysis impossible. This subgroup of patients with distinct worse outcome is missing in the analysis.In summary, the present study highlights the importance of technical aspects during mechanical recanalization of acute LVO. With the use of DTI, it was shown that achieving complete reperfusion in a setting of BGC usage minimizes microstructural tissue damage in the salvaged penumbra and improves long-term outcome. This reinforces the importance of using BGC in endovascular stroke treatment.ESM 1(DOCX 32 kb)"}
{"text": "Growth behavior, of both shoots and roots, was recorded and correlated to plant physiological responses. 11CO2 fixation, leaf export of [11C]-photosynthates, and their rate of transport increased systematically with increasing BA concentrations, while the fraction of [11C]-photosynthates delivered to the roots under 0 mM and 0.5 mM BA treatments was lower than under 0.05 mM BA treatment, likely due to changes in root growth. Additionally, solid-phase extraction coupled with gamma counting, radio-fluorescence thin layer chromatography, and radio-fluorescence high-performance liquid chromatography techniques applied to tissue extracts provided insight into the effects of BA treatment on \u2018new\u2019 carbon (as 11C) metabolism. Most notable was the strong influence reducing boron levels had on raising 11C partitioning into glutamine, aspartic acid, and asparagine. Altogether, the growth of maize under different regimes of boron affected 11CO2 fixation, its metabolism and allocation belowground, and altered root growth. Finally, inductively coupled plasma mass spectrometry provided insight into the effects of BA treatment on plant uptake of other essential nutrients. Here, levels of boron and zinc systematically increased in foliar tissues with increasing BA concentration. However, levels of magnesium, potassium, calcium, manganese, and iron remained unaffected by treatment. The rise in foliar zinc levels with increased BA concentration may contribute to improved 11CO2 fixation under these conditions.In agriculture, boron is known to play a critical role in healthy plant growth. To dissect the role of boron in maize metabolism, radioactive carbon-11 (t Crop yields are negatively impacted by various biotic and abiotic stresses and by nutrient deficiencies. Boron is a micronutrient essential for plant growth and development , affectiBoron deficiency primarily leads to a cessation of growth in plant meristems (groups of undifferentiated stem cells) ,11,12. BZea mays (maize), have a lower boron requirement compared to dicotyledonous species -photosynthates -Physiology Measurements: After 11CO2 pulsing, plants were incubated for 3 h before separating the load leaf from shoots and roots. During that time, levels of radioactivity were monitored using two radiation detectors  affixed to the plant 8 cm above the base of the stem and below the base of the stem . Measurement of 11C radioactivity was performed using gamma counting and data was decay-corrected to end of bombardment. The individual components were summed together for total plant 11C radioactivity and normalized based on fresh tissue weight. Individual components were used to calculate leaf export and root allocation fractions.Whole-Plant [the stem  which pr11C]-Metabolite Analyses: After 11CO2 pulsing, the load leaf was removed 20 min later and subjected to metabolite analyses following published procedures -sugars were measured by radio thin layer chromatography (TLC) using glass-backed NH2-silica HPTLC plates  purchased from Sorbent Technologies  according to published procedures -sugar content was related to the 11C radioactivity quantified along the sample lane of the TLC plate and then corrected to percent total fixed 11CO2 using gamma count data from the insoluble and soluble fractions.Total soluble [ocedures . A mobil11C]-amino acids were analyzed following published procedures [v/v with 0.5 M sodium acetate:methanol) and Solvent B (70:30 methanol:18 M\u2126 water) starting at 75:25 and switching to 25:75 within 30 min at a flow rate of 0.7 mL min\u22121. On-line fluorescence detection  was used for correlating retention times of standards with those of [11C]-amino acids in biological samples. A NaI gamma radiation detector  enabled direct measurement of radiolabeled metabolites. Data was acquired using PeakSimple\u2122 chromatography software  and radioactivity assigned to peaks was corrected for radioactive decay, summed for a total [11C]-amino acid value, and related back to the amount of 11C radioactivity fixed by the plant at the start of the study  using 1 N NaOH, and the total volume was processed through an Accell QMA Plus Light Sep-Pak\u2122  followed by rinsing of the contents with 10 mL of DI water. Cartridges were then counted for 11C radioactivity with a NaI gamma counter.For [11C]-basic metabolite analysis, 200 mL of tissue extract was processed through a strong cation exchange cartridge  followed by rinsing of the cartridge using 10 mL of DI water. The cartridge was subjected to gamma counting for quantification of this metabolite pool.For [Inductively Coupled Plasma-Mass Spectrometry (ICP-MS): For ICP-MS analyses of nutrients, leaf 2 samples were harvested at the V2 developmental stage from plants, and 1 cm of leaf tips and lower sheath regions were removed. Tissues were air dried at 70 \u00b0C for 1 week and ground to a powder using a mortar and pestle. Ground tissue was weighed and digested in 3.0 mL of concentrated nitric acid at 190 \u00b0C using a Milestone Ethos Plus  microwave digestion system, then diluted to 50 mL with ultrapure water followed by gravimetric dilution by a factor of 10 with 0.45 N nitric acid. Samples were analyzed with a Perkin-Elmer NexION ICP-MS in Kinetic Energy Discrimination mode. Reference materials included NIST SRM 1570 spinach leaves and NIST SRM 1573 tomato leaves prepared in the same way. Internal standards at known concentrations were prepared from stock solutions  and used to calibrate instrument response.p < 0.05.Statistical Analysis: Data was subjected to the Shapiro\u2013Wilk Normality Test to identify outliers, so all data groups reflected normal distributions. Data was then subjected to one-way analysis of variance (ANOVA) in R using the multcompview package . Tukey\u2019s11C-radiotracing allowed us to quantify changes in the plants\u2019 physiological status by examining changes in 11CO2 fixation, leaf export of 11C-photosynthates, their rate of transport, and the amount of photosynthates that were allocated to roots. Furthermore, these biological functions were correlated with the growth performance of plants, both aboveground and belowground, as well as correlated with plant uptake of essential nutrients. Finally, 11C-radiotracing provided a way to examine changes in metabolic regulation by measuring 11C-partitioning across all the plant metabolite pools and by measuring certain individual metabolites within these pools. Our study therefore provides unprecedented insight into the effects of boron on maize physiological and metabolic processes. By examining the flux of \u2018new\u2019 carbon (as 11C) into metabolites, one can often gain greater insight into boron\u2019s influence on plant metabolism and its regulation than by simply profiling the endogenous metabolite concentrations. Most notable here were the strong influence that reducing boron levels had on raising 11C partitioning into glutamine, aspartic acid, and asparagine, which may be part of a plant stress response to boron deficiency.Using a combination of radiotracer tools and ICP-MS, we were able to gain new insight into the physiological and metabolic responses of maize plants subjected to different levels of BA treatment during growth spanning low to high boron levels. 11CO2 fixation under these conditions. Altogether, boron appears to have a selective effect on the uptake of only certain plant micronutrients. Further studies will be needed to elucidate the mechanisms for this behavior.Finally, inductively coupled plasma mass spectrometry provided another layer of insight into the effects of boron on plant uptake of essential micronutrients. Here, levels of boron and zinc were seen to systematically increase in foliar tissues with increasing BA concentration, while levels of magnesium, potassium, calcium, manganese, and iron remained unaffected by treatment. This rise in foliar zinc levels with increased BA concentrations may contribute to improved"}
{"text": "Staphylococcus spp. isolates recovered from raw goat milk in Brazil.Antimicrobial resistance poses a major threat to global public health. Foodstuff of animal origin can serve as potential vehicles for the dissemination of antimicrobial-resistant bacteria and resistance genes to consumers. In view of the lack of knowledge about antimicrobial resistance in bacteria associated with goat milk, the aim of this study was to report species-level identification and antimicrobial susceptibility profiles of a large collection of Staphylococcus spp. isolates originated from 510 goat milk samples in Northeast Brazil were investigated. The isolates were obtained by conventional microbiological methods. Species identification and antimicrobial susceptibility testing were performed by means of a semi-automated system using a panel for biochemical tests and broth microdilution method for 19 antimicrobial drugs.A total of 434 Staphylococcus aureus (22.6%) accounted for the majority of the isolates, a total of 13 different non-aureus staphylococci spp. were identified. High resistance rates against erythromycin (40.8%), and the beta-lactams ampicillin (45.9%) and penicillin (42.9%) were observed among S. aureus isolates. The most significant findings were related to the resistance against quinupristin-dalfopristin, a drug of last resort used in human medicine to treat infections caused by vancomycin-resistant S. aureus and enterococci.Although Staphylococcus spp. showing phenotypic resistance against different antimicrobial drugs encourages further investigations on the real impact of these bacteria as reservoirs of antimicrobial resistance genes to consumers. Furthermore, the potential impact of technological processes, such as pasteurization, fermentation, and maturation, on the maintenance and dissemination of antimicrobial resistance among the microbial populations in milk and dairy products must also be investigated.The high diversity of  Opuntia ficus) as fodder [Paraiba is the leading goat milk producer state in Brazil, with a total production of 5.63 million litters, representing 23.9% of the revenue attributed to the whole Brazilian goat milk sector . In ParaStaphylococcus spp. are of public health concern. Although the extensive use of antimicrobials in human medicine is a major driver for the emergence and dissemination of antimicrobial resistance [Staphylococcus spp., the most common disease affecting the dairy industry worldwide [Staphylococcus associated with intramammary infections in goats is limited [Staphylococcus associated with milk and dairy products, as the majority of the studies identify isolates as Staphylococcus aureus and non-aureus staphylococci (NAS) spp. The identification of the most frequent Staphylococcus spp. and the determination of their antimicrobial susceptibility rates are important for controlling and prevention purposes at both farm and industry levels [Although dairy goat production plays an important socioeconomic role in low-income Northeast Brazil, very little is known regarding staphylococcal contamination in milk produced by smallholders in semi-arid Brazil. Besides, the fact that staphylococci are leading foodborne pathogens worldwide due to the capacity of some strains to produce toxins ,5, inclusistance , foodstusistance . The emesistance . Subclinorldwide , are assorldwide . However limited . Anothery levels .Staphylococci are highly capable of acquiring resistance to several classes of drugs, including the highest priority critically important antimicrobials (HPCIA), which are drugs of last resort to treat serious bacterial infections in people. EstimatStaphylococcus contaminating goat milk and to assess their susceptibility profiles against antimicrobials, including some drugs commonly used in human medicine to treat staphylococcal infections.The aim of the present study was to investigate the most common spp. of Ethics approval was not required for this study. Verbal consent was obtained from farmers allowing milk samples to be collected during routine milking procedures.The milk samples were collected from Cariri region, Para\u00edba State, Northeastern Brazil from March 2015 to February 2016. Milk samples were processed at the Laboratory for Animal-derived Foods (LAPOA) of the College for Agricultural Sciences, Federal University of Para\u00edba (CCA/UFPB).Staphylococcus spp. isolates originated from goat milk (n=434) were investigated in this study. The isolates were recovered from 510 milk samples \u00adcollected in Cariri, a semi-arid region in Paraiba State, Northeast Brazil. Samples (10 mL) were streaked onto Baird Parker agar  and incubated at 37\u00b0C for 24-48 h Colonies showing typical and non-typical Staphylococcus characteristics were tested for catalase, oxidase and examined microscopically after conventional Gram staining procedure. Confirmed Staphylococcus isolates were further identified by means of a biochemical panel , including the following tests: Crystal violet reaction, nitrate reduction, novobiocin resistance, Voges\u2013Proskauer, sensitivity to optochin, alkaline phosphatase, bile esculin, production of pyrrolidone, dehydrolyzation of arginine, breakage of arginine, glycosidase production, hydroxylation of indoxyl phosphatase, urea, phenol red, growth at 6.5% NaCl, bacitracin resistance, use of pyruvate, presence of \u03b2-lactamase, and hemolysis. Species identification was based on automatic interpretation of the biochemical test results by AutoScan4 software .S. aureus/Staphylococcus lugdunensis); penicillin (Pen) (\u22650.25); rifampin (Rif) (\u22654); trimethoprim/sulfamethoxazole (Sxt) (\u22654/76); and tetracycline (Tet) (\u226516). The interpretation of the results was performed according to the Clinical and Laboratory Standards Institute criteria [S. aureus ATCC 25923 was used as control.Phenotypic antimicrobial susceptibility was assessed by means of a semi-automated microdilution method  for the following drugs and their respective cutoff values (\u03bcg/mL): Ampicillin/sulbactam (Sam) (\u226532/16); ampicillin (Amp) (\u22650.5); amoxicillin/clavulanate de K (\u22658/4); ceftriaxone (\u226564); clindamycin (Cli) (\u22654); ciprofloxacin (Cip) (\u22654); daptomycin (Dap) (\u22652); erythromycin (Eri) (\u22658); gentamicin (Gen) (\u226516); nitrofurantoin (\u2265128); quinupristin/dalfopristin (QD) (\u22654); levofloxacin (Lvx) (\u22658); linezolid (Lzd) (>4); moxifloxacin (Mxf) (\u22658); oxacillin (Oxa)  isolates .S. aureus  and Staphylococcus epidermidis  were the most frequent spp. cultured from raw goat milk  is noteworthy. S. aureus and NAS. Pan-susceptibility was observed in 155 (35.7%) isolates. Interestingly, we detected NAS showing resistance to the highest priority critically important antimicrobials that are used exclusively to treat skin and urinary tract infections in humans [oat milk . A totaln humans ,21.S. aureus and 6.3% of NAS isolates were resistant to amoxicillin/clavulanate. We also detected resistance against QD among the NAS. This group showed higher resistance rates against Cli and Eri (p<0.05) than S. aureus.In our study, 4.1% of Staphylococcus spp. isolated from goat milk. A total of 134 (30.9%) MDR isolates were detected. One S. aureus isolate was resistant to 14 drugs .S. aureus in our study was expected as this species is commonly isolated from dairy animals worldwide. Nonetheless, this finding is of public health significance, as S. aureus is among the main Staphylococcus species causing food poisoning in humans due to the production of heat-stable enterotoxins [S. hyicus among the goat milk samples in the present study is surprising, since S. hyicus has not been commonly detected in milk. Considering that S. hyicus originates from skin sources [Staphylococcus xylosus, Staphylococcus schleiferi, and Staphylococcus simulans, and have been reported as emerging zoonotic pathogens [The high occurrence of rotoxins . The obs sources , the hig sources  that can sources  were detathogens -27.S. aureus, S. epidermidis, and S. hyicus are leading pathogenic staphylococci in veterinary medicine [S. epidermidis, S. simulans, and S. xylosus have been commonly isolated from intramammary infections [S. epidermidis in our study is in accordance with a recent study in Mexico [Staphylococcus spp. cultured from goat milk. S. epidermidis high frequency in goat milk has also been reported in previous studies, possibly associated with subclinical mastitis in dairy goats [medicine . In dairfections ,29,30. Tn Mexico  reportinS. aureus from foodstuffs (1150 samples), the highest resistance rates were observed against Pen (83%), Lzd (67.7%), and Eri (52.1%); however, significant differences were observed according to the type of food [According to a previous investigation on the antimicrobial susceptibility of a large set of  of food .S. aureus and NAS resistance to amoxicillin/clavulanate suggests that some Staphylococcus strains found in raw goat milk can harbor different resistance mechanisms, such as serine-\u03b2-lactamase production, including extended-spectrum \u03b2-lactamases that could hydrolyze later-generation cephalosporins and carbapenemases. Thus, NAS can be considered reservoirs of antimicrobial resistance, although S. aureus has been considered the key spp. involved in both food poisoning outbreaks and clinical infections in humans. This is also corroborated by the high resistance rates observed in NAS isolated from food animal sources, particularly resistance against macrolides, Tet, and streptogramins [The detection of ogramins .S. aureus and enterococci, and these are among the most rapidly growing multidrug-resistant bacteria affecting humans [Considering that quinupristin is a drug of last resort used to treat infections caused by vancomycin-resistant g humans , furtherTet has been commonly used for therapeutic purposes on farm animals for decades and high rates of resistance against this drug have been observed among different organisms, including pathogenic bacteria .Staphylococcus spp. contaminating goat milk on the putative dissemination of antimicrobial resistance to humans is beyond the scope of this study. However, considering that pasteurization is a mild heat treatment, and the fact that milk is a suitable medium for the maintenance and growth of bacteria, it is plausible to consider that milk can serve as source of antimicrobial resistance genes to the human gut microbiota, supporting previous assumptions [The real impact of antimicrobial-resistant umptions ,37. The Finally, staphylococci can serve as reservoirs of antimicrobial resistance genes to milk microorganisms. A large number of antimicrobial resistance genes have been identified in lactic acid bacteria ,39. IncrS. aureus and NAS in goat milk produced in the investigated region reveal the potential role of milk as a reservoir of antimicrobial resistance genes. The findings encourage further investigation of putative effects of technological processes, such as pasteurization, fermentation, and maturation, on the maintenance and dissemination of antimicrobial resistance genes in the microbiota of goat milk and dairy products. Such knowledge could be important to mitigate the spread of antimicrobial resistance in the dairy industry. Finally, our results highlight the importance of on-herd hygiene measures to reduce microbial contamination in milk produced by smallholders and the continuous awareness of producers toward antimicrobial stewardship.The high antimicrobial resistance rates in CJBO and LSF: Study conception and design. AESJ, MMSS, NMVS, and PCV Acquisition of data, analysis and interpretation of data. PENG, AESJ and LSF: Drafting of the manuscript: PENG, NMVS and CJBO: Supervision. PENG and CJBO: Critical revision. All authors read and approved the final manuscript."}
{"text": "On August 12, 2021, the Food and Drug Administration (FDA) amended Emergency Use Authorizations (EUAs) for the Pfizer-BioNTech and Moderna COVID-19 vaccines to authorize administration of an additional dose after completion of a primary vaccination series to eligible persons with moderate to severe immunocompromising conditions . Persons who reported receiving a primary series from different manufacturers or a manufacturer that was unknown or unavailable in the United States, or 2 doses of vaccine after receipt of a Janssen (Johnson & Johnson) single-dose vaccine (150) were excluded from the analysis of adverse reactions after receipt of the additional dose. Time elapsed from completion of the primary vaccination series to receipt of an additional dose was described by pattern of vaccination. Adverse event profiles after doses 2 and 3 were compared for registrants who received mRNA vaccine from the same manufacturer for all 3 doses.**During August 12\u2013September 19, 2021, a total of 22,191 v-safe registrants reported receipt of an additional dose of COVID-19 vaccine after completing the primary series . Among tAmong the 22,191 v-safe registrants, the median interval from completion of the primary COVID-19 vaccination series to receipt of an additional dose was 182 days (interquartile range [IQR]\u00a0=\u00a0160\u2013202 days) . Among tLocal  and systemic  reactions were frequently reported during the week after an additional dose of COVID-19 vaccine, most commonly on the day after vaccination. Frequently reported reactions were injection site pain , fatigue , and headache .Among 22,191 additional dose recipients, a total of 7,067 (31.8%) reported health impacts, and approximately 28.3%  reported they were unable to perform normal daily activities, most commonly on the day after vaccination. Medical care was sought by 401 (1.8%) registrants, and thirteen (0.1%) were hospitalized. Reasons for receiving medical care or hospitalization were not identified in the v-safe survey; however, registrants who indicate that medical attention was sought after vaccination are contacted by VAERS staff and encouraged to complete a VAERS report.Among 21,658 v-safe registrants who received the same mRNA vaccine for all 3 doses, 12,591 (58.1%) completed at least one health check-in survey on days 0\u20137 after all 3 doses; 79.4% and 74.1% reported local or systemic reactions, respectively, after dose 3, compared with 77.6% and 76.5% who reported local or systemic reactions, respectively, after dose 2. Among registrants who received 3 doses of Moderna , local reactions were reported more frequently after dose 3 than dose 2  . SystemiAs of September 19, 2021, approximately 2.21 million persons in the United States had received additional doses of COVID-19 vaccinesThe number of registrants who indicated that they received 2 doses of Janssen vaccine or received their additional dose from a manufacturer different from that of their primary series was small, limiting any conclusions. Data on the safety or effectiveness of vaccination with COVID-19 vaccine products from different manufacturers are limited; the Advisory Committee on Immunization Practices (ACIP) recommends that persons with moderately to severely immunocompromising conditions receive a third dose of mRNA COVID-19 vaccine from the same manufacturer as their primary series (During the period covered by this study, ACIP recommendations for an additional dose of COVID-19 vaccine were limited to persons with moderately to severely immunocompromising conditions who had received 2 doses of an mRNA vaccine.The findings in this report are subject to at least four limitations. First, enrollment in v-safe is voluntary and likely not representative of the vaccinated U.S. population; the majority of participants identified themselves as White and non-Hispanic. Second, during this study period, additional dose recommendations were limited to persons with immunocompromising conditions who completed a primary mRNA COVID-19 vaccination series; however, v-safe does not include information about immune status. Additional-dose recipients likely include persons with and without immunocompromising conditions. Third, a causal relationship between a vaccine and clinically serious adverse event reported after vaccination cannot be established using v-safe data. Finally, insufficient data were available to determine patterns of adverse reactions after receipt of an additional dose from a manufacturer different from the primary series or for the Janssen vaccine.An additional dose of mRNA COVID-19 vaccine is recommended for persons with moderately to severely immunocompromising conditions (Among 306 Pfizer-BioNTech clinical trial participants, adverse reactions after dose 3 were similar to those after dose 2.During August 12\u2013September 19, 2021, among 12,591 v-safe registrants who completed a health check-in survey after all 3 doses of an mRNA COVID-19 vaccine, 79.4% and 74.1% reported local or systemic reactions, respectively, after the third dose; 77.6% and 76.5% reported local or systemic reactions after the second dose, respectively.Voluntary reports to v-safe found no unexpected patterns of adverse reactions after an additional dose of COVID-19 vaccine. CDC will continue to monitor vaccine safety, including for additional COVID-19 doses."}
{"text": "Vaccination against SARS-CoV-2 (the virus that causes COVID-19) is highly effective at preventing hospitalization due to SARS-CoV-2 infection and booster and additional primary dose COVID-19 vaccinations increase protection  recommended that moderately or severely immunocompromised recipients of an mRNA COVID-19 vaccine (Pfizer-BioNTech or Moderna) primary series receive a homologous additional primary dose \u226528 days after the second dose in the primary series . Tests for statistical significance were not conducted because these data are reflective of the U.S. population aged \u226565 years and were not based on population samples. This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.Among 42.5 million eligible persons aged \u226565 years, 18,745,803 (44.1%) received a booster or additional primary dose of COVID-19 vaccine during August 13\u2013November 19, 2021 , includiAmong Pfizer-BioNTech recipients, the daily number of persons who received a booster or additional primary dose peaked 5 days after release of the Pfizer-BioNTech booster recommendations  with 341,395 recipients vaccinated . After rAs of November 19, 2021, 44.1% of 42.5 million eligible adults aged \u226565 years had received a booster or additional primary dose, leaving an estimated 23.8 million eligible adults in this age group in need of a booster or additional primary dose. Coverage with booster or additional primary COVID-19 vaccine doses varied by the primary series vaccine product and race/ethnicity; approximately one third of eligible American Indian or Alaska Native persons, Hispanic or Latino persons, and Native Hawaiian or Other Pacific Islander persons received a booster or additional dose, compared with approximately one half of non-Hispanic White and multiple/other race persons. All adults aged \u226565 years should be vaccinated against COVID-19, including receiving an additional primary dose if they are immunocompromised and/or a booster dose after the minimum recommended interval after primary series completion.Differences in coverage between recipients of different primary series vaccine products can partially be explained by the staggered timing of ACIP recommendations, which also lowered overall coverage because not all persons represented in these results had an equal amount of time to receive a booster or additional primary dose. For example, based on timing of recommendations, Pfizer-BioNTech primary series recipients had 28 days longer to receive a booster dose than did Moderna or Janssen recipients. In addition, Moderna and Pfizer-BioNTech coverage includes recipients of additional primary doses administered since August 13, whereas Janssen coverage does not. However, only 10.7% of booster or additional primary dose recipients received an additional primary dose before booster dose recommendations, so inclusion of these recipients in mRNA primary series coverage cannot account for the differences observed. Certain groups  were recommended to receive a homologous dose, while the first recommendations for Janssen recipients allowed a heterologous booster dose. Although 61.5% of Janssen recipients received a heterologous booster dose, this represents only 227,079 persons, or 1.2% of overall booster or additional primary dose recipients.The findings in this report are subject to at least five limitations. First, the use of a composite measure for coverage and absence of information on the immunocompromise status of vaccine recipients limits the conclusions that can be drawn from the analysis and might also have resulted in underestimation of the eligible population because immunocompromised persons who completed a primary series after May 19, 2021, could not be identified. Second, identification of booster or additional primary dose recipients depends on linkage between vaccination records in jurisdiction-specific immunization information systems or other data systems. Persons who received a booster or additional primary dose in a different jurisdiction from that of their primary series, or who for other reasons were not able to be linked back to their primary series, might not be represented in these results. Third, restricting the source population to persons aged \u226565 years at the time of primary series completion might have excluded some valid recipients, including those who reached age 65 years between completion of the primary series and administration of the booster or additional primary dose, and persons with a missing, incorrect, or incomplete date of birthA booster or additional primary dose of COVID-19 vaccine provides a robust immune response (Although COVID-19 vaccines are highly effective, vaccine effectiveness wanes over time, and adults aged \u226565 years are at increased risk for severe COVID-19\u2013associated illness. Booster and additional primary vaccine doses increase protection.During August 13\u2013November 19, 2021, 18.7 million persons aged \u226565 years received a booster or additional primary dose of COVID-19 vaccine, constituting 44.1% of eligible persons aged \u226565 years. Coverage differed by primary series vaccine product and race/ethnicity.Strategic efforts are needed to encourage eligible persons aged \u226518 years, especially those aged \u226565 years and those who are immunocompromised, to receive a booster and/or additional primary dose to ensure maximal protection against COVID-19."}
{"text": "Vector-borne diseases (VBDs), such as dengue, Zika, West Nile virus (WNV) and tick-borne encephalitis, account for substantial human morbidity worldwide and have expanded their range into temperate regions in recent decades. Climate change has been proposed as a likely driver of past and future expansion, however, the complex ecology of host and vector populations and their interactions with each other, environmental variables and land-use changes makes understanding the likely impacts of climate change on VBDs challenging. We present an environmentally driven, stage-structured, host\u2013vector mathematical modelling framework to address this challenge. We apply our framework to predict the risk of WNV outbreaks in current and future UK climates. WNV is a mosquito-borne arbovirus which has expanded its range in mainland Europe in recent years. We predict that, while risks will remain low in the coming two to three decades, the risk of WNV outbreaks in the UK will increase with projected temperature rises and outbreaks appear plausible in the latter half of this century. This risk will increase substantially if increased temperatures lead to increases in the length of the mosquito biting season or if European strains show higher replication at lower temperatures than North American strains. In 2017nce 1990 , though nce 1990 \u20137. Numernce 1990 . For exance 1990 ,9 to shaAedes albopictus, a vector of dengue, chikungunya and Zika, established in Italy in 1990 and has since spread across much of the Mediterranean basin, with rising temperatures, trade and travel implicated in its introduction and subsequent dispersal [Disentangling the impacts of climatic and non-climatic drivers on expansion of VBDs is challenging, creating difficulties in forecasting potential effects of climate change ,10. For ispersal . Historiispersal . Howeverispersal  and estaispersal .et al. [A recent review by Sadeghieh et al.  found thtability . Novel atability ,19. In dtability .et al. [et al. [R0 values for WNV in Europe could change by a factor of approximately 6 across the range of predicted vector\u2013host ratios. Similarly, temperature-dependence of vector biting and mortality, and pathogen latency, resulted in an approximate threefold increase in R0 values of WNV across a temperature gradient of 18\u201328\u00b0C. Given this, there is a clear need for modelling approaches which capture the seasonal nature of drivers of VBDs.Despite their wide applicability, existing mathematical models of VBDs generally make simplifying assumptions regarding vector or pathogen dynamics, as summarized by Reiner et al.  in a revf models ). This if models . Likewiss et al.  estimateflavivirus primarily transmitted in a cycle between Culex pipiens mosquitoes and birds [We propose a novel approach that explicitly models temperature effects on the timing and seasonal coincidence of events in the pathogen, host and vector life cycle. In doing so, we aim to improve prediction of the risk of VBD establishment in marginal temperate environments under climate change. Using delay-differential equations (DDEs) with environmentally driven delays we incorporate realistic, climate-dependent representations of vector vital rates and pathogen latency, thus capturing the impacts of climate on seasonal variations in vector\u2013host ratios on transmission risk. We apply our modelling approach to the prediction of establishment risk of WNV in the UK, as WNV is a VBD that exhibits high seasonality and which is currently expanding its distribution into Northern Europe, having recently been reported in both Germany and the Netherlands ,23,24. Wnd birds  that cannd birds . As WNV nd birds ,28. Howe. 2Cx. pipiens seasonal dynamics [We developed a susceptible\u2013exposed\u2013infectious\u2013recovered (SEIR) vector\u2013host mathematical model with compartments for each life stage of the vector population and for each possible infection status of hosts and adult vectors . The moddynamics , which hdynamics . It is bdynamics ,30). Adudynamics . Diseasedynamics . For simdynamics . Insteaddynamics .Figure The explicit temperature-dependence in vector development and survival rates and pathogen replication rates enables predictions of whether projected temperatures are sufficiently high to sustain transmission cycles. By coupling the seasonal vector model with a seasonal host model, we can control the timing of pathogen introduction and make predictions about how timing of the pathogen introduction will affect outbreak risk. The use of temperature-dependent, stage-structured DDEs allows us to capture developmental lags in both the vector population dynamics and the transition of exposed to infectious mosquitoes, facilitating understanding of whether the time between pathogen introduction and the cessation of the active vector season is sufficient to allow amplification of the pathogen to levels which may cause spillover into humans. The flexibility of our mechanistic modelling framework, which is based upon the fundamental biological processes, means that it is straightforward to change parameters, such as the season start and end timings, to understand how risk may change under future climate. While this model is applied to WNV in this case, the underlying mechanistic model framework could readily be adapted to study other pathogens.Cx. pipiens is substantially less than 12 km [We simulated the WNV model under various warming scenarios provided as part of the UK Climate Projections 2018 (UKCP18) report produced by the Met Office . We usedan 12 km . Each siet al. [Risk maps showing MIR for each grid square for each of the 12 sets of predicted temperature data were produced for the last year of each decade from 2019 until 2079. Under our model, the vector seasonal abundance patterns and pathogen replication rates will vary temporally across years and spatially across the UK as a result of differences in input temperature and photoperiod (the number of daylight hours per day). Three different times of WNV introduction times were considered: introduction at the end of March, the end of April and the end of May. These timings are consistent with the arrival times of a range of potentially competent migratory hosts such as the swallow, chiffchaff and willow warbler , among oet al. . We alsoet al. . To undeet al. [Details of the mathematical model used to simulate vector and host population dynamics and on the assumptions regarding WNV transmission between the mosquito and bird populations are given in full in electronic supplementary material, S1. The following further information is given in electronic supplementary material: parameterization of the extrinsic incubation period/viral replication rate S2.1) , parametet al. . In the .1 , para. 3. 3.1The predicted MIR following WNV introduction via infected birds arriving at the end of March was never projected to exceed 0.25 because this was earlier than termination of mosquito diapause. Likewise, the predicted risk for late April introduction was low for all climate projections with seven of the 12 simulations in the year 2079 predicting MIR values below 0.25 across the entire UK . In those runs for which the MIR exceeds 0.25 in some areas risks are isolated to a very small area and the predicted MIR is generally only very slightly above 0.25. Risks are predicted to be low for these earlier introduction times because we assume that adult females exiting diapause only take one blood meal before laying an egg raft and dying due to the energetic demands of diapause . TransmiThe predicted MIR following introduction via migratory birds at the end of May is substantially higher, as there has been sufficient time for mosquitoes to exit diapause and for the first spring generation to develop prior to arrival of the virus. Of the 12 sets of climate projections available, 11 of them resulted in predicted MIR values above 0.25 in at least one year in a least one location in the UK. In Tmin. Due to a lack of data linking temperature to WNV replication rates in a European context, we use a laboratory-derived relationship based on a North American strain of WNV, increasing the importance of understanding the sensitivity of this parameter in particular. In Tmin for viral replication inside the vector, which is 7.3\u00b0C  [A sensitivity analysis of the more uncertain parameters regarding WNV transmission between vectors and hosts is presented in electronic supplementary material, S2.8. However, one parameter which is likely to be particularly influential in the determination of WNV risk under future climate scenarios is the thermal minimum for WNV replication,  11.4\u00b0C) . It is c. 3.2The general pattern of increase in WNV risk and the prediction that outbreaks will be unlikely until the middle of the twenty-first century stems from changes to a range of processes affecting virus transmission across the mosquito biting and development season. Specifically, three of the main processes known to drive WNV transmission  are the biting rate, the vector\u2013host ratio and the viral replication rate . In To understand the relative importance of each of the modelled transmission processes  and the interactions between these processes in determining the MIR, a series of Besag\u2013York\u2013Molli\u00e9 2 (BYM2) spatial models were fitted to the results of all model runs using INLA   to larger more permanent sites  [We have made the simplifying assumption in this work that there is no spatial variability in the extent of vector breeding habitat. This is clearly untrue, however translating the effect of changing rainfall, a known predictor of habitat , into a r butts) . Human br butts) , as theyr butts) . Indeed,r butts) . Given tr butts) , should We highlight that our modelling framework, combining environmentally forced stage-structured DDEs capturing the vector life cycle with equations representing host and pathogen dynamics, could be applied to study establishment risk of a wide range of VBDs across temperate regions under projected climate scenarios. While the model presented here focusses specifically on WNV, modification of the pathogen replication rate would readily allow other mosquito-borne diseases of risk in Europe, such as Usutu, to be studied  and smalThe model presented predicts that WNV outbreaks in the UK are unlikely given current temperatures, though the outbreak risk is predicted to increase as temperatures warm throughout the century. Nonetheless, these predictions are based upon temperatures simulated under RCP8.5, which is intended to describe a worst-case scenario with regards to continued investment in fossil fuels and no effort to reduce greenhouse gas emissions . Further"}
{"text": "Here, we demonstrate the supreme adjuvant effect of the recently developed and pharmaceutically defined double-stranded (ds)RNA adjuvant Riboxxim especially when incorporated into PLGA particles. Encapsulation of Riboxxim together with antigens potently activates murine and human dendritic cells, and elevated tumor-specific CD8+ T cell responses are superior to those obtained using classical dsRNA analogues. This PLGA particle vaccine affords primary tumor growth retardation, prevention of metastases, and prolonged survival in preclinical tumor models. Its advantageous therapeutic potency was further enhanced by immune checkpoint blockade that resulted in reinvigoration of cytotoxic T lymphocyte responses and tumor ablation. Thus, combining immune checkpoint blockade with immunotherapy based on Riboxxim-bearing PLGA particles strongly increases its efficacy.With emerging supremacy, cancer immunotherapy has evolved as a promising therapeutic modality compared to conventional antitumor therapies. Cancer immunotherapy composed of biodegradable poly(lactic- PLGA based cancer immunotherapy incorporating antigen and TLR ligands has resulted in enhancement of the anti-tumour response. Here, the authors explore the use of a defined double stranded RNA adjuvant, Riboxxim, and test its incorporation with PLGA immunotherapy in the context of in vivo tumour models and show enhanced induction of the anti-tumour response. Due to the unique ability of dendritic cells (DCs) to prime and activate naive T cells, effective antigen charging and stimulation of DCs are key goals in immunotherapy. We and others have established the use of PLGA-MP (microparticles) and NP (nanoparticles) as efficient antigen delivery systems for targeting DCs in situ2. The aliphatic copolymer PLGA is extensively used for optimizing controlled and joint release of immune stimulants and antigens in cancer immunotherapy in preclinical settings3. Due to its biodegradability and proven safety profile, PLGA and its formulations have been licensed by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) for pharmaceutical applications via parenteral and mucosal routes4. Especially, PLGA-MP exhibit ideal properties for facilitated uptake by antigen-presenting cells (APCs). Concomitant delivery of antigens and adjuvants to the same APC leads to efficient DC or macrophage activation and simultaneous activation of both, CD4+ T helper cells and CD8+ cytotoxic T lymphocytes (CTL), via cross-presentation5.Cancer still represents one of the most prevalent and challenging malignancies worldwide as a failure of standard therapies results in tumor relapse and metastasis formation. Hence, there is an urgent need for the development of alternative therapies such as immunotherapy with cancer vaccines6. The dsRNA analog poly(I:C) (polyinosinic:polycytidylic acid) is the most widely used TLR3 agonist but also engages RIG-I and preferably MDA57. Its adjuvant properties result from induction of type I interferons and pro-inflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF), and interleukin (IL)\u22121\u03b28 thus enabling an improved efficacy of cancer immunotherapy in murine tumor models11. However, its ill-defined macromolecular structure and heterogeneous composition prevented polyI:C from extensive clinical use due to unpredictable pharmacokinetics and toxicity issues. The TLR3/RIG-I ligand Riboxxim\u2122, in contrast, is characterized by its well-defined chemical structure as double-stranded RNA and nucleotide composition of 100\u2009bp for effective TLR3 triggering. An uncapped triphosphate moiety at the 5\u2032 end of Riboxxim also enables the activation of RIG-I12. Riboxxim exhibits very good solubility in water, prolonged stability in solution at 4\u2009\u00b0C and in serum.Successful immunotherapy against cancer also requires an ideal adjuvant. Multiple immunostimulatory RNA duplexes with agonistic activity for pattern-recognition receptors (PRR) have been reported as candidates for adjuvants in cancer immunotherapy. Double-stranded RNA molecules classically trigger endosomal TLR3 (Toll-like receptor 3). Depending on the structural characteristics and length of the viral RNA mimetic, it may also activate cytosolic RIG-I-like receptors (RLRs), including retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5). TLR3 and RIG-I downstream signaling results in nuclear translocation of the key transcription factors nuclear factor \u03ba/light-chain enhancer of activated B cells (NF-\u03baB) and interferon regulatory factor (IRF)3/7 culminating in secretion pro-inflammatory cytokines and of type I interferons (IFN), respectively. Co-encapsulation of TLR3 ligands with antigen greatly enhanced PLGA particle-mediated cancer vaccine efficacy by inducing reliable maturation of DCs13. Monoclonal antibodies directed against these checkpoint receptors have achieved efficient remission of several tumor types, which resulted in FDA and EMA approval of immune checkpoint blockade for adjuvant treatment of advanced melanoma, head and neck squamous cell carcinoma, and metastatic bladder carcinoma14. However, the majority of cancer patients did not benefit from this therapy with only up to 20% complete responses in patients with solid cancer. The therapeutic success of immune checkpoint blockade necessitates the preexistence of cancer-specific CTLs at the tumor site. Thus, there is an urgent medical need to develop defined immunotherapeutic approaches that would elicit tumor-specific CTLs and boost the success of checkpoint blockade.Cancer immunotherapy has been greatly improved and promoted by pharmacological blockage of so-called immune checkpoint receptors such as CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), PD-1 (programmed cell death protein 1), and its ligand PD-L1. These molecules can be co-opted as immune evasion mechanism by tumors to inhibit CTL activity and suppressing antitumor immune responses18. With respect to a possible clinical translation, this study demonstrates the immune-potentiating capacity of GMP-grade Riboxxim contained in PLGA-MP and the synergistic therapeutic effect of combining this approach with immune checkpoint blockade.Previously, we have shown that PLGA-MP mediated co-delivery of tumor antigens or lysates along with several experimental TLR ligands yielded potent and long-lasting anti-cancer immune responses in vivo affording suppression of tumor growth+ CTL induction with PLGA-NP or MP using different administration routes. PLGA particles containing ovalbumin (OVA) and GMP-certified pyrogen-free Riboxxim were injected either intraperitoneally (i.p.), intramuscularly (i.m.), intranodally (i.nd.), or subcutaneously (s.c.) into C57BL/6J mice. The maximal cytokine response on day 6 after vaccination was achieved after s.c. application  images show uniform spherical PLGA-NPs with a smooth surface of an average size of 250\u2009nm in diameter. MPs produced by spray-drying possess a relatively broad size distribution ranging from nanosized up to 1.5\u2009\u00b5m sized particles, notoriously suitable for cellular uptake and internalization by different APCs Fig.\u00a0. Since tion Fig.\u00a0. We deci+ bone marrow-derived DCs (BMDCs) and peritoneal macrophages (pM\u00d8), as well as by human CD14+ monocytes, that have been in vitro differentiated into monocyte-derived DCs (MoDCs) or macrophages (MoM\u00d8). Efficient particle engulfment of about 35% could also be demonstrated with CD1c+ and CD141+ primary human myeloid dendritic cells isolated from peripheral blood (mDCs). Of note, no in vitro cytotoxicity was detected after engulfment of PLGA-MP by BMDCs over several days  analysis showed unimodal size distribution with a mean particle size of ~1\u2009\u00b5m in diameter and negative surface charge  and BDCA3+ (CD141+) cells, found in low numbers in human peripheral blood21.A proper activation of DCs is crucial for T-cell targeted cancer vaccines. Whether Riboxxim promotes upregulation of maturation markers and the release of cytokines by TLR3-expressing murine and human DCs was analyzed in comparison to conventional polyI:C (pIC) or a pyrogen-free polyI:C variant (pIC pyf). Endotoxin levels of \u22640.5 EU/ml further demonstrated safety of Riboxxim, while the most widely used conventional polyI:C exhibited a high level of endotoxin and pyrogen contaminations, which leads to a potential contribution of TLR4 signaling after addition of polyI:C , stimulation with Riboxxim achieved efficient elevation of co-stimulatory molecules, maturation markers, and MHC-I equivalent to the stimulation by polyI:C Fig.\u00a0. This re+ and CD141+ DCs . TLR3-dependent upregulation of these markers was comparable to Riboxxim stimulation in WT cells. BMDCs deficient for the adapter molecule MAVS failed to release type I IFN with Riboxxim  into PLGA microparticles enabled the induction of peptide-specific CTL activation and cytotoxic effector function in chimeric human HLA-A*0201 transgenic AAD mice18. Encapsulation of STEAP1262\u2013270 into PLGA-MP together with Riboxxim enabled even more potent CTL responses  epitope STEAP1ses Fig.\u00a0. Notably+ T-cell responses by MP-OVA/R848 to MP-OVA/Riboxxim, a therapeutic administration induced only a mediocre antitumor response against established E.G7 tumors, which was similar to the antitumor efficacy of MP-OVA/polyI:C . Animals that had received a single vaccination of MP-OVA/Riboxxim or MP-OVA/polyI:C remained tumor-free throughout the whole experiment of 60 days Fig.\u00a0. We then24 in order to direct the antitumor response towards the lung. A PLGA-MP booster application into the tumor microenvironment seems to recruit and amplify the antitumor response by systemic and local clonal expansion of tumor-specific T cells  was added to the therapeutic regimen 31 while mismatched uracil and guanosine residues were added to the polyI:C structure to decrease toxicity issues in Ampligen\u2122 32. The adjuvant effect of polyIC analogs includes dose-dependent stimulation and activation of DCs expressing pro-inflammatory cytokines and type I IFNs to promote Th1 polarization and activation of antigen-specific cytotoxic T cells, with too high doses leading to inhibition of the immune activity. The additional activation of MDA5 might also lead to overshooting immune responses. Over 60 clinical studies involve the administration of the dsRNA analog Hiltonol\u00ae in solid tumors in combination with peptide vaccines, fusion proteins, or immune checkpoint blockade. However, the therapeutic effect largely depends on multiple injections using high doses of polyICLC. Repeated systemic administration of TLR agonists may induce immune hypo-responsiveness, which is commonly known as TLR tolerance33. Only a few clinical studies encompassed Ampligen for cancer therapy by inducing DC maturation and Th1-type T-cell immunity34. The multimodal immunomodulatory properties of Ampligen are not extensively characterized, which marks Ampligen as a bona fide TLR3 agonist35.The importance of dsRNA agonists in cancer vaccines has been demonstrated in several studies as it facilitates potent DC activation and subsequent priming of tumor-specific CTLs with induction of a tumor-suppressive microenvironment36. The induction of a type I IFN response and apoptotic tumor cell death was enabled by targeted delivery of Riboxxol coupled to an anti-prostate stem cell antigen (PSCA) antibody derivative36. Matsumoto and colleagues created the chimeric TLR agonist ARNAX, which exclusively triggers TLR3 by the addition of a 5\u2032phosphorothioate oligodeoxynucleotide (ODN)-cap of GpC DNA to the 140\u2009bp dsRNA that specifically targets ARNAX to the endosome. ARNAX therapy-induced robust antitumor immune responses without the induction of systemic inflammation37. This non-inflammatory TLR3 agonist was shown to synergize with PD-L1 blockade in tumor eradication in immunocompetent hosts to overcome PD-1/PD-L1 resistance and provide long-term protection from tumor relapse. The authors highlight the necessity of both proper DC priming by a TLR3 ligand and checkpoint inhibitor blockade for efficient cancer immunotherapy43.In contrast to multiple prime-boost injections, PLGA particles carrying Riboxxim show antitumor responses after a single treatment. Compared to other dsRNA mimetics, Riboxxim displays superior properties as immune adjuvant due to the absence of systemic cytokinemia or adjuvant toxicity. The 50\u2009bp derivative of Riboxxim, designated Riboxxol (RGIC\u00ae50), potently improves murine and human cDC1 activation to enhance T-cell proliferation+ CTLs and antitumor immunity in mouse models of melanoma or colorectal carcinoma45 and in human ovarian cancer46. The addition of 5\u2032pppRNA to a cancer-peptide containing NP was shown to specifically boost CD8+ T-cell population while not inducing suppressive immune cell types45.Therapeutic targeting of RIG-I has recently emerged as another aim in antitumor immunotherapy and is currently explored in clinical trials. A combinatory vaccination scheme using 5\u2032-triphosphorylated RNA-driven RIG-I stimulation with CTLA-4 or PD-1 blockade already has been demonstrated to induce potent cross-priming of CD8+ and CD141+ DCs as well as the induction of IFN\u03b1/\u03b2 and IL-6/TNF , which has been most widely used in preclinical studies, probably originated from pyrogen contaminations, which was markedly reduced in pyrogen-free polyI:C (Invivogen) or in GMP-grade Riboxxim  2Enge/J (AAD) mice and B6;129-Mavstm1Zjc/J (Mavs\u2212/\u2212) mice were obtained from The Jackson Laboratory . B6;129S1-Tlr3tm1Flv/J (Tlr3\u2212/\u2212) mice were obtained from the Laboratory Animal Services Center (LASC)-Vivaria, University of Zurich, Switzerland. Transgenic animals are verified by genotyping of ear biopsies. Primers are listed in Supplementary Table\u00a0+ T cells in blood after vena facialis puncture. Animals were further bred in the accredited animal facility of the University of Konstanz under specific pathogen-free conditions on a 12-h light/dark cycle with lights on at 7\u2009a.m. Mice were kept in air-conditioned rooms with controlled temperature (22\u2009\u00b0C), 55% relative humidity, and constant ventilation (17 air changes/h). Animals were provided ad libitum access to standard, autoclaved laboratory animal diet, and tap water. Male and female mice were used at 8\u201312 weeks of age. The number of animals in each group was determined according to statistical verification and previous studies.C57BL/6J mice and BALB/cAnNCrl (BALB/c) mice were originally purchased from Charles River . C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-1) mice and B6.Cg-Animal experiments have been conducted in compliance with ethical standards of German and EU guidelines after approval by the animal experimentation ethics committee of the Review Board of Governmental Presidium Freiburg, Germany . Human specimens were used in accordance with the requirements of the institutional ethics committee and national policies. Blood donations were approved by the Ethics Committee of the University of Konstanz, Germany. Written informed consent was obtained from each of the randomly enrolled healthy volunteers before blood donation following guidelines of the Review Board protocol of the University of Konstanz. Blood donations were performed by the transfusion unit at the Klinikum Konstanz in accordance with the precepts of the transfusion unit at the hospital and of the DRK (German Red Cross) with all applicable regulations, guidance, and local medical policies.+/GFP+ was kindly provided by Prof. Olaf van Tellingen . To maintain Luciferase expression, respective growth media were supplemented with 0.4\u2009mg/ml G418 Sulfate . Positive expression of luciferase was verified before every injection into animals by in vitro bioluminescence imaging on the IVIS\u00ae Spectrum 200 platform (Perkin Elmer). Expression of ovalbumin was authenticated via immunoblotting using anti-chicken egg albumin antibody . The immature dendritic cell line DC2.4  was provided by Kenneth Rock  and maintained in RPMI 1640. The CD8+ splenic DC cell line MutuDC211459 was a kind gift of Prof. Hans Acha-Orbea  and cultured in IMDM with 10% (v/v) FCS, 100 IU/ml Penicillin, and 100\u2009\u00b5g/ml Streptomycin. The B3Z T-cell hybridoma60 was a kind gift from Prof. Nilabh Shastri  and cultured in IMDM supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin, and 100\u2009\u00b5g/ml Streptomycin. The CD4+ T-cell hybridoma DOBW61 was kindly contributed by Prof. Clifford V. Harding  and maintained in DMEM supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin and 100\u2009\u00b5g/ml Streptomycin, 1\u2009mM sodium pyruvate, 10\u2009mM HEPES and 0.5\u2009mM \u03b2-mercaptoethanol. The human monocytic cell line THP-1 (#TIB-202\u2122) was purchased by ATCC\u00ae  and maintained in RPMI 1640. All culture media were supplemented with GlutaMAX\u2122. Media and standard additives were purchased from Gibco\u00ae (ThermoFisher Scientific). Cell lines were regularly tested negative for Mycoplasma by PCR analysis of cell culture supernatants by Microsynth, Switzerland. Cell cultures were maintained at 37\u2009\u00b0C with 5% CO2 in a humidified atmosphere. Adherent cells were detached by incubation with 0.05% trypsin-EDTA for 5\u2009min at 37\u2009\u00b0C. Primary DCs were detached by 5\u2009mM EDTA/PBS for 15\u2009min at 37\u2009\u00b0C.The murine lymphoma cell line stably expressing OVA and Luciferase, E.G7-OVA-luc+ cells were kindly provided by Prof. Thomas Blankenstein  and maintained in RPMI 1640 plus 10% (v/v) FCS, 100 IU/ml Penicillin and 100\u2009\u00b5g/ml Streptomycin. MO5 cells, murine melanoma cells stably expressing OVA, were provided by Dr. Antje Heit  and cultured in DMEM supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin, and 100\u2009\u00b5g/ml Streptomycin. Luciferase-positive, OVA-expressing B16BL6 cells were generated by lentiviral transduction of cytosolic ovalbumin (amino acids 51-386) and cultured in DMEM and 10% (v/v) FCS, 100 IU/ml Penicillin, and 100\u2009\u00b5g/ml Streptomycin. The original cell line B16BL6-luc62. 5\u2009mg ovalbumin  and 0.05\u2009mg adjuvant in 100\u2009\u00b5l 0.1\u2009M NaHCO3 were dissolved in the primary emulsion (100\u2009mg PLGA in 3\u2009ml dichloromethane (DCM)) and mixed with 25\u2009ml of 2% poly . The mixture was stirred overnight at 4\u2009\u00b0C to evaporate DCM. NPs were collected by ultra-centrifugation and lyophilized at \u221280\u2009\u00b0C for 48\u2009h. PLGA microparticles (MP) were prepared by spray-drying. For antigen/TLR3L co-encapsulation, 50\u2009mg OVA or 10\u2009mg of peptide antigens and 0.5\u2009mg adjuvants  (HMW), InvivoGen; Riboxxim\u2122, Riboxx Pharmaceuticals; MPLA-SM VacciGrade\u2122, InvivoGen; R848 VacciGrade\u2122, InvivoGen; \u03b1-Galactosylceramide ) were dissolved in 1\u2009ml of 0.1\u2009M NaHCO3 (aqueous phase) and emulsified with 1\u2009g PLGA in 20\u2009ml dichloromethane (organic phase) using a digital microtip sonicator. Riboxxim is provided at a 1\u2009mg/ml solution in water for injection and is certified as GMP material sterile and endotoxin-free (European guidelines ICH-6). The HLA-A*0201 restricted tumor-associated peptides STEAP1262\u2013270 (LLLGTIHAL); STEAP1292\u2013300 (MIAVFLPIV); PAP135\u2013143 (ILLWQPIPV); PSMA469\u2013477 (LMYSLVHNL); Her2689\u2013697 (RLLQETELV); TRP-2180\u2013188 (SVYDFFVWL) and NY-ESO1157\u2013165 (SLLMWITQV) were synthesized by the Institute for Biochemistry, Charit\u00e9 Berlin or were purchased by peptides&elephants, Germany. The obtained dispersion was immediately spray-dried with the Mini Spray-Dryer 290 (B\u00fcchi Labortechnik AG) at a flow rate of 1\u2009ml/min and inlet/outlet temperatures of 25\u2009\u00b0C/23\u2009\u00b0C. Spray-dried microparticles were washed out of the spray-dryer\u2019s cyclone with 0.05% Poloxamer 188  and collected on a cellulose acetate membrane filter. PLGA-MP were dried under vacuum at room temperature and subsequently stored under desiccation at 4\u2009\u00b0C.PLGA particles were prepared from Resomer RG502H (Evonik Industries). Nanoparticles (NP) were produced by the double emulsification solvent evaporation method as previously describedThe amounts of encapsulated antigens and TLR ligands were determined after dissolving the PLGA particles in DMSO for 10\u2009min. Phase separation of precipitated polymer and soluble compounds was achieved by the addition of 0.1\u2009M NaOH. Antigen release was quantified using Pierce\u2122 MicroBCA Protein Assay Kit (Thermo Fisher Scientific) after incubating particles in PBS pH 7.4 at 37\u2009\u00b0C under continuous orbital agitation.The particle size and polydispersity index (PDI) were analyzed by cumulative analysis of dynamic light scattering (DLS) using the Zetasizer\u00ae Nano ZSP particle size analyzer and Zetasizer software version 7.12 . The \u03b6 (zeta) potential of particles was measured by laser Doppler velocimetry in combination with phase analysis light scattering using the same device. Particles were dispersed in ultrapure water prior to measurement at 25\u2009\u00b0C with the Helmholtz\u2013Smoluchowski model.To characterize surface morphology using scanning electron microscopy (SEM) particles were mounted on aluminum stubs and sputter-coated with a 6\u2009nm thick layer of platinum by an argon beam sputter coater . Micrographs were produced on a Zeiss Auriga 40 at 1, 3, or 5\u2009kV regarding the sample with SmartSEM v6.0 .Endotoxin levels of PLGA particles were detected by ToxinSensor\u2122 Chromogenic LAL Endotoxin Assay according to the manufacturer\u2019s instructions .6 cells/well. Particle uptake was performed for 6\u2009h at 37\u2009\u00b0C or at 4\u2009\u00b0C as a control (no uptake). Phase-contrast microscopy was performed for semi-quantitative estimation of particle uptake. After incubation, cells were harvested and incubated for 1\u20133\u2009s in pure DMSO to remove unbound particles. After that, cells were washed three times with 1\u00d7 DPBS. QD-positive fluorescent signals were assessed for each sample by flow cytometry.In vitro particle internalization by primary DCs and cell lines was determined using fluorescently labeled particles (PLGA-MP-OVA/Riboxxim/QD) . QD-loaded MPs were added to cells at a concentration of 10\u2009\u00b5g/ml per 1 \u00d7 1063. Isolated bone marrow cells were cultured for 9 days in 10\u2009cm dishes at 2 \u00d7 106 cells in RPMI 1640, supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin, 100\u2009\u00b5g/ml Streptomycin, 1\u2009mM sodium pyruvate, 55\u2009\u00b5M \u03b2-mercaptoethanol and 20\u2009ng/ml recombinant murine GM-CSF . Peritoneal macrophages were elicited by i.p. injection of 0.5\u2009ml of 3% Brewer thioglycolate medium (w/v)64. Cells were harvested on day 3 after injection by washing cells from the peritoneal cavity by lavage with 5\u2009ml of ice-cold, sterile PBS/3%FCS. After a centrifugation step at 300 \u00d7 g for 5\u2009min, cells were seeded at 1 \u00d7 106 cells/well in RPMI 1640, supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin, 100\u2009\u00b5g/ml Streptomycin, 55\u2009\u00b5M \u03b2-mercaptoethanol. Non-adherent cells were discarded after 6\u2009h. Adherent macrophages were used after overnight cultivation.DCs from proliferating mouse bone marrow progenitors were isolated from femur and tibia of 8\u201310-weeks-old wild-type C57BL/6J mice and differentiated according to Lutz et al.6 cells. If required, cells were stained after RBC\u2010lysis with 1.66% NH4Cl w/v. Live/Dead cell discrimination was performed by the addition of Zombie Green\u2122 Fixable Viability Dye . Flow cytometry was performed using BD LSRFortessa\u2122 or BD FACSLyric\u2122 instruments. Data were acquired using BD FACSDiva\u2122 Version 8.0.1 and BD FACSuite\u2122 1.2.1 (BD Biosciences). Data were analyzed using FlowJo\u2122 v10.7.1 software (BD Biosciences).Antibodies and dilutions used in this study are listed in Supplementary Table\u00a05 THP-1/ml in the presence of rhGM-CSF (20\u2009ng/ml) and rhIL-4 (20\u2009ng/ml) for 5 days65. THP-1 cells were differentiated into monocyte-derived macrophages (MoM\u00d8) using 100\u2009nM of phorbol 12-myristate 13-acetate  for 72h66. Blood samples from healthy donors were collected as whole blood donation in 500\u2009ml blood bags supplemented with CPDA-1 stabilizer. Human peripheral blood mononuclear cells (PBMC) were isolated within the first hour of acquisition by Ficoll\u00aePaque\u2122 PLUS  centrifugation of 35\u2009ml fresh blood in 50\u2009ml LeucoSep (Greiner) tubes at 800\u00d7g for 15\u2009min at RT without brake. Mononuclear cells at the interphase were purified by multiple washing steps with 1\u00d7 DPBS. Human primary cells were cultured in AIM-V medium  supplemented with 2% heat-inactivated human AB serum  and 50\u2009\u03bcM \u03b2-mercaptoethanol (complete AIM-V). CD14+ monocyte-derived DCs (MoDCs) were enriched by positive magnetic selection using anti-CD14 microbeads according to the manufacturer\u2019s instructions . Purified CD14+ monocytes were differentiated into MoDCs by culturing 2 \u00d7 106 cells/ml in complete AIM-V medium supplemented with 500 U/ml recombinant human GM-CSF  and 250 U/ml recombinant human IL-4  for 5 days. CD14+ monocytes were differentiated into CD68\u2009+\u2009MoM\u00d8 by culturing 2 \u00d7 106 cells/ml in a complete AIM-V medium supplemented with 50\u2009ng/ml rhGM-CSF for 5 days67. CD1c+ (BDCA-1+) and CD141+ (BDCA-3+) myeloid DCs were purified from CD14-negative PBMCs using MACS isolation kits  or magnetic MicroBeads , respectively. CD1c+ and CD141+ cells were used directly.The human monocytic leukemia cell line THP-1 was differentiated into immature DC with similar characteristics to human monocyte-derived DCs (MoDCs) by culturing 2 \u00d7 10\u22126\u2009M SIINFEKL peptide for 5\u2009h in the presence of 10\u2009\u00b5M/ml Brefeldin A . For surface staining, cells were incubated with anti-CD8\u03b1 APC  for 30\u2009min at 4\u2009\u00b0C; then cells were fixed and permeabilized using a Cytofix/Cytoperm Kit  according to the manufacturer\u2019s instructions as to preparation for intracellular staining using anti-IFN\u03b3 BV421 antibody  in BD Perm/Wash\u2122 buffer . CD8+IFN\u03b3+ cells were analyzed 16\u2009h later by flow cytometry.Intracellular cytokine staining (ICS) was performed to detect intracellular IFN\u03b3 as a measure of cytotoxic T lymphocyte activation. Splenocytes or tumor-infiltrating lymphocytes (TILs) were cultured with or without 105 cells per well onto pre-coated MultiScreen\u00aeHTS Filter Plates (Merck Millipore) and re-stimulated with 10\u2009\u03bcM of the respective peptide for 20\u2009h. After incubation with the biotinylated secondary antibody specific for IFN\u03b3, a streptavidin-alkaline phosphatase enzyme conjugate was added. After the addition of the BCIP\u00ae/NBT substrate solution , a purple precipitate is formed as spots at the sites of captured IFN\u03b3. Automated spot analysis and quantification were performed using the ImmunoScan\u00ae analyzer and Immunospot software v.6.0.0.2 .IFN\u03b3 production of OVA-specific cells was analyzed using a commercially available mouse ELISPOT antibody pair according to the manufacturer\u2019s protocol . Briefly, splenocytes of immunized mice were seeded at 5 \u00d7 10Equal amounts of supernatants of PLGA-MP release assays were resolved in reducing conditions on 10% SDS-polyacrylamide gels for electrophoresis. Silver Staining was performed according to the manufacturer\u2019s instructions .Supernatants of DC cultures and BMDC-DC co-cultures were analyzed for different cytokines by ELISA according to the manufacturers\u2019 instructions. Commercially available ELISA Kits were purchased from Thermo Fisher Scientific (Invitrogen\u2122 eBioscience\u2122 ELISA Ready-SET-Go!\u2122) except for mIFN\u03b3  and mIFN\u03b2 . Mouse IFN\u03b1, as well as human IFN\u03b1 and IFN\u03b2 were purchased from PBL .+ T-cell hybridoma, specific for the SIINFEKL peptide of ovalbumin (OVA257\u2013264) and the CD4+ T-cell hybridoma DOBW specific for the ovalbumin class II epitope ISQAVHAAHAEINEAGR (OVA323\u2013339). 1 \u00d7 105 hybridoma cells were co-cultured overnight with 2 \u00d7 105 DCs loaded with PLGA particles. To determine the relative maximum of T-cell activation, DCs were pulsed with SIINFEKL peptide . 100\u2009\u03bcl of lacZ buffer ) was added for an additional 4-h incubation step68. The absorbance of the chromogenic reaction was measured at 570\u2009nm with background subtraction at 620\u2009nm using a spectrofluorometer . Presentation on MHC class II was similarly assessed as described above, except for using the OVA323\u2013339-specific hybridoma cell line DOBW. IL-2 secretion was measured from supernatants of DOBW-DC co-cultures using a mouse IL-2 ELISA kit  according to the manufacturer\u2019s protocol.Antigen (cross-) presentation of SIINFEKL peptide was assessed using the B3Z CD8+ T cells were isolated from OT-I spleens and LN using mouse CD8a (Ly-2) MicroBeads , and then labeled with 1\u2009\u03bcM of the proliferation and cell tracking dye CFSE . 1 \u00d7 105 labeled CD8+ cells were co-cultured with an equal number of PLGA particle loaded DCs or splenocytes of PLGA-MP immunized mice in complete medium. After 72\u2009h, cells were recovered, and OT-I proliferation was measured by CFSE dye dilution of CD8+ T cells using flow cytometry. Additionally, IFN\u03b3 secretion of culture supernatants was measured by mouse IFN\u03b3 ELISA kit  according to the manufacturer\u2019s protocol.CD869. On day 5 post-vaccination, splenocytes from congenic C57BL/6J mice were pulsed with 1\u2009\u00b5M SIINFEKL peptide (vaccine-specific target cells) or left unpulsed (non-specific control cells). Immunized mice were i.v. injected with a 1:1 mixture of 107 cells labeled with either 5\u2009\u00b5M CFSE  (peptide-pulsed) or 0.5\u2009\u00b5M CFSE (unpulsed). The following day, ratios of the CFSE bright to dim fluorescent peaks of CD45+ splenocytes were analyzed. OVA-specific cytotoxicity was calculated using the following equation: {1\u2212(PLGA-MP immunized mice) [CFSEhigh(%)/CFSElow(%)]/naive mice [CFSEhigh(%)/CFSElow(%)]} \u00d7 100Cytotoxic activity of OVA-specific CTLs in PLGA-MP immunized mice were analyzed in vivo4 cells per well for 48\u2009h at 37\u2009\u00b0C. The number of live  cells was evaluated by the addition of 10% of Deep Blue Cell Viability\u2122 Kit  according to the manufacturer\u2019s protocol. The reduction of resazurin into resorufin was photometrically detected using a spectrofluorometer  at 550\u2009nm. To demonstrate cytotoxicity, cells were treated with 10% (w/v) Triton\u00aeX-100 (Merck) for the last 4\u2009h as a positive control.The viability of murine BMDCs was determined by incubation of DCs with varying concentrations of PLGA-MP in 96-well plates at 5 \u00d7 10Mavs\u2212/\u2212, Tlr3\u2212/\u2212 or AAD mice were immunized either subcutaneously at the base of the tail or via the intraperitoneal route using 5\u2009mg PLGA particles loaded with antigen  and dsRNA analogs (2.5\u2009\u00b5g/mouse) in PBS. The following applications were performed under isoflurane anesthesia. For intranasal immunizations, mice were vaccinated with a suspension of 2.5\u2009mg PLGA particles  in 50\u2009\u03bcl PBS (25\u2009\u03bcl per nostril). 50\u2009\u00b5l of a 1\u2009mg/ml PLGA particle solution in PBS  were administered intramuscularly into the hind limb thigh muscle. The intranodal injection was also performed by administering 10\u2009\u00b5l of a 1\u2009mg/ml PLGA particle suspension in PBS  into the inguinal lymph node. Control mice were treated with respective amounts of empty PLGA particles or particles containing only dsRNA analogs. On day 6 after immunization, target organs were resected and homogenized into single-cell suspension by smashing through 70\u2009\u00b5m cell strainers  for further analysis. Lung tissue was dissociated using the mouse Lung Dissociation Kit  with a gentleMACS\u2122 octo Dissociater according to the manufacturer\u2019s protocol.For immunization studies, 8-to-12-week-old female and male C57BL/6J, Female BALB/c mice were s.c. injected with 5\u2009mg of QuantumDot containing PLGA-MP-OVA/Riboxxim into the left flank. The left non-injected flank served as a negative control. Fluorescence signals were acquired at indicated time points with the IVIS\u00ae Spectrum 200 In Vivo Imaging System (Perkin Elmer) using the Far-red filter set. Popliteal and inguinal LNs were dissected at different time points post-immunization and incubated in 10% neutral formalin for subsequent paraffin-embedding and histology.Organs were excised and immediately fixed in 10% neutral-buffered formalin for 24\u2009h followed by paraffin-embedding and sectioning to 5-\u00b5m-thick slides. H&E staining was performed according to standard protocols. For IHC staining, tumor-bearing tissues were collected and processed as described before. Paraffin-embedded tissue sections of 3\u2009\u00b5m thickness were rehydrated and subjected to heat-induced antigen retrieval (HIER) in 10\u2009mM citrate buffer (pH 6). Blocking was performed with 10% goat serum. Sections were incubated with rat primary antibodies specific for mouse CD8a (1:200)  at 4\u2009\u00b0C overnight and then stained with an avidin-biotin-based peroxidase system . Positive immunoreactions were visualized using peroxidase substrate kits . Sections were counterstained with hematoxylin. Results of three random fields per section were recorded with AxioObserver or AxioImager microscopes and ZEN 3.2 (blue edition) imaging software (Carl Zeiss Imaging Inc.) and analyzed in a blinded manner.n\u2009=\u20095). The establishment of lung metastases was performed by i.v. injection of 105 MO5 melanoma cells. To direct antitumor responses to the site of tumor formation, mice were immunized with PLGA particles in a prime-boost setting as previously described24. To this aim, mice received a subcutaneous prime immunization followed by an intranasal boost 14 d later. Therapeutic PLGA-MP treatment was performed by simultaneous s.c. and i.n. double route-immunization. Solid tumors were established by s.c. injection of 5 \u00d7 105 syngeneic E.G7-OVA-luc+ cells into the left flank of each mouse. Tumor volumes were measured at regular intervals by using bioluminescence and caliper measurements of two perpendicular tumor dimensions. The tumor volume was calculated using the following formula: Tumor volume (mm3) = [(long diameter) \u00d7 (short diameter)2] \u00d7 0.5. A single dose of PLGA-MP treatment was administered either in a protective manner 6 days before tumor challenge or therapeutically when tumor nodules became palpable. To deplete CD8+ T cells, mice were i.p. injected with 100\u2009\u00b5g of InVivoMAb anti-mouse CD8\u03b2/Lyt 2.1 mAb (clone 116-13.1 (HB129), #BE0118, BioXCell) 3 days before and on the day of tumor cell inoculation followed by weekly injections until the end of the experiment. Depletion of CD8+ T cells in peripheral blood was validated by flow cytometric analysis with 100% loss of CD8+ T cell at the peak of PLGA-MP-mediated antitumor CTL response (day 6 post-vaccination). Immune checkpoint blockade was achieved by multiple i.p. injections of GoInVivo\u2122 purified anti-mouse CD152  with a cumulative dose of 250\u2009\u00b5g anti-CTLA-4 per mouse or a total of 1500\u2009\u00b5g purified anti-PD-1 mAb per mouse ). Detailed treatment regimens of immune checkpoint blockade and PLGA-MP vaccinations are outlined in schemes and figure legends. The tumor size was measured every 3 days using a caliper and bioluminescent signals were acquired at indicated time points using the IVIS\u00ae 200 imaging system (Perkin Elmer). Mice were anesthetized by isoflurane inhalation and D-Luciferin*K  was administered intraperitoneally at 150\u2009mg/kg prior to luminescence imaging. Luminescent signals were acquired using a 20\u2009cm field of view, small binning, and 120\u2009s exposure time. Regions of interest (ROI) were pre-set around the signal on pseudo-color luminescent images using Living Image\u00ae v4.1 Software (Perkin Elmer). The emitted signal intensity (photon flux) was integrated over these ROIs and it is expressed as relative light units  with the lower signal threshold set to 5% of the maximum signal value. Body weights were monitored three times a week. Tumor-bearing mice were euthanized in case s.c. tumors exceeded 1500 mm3 or exhibit necrotic or ulcerative pathologies and when animals showed signs of distress or rapid weight loss of 20% of the initial weight.C57BL/6J mice were randomized into different groups  followed by Tukey\u2019s or Sidak\u2019s post-hoc tests was used for comparison of multiple groups. The Kaplan\u2013Meier survival analysis was used to estimate statistical significance in overall survival distribution between the groups and Log-rank (Mantel\u2013Cox) tests were applied to compare survival rates. Unless otherwise stated in the figure legends, all data were derived from at least three independent experiments and are presented as means \u00b1 standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8.4.1 . Statistical significance was achieved when P\u2009<\u20090.05.Statistical significance was determined by applying a two-tailed Student\u2019s Further information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationPeer Review FileReporting summary"}
{"text": "Background: Cardiovascular disease, and more recently, subclinical cardiac dysfunction have both been implicated as important risk factors for cognitive decline. Several measures have been used to detect subclinical cardiac dysfunction, with global longitudinal strain (GLS) emerging as an important and more sensitive indicator than traditional measures. Yet, the association of GLS with cognitive function remains relatively unexplored. Objective: The aim of this review is to systematically summarize the literature exploring the association between GLS and cognitive function. Methods: We conducted a systematic review of the literature following PRISMA guidelines using the following databases: PubMed, OVID Medline, Embase, Web of Science, and CINAHL. Inclusion criteria were observational studies published in English, measuring GLS and assessing cognitive function through neuropsychiatric tests or brain imaging. Quality assessment was done using the Newcastle Ottawa Scale. Results: The initial search revealed 394 studies, of which three met inclusion criteria and were included for final review. The three studies included were cross-sectional and of high quality. They all reported that lower GLS scores were associated? with worse cognitive function and more brain abnormalities in both bivariate and multivariable analysis. Conclusion: Subclinical cardiac dysfunction, identified by GLS, was associated with worse cognitive function and presence of cerebral abnormality on brain imaging. The underlying mechanism could be attributed to dysfunctional autoregulatory and microvascular processes occurring in the brain vasculature. Further longitudinal studies are needed to better delineate the relationship between GLS and cognitive function."}
{"text": "The obtained results show that the energy levels can be modulated by changing the strength of the acceptor unit. Among the three investigated end-groups, 1,1-dicyanomethylene-3-indanone exhibited the largest bathochromic shift and the lowest band gap suggesting the strongest electron-withdrawing character. Moreover, the emissive properties of the investigated systems vary greatly with the nature of the terminal group and are generally lower compared to their precursor aldehyde derivatives.The synthesis of some novel donor-acceptor and acceptor-donor-acceptor systems containing a 2,2\u2032-bi[3,2- I\u2013III, while compounds with a core structure IV showing a S(IV) atom are not stable and could not be isolated.Among the aromatic-fused 5,5 heterocyclic systems with one heteroatom in each ring, fused bithiophenes stand out due to their versatile properties and vast applications ,4,5,6,7.b]thienothiophene  is the most investigated -TT derivatives are also connected to converting solar energy into electricity. Thus, they were employed for the fabrication of efficient dye-sensitized solar cells (DSSCs) -TT derivatives, we considered it of interest to expand our research in the field of molecules with exciting optoelectronic properties [b]thienothiophene derivatives belonging to D-A (V) or A-D-A (VI) systems  with the hexyloxy (compound 2) groups was mandatory to ensure good processability for the structure and properties investigations of the targeted new derivatives with 2,2\u2032-bithienothiophene. The homocoupling of 2, leading to 3,3\u20326,6\u2032-tetrakis(hexyloxy)-2,2\u2032-bithienothiophenes 4  or diformyl (6) derivatives  molar ratio.The first step to accessing the target compounds of types reported . We starorted -TT central unit.Concerning the low-energy absorption bands, their theoretical absorption maxima ranges from 546 nm to 645 nm within the O) Egap,  trend foO) Egap, . More pr(A-)D-A motifs, with a lower coefficient on the acceptor moiety in the case of the DCV- and InOCN-based compounds. Similarly, LUMO is delocalized over both the donor and acceptor units within the A-D-A molecules, but with a considerably higher coefficient on the acceptor part in the case of the D-A counterparts  and internal charge transfer (ICT), while in the case of the A-D-A counterparts, only local transitions take place. To further prove these statements, we plotted the electron density difference between first excited state and ground state for each molecule  the absence of a \u03c0-spacer between D and A that could facilitate the localization of the HOMO on the donor side and the LUMO on the acceptor moiety ; (ii) the presence of two acceptor moieties, which pull the electron density in opposite direction and do not allow its polarization toward one EWG; and (iii) the imbalance between the donation ability of the -TT platform and accepting ability of EWGs.As HOMO and LUMO have the strongest impact on the optoelectronic properties of our studied compounds, it is useful to depict their spatial distribution in order to obtain more information about the nature of the electronic transitions. On this note, careful inspection of the wave functions shows a distribution of HOMO over the entire molecule in all terparts . This ormolecule . Upon ex1H NMR (400 or 600 MHz) and 13C NMR (100 or 150 MHz) spectra were recorded in CDCl3 or CD2Cl2 at room temperature using the solvent line as a reference.254 TLC plates. All plates were visualized by UV irradiation at 254 and 365 nm.Thin layer chromatography (TLC) was conducted on silica gel 60 FSolvents were dried and distilled under argon using standard procedures. Chemicals were purchased from TCI Chemicals, and Alfa Aesar and Sigma Aldrich and were used without further purification.Melting points were determined with a Kleinfeld apparatus and are uncorrected.HRMS were recorded using an LTQ XL OBITRAP mass spectrometer equipped with ESI/APCI sources. UV-vis optical data in solution and in films were recorded with an UV-vis 1900 Shimadzu spectrometer. Fluorescence spectra were recorded on a JASCO FP-8300 spectrofluorometer using glass cuvettes (1 cm). Solutions for UV-vis, CV, HRMS and fluorescence measurements were prepared in HPLC grade solvents .Films were obtained from chloroform solutions using a classical spin coater.Fluorescence quantum yields were calculated using fluoresceine  or rhodamine B  as standards.5 and 6; 520 nm for 7, 8, 10, 11; and 640 nm for 9) and at a 40 MHz repetition rate. The signal was collected from the compounds in a CHCl3 solution with a UPlanSApo 60 \u00d7/NA = 1.2 water immersion objective and filtered thereafter with a 50 \u03bcm pinhole and adapted long-pass emission filters.Fluorescence lifetime measurements were performed on a MicroTime200 time-resolved confocal fluorescence microscope system , described in detail elsewhere . The exc4NPF/CHCl3 at 100 mV/s, Pt electrode references, SCE.Cyclic voltammetry (CV) measurements were carried out with a Biologic SP-150 potentiostat using a three-electrode cell equipped with a platinum electrode, a calomel reference electrode (SCE) and a platinum wire counter electrode. All experiments were performed in 0.10 M Bun-butyllithium (1.5 M in hexane)  was added dropwise over 10 min at \u221278 \u00b0C to a solution of 3,6-bis(hexyloxy)thienothiophene  in dry THF (65 mL) and the stirring was continued for another 2 h at the same temperature. Then, CuCl2  was added in one portion and the reaction mixture was gradually warmed up to room temperature and then stirred overnight. After adding water (30 mL) and triethylamine (10 mL), the mixture was extracted with dichloromethane (3 \u00d7 30 mL) and the combined organic layers were dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure. The crude product was purified with silica gel gradient column chromatography (elution system started with pentane/CH2Cl2 = 2/0.2 and ended with pentane/CH2Cl2 = 2/0.4) to afford a yellow solid .Under stirring and in an argon atmosphere, 4  in dry 1,2-dichloroethane (10 mL) was purged with argon and cooled to 0 \u00b0C. Then, DMF  and phosphoryl chloride  were slowly added and the reaction mixture was additionally stirred for 1 h at 0 \u00b0C. Then, the reaction mixture was heated under stirring at 60 \u00b0C for 18 h. Afterwards, the mixture was poured into a NaOAc solution  and vigorously stirred for 2 h at rt. The final solution was extracted with dichloromethane (3 \u00d7 30 mL) and the combined organic layers were dried over magnesium sulfate. The solvents were removed under reduced pressure and the crude product was purified by silica gel gradient column chromatography (elution system started with pentane/CH2Cl2/Et3N = 2/1/0.02 and ended with pentane/CH2Cl2 = 2/1.5) to afford an orange solid for 5  or a red solid for 6 .A solution of 7: 47 mg, 0.71 mmol; 10: 107 mg, 1.62 mmol) and triethylamine (0.1 mL) were subsequently added to an argon purged solution of 5  or 6  in toluene (10 mL) and the reaction mixture was stirred at room temperature for 18 h. Then, the solvent was removed under reduced pressure, the crude product was triturated with ethanol, and the precipitate was filtered off to afford a metallic green solid .Malononitrile  and piperidine  were subsequently added to an argon-purged solution of 5  or 6  in toluene  and the reaction mixture was stirred at 70 \u00b0C for 20 h. Then, the solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography . Pure samples were obtained with further purification by crystallization through slow evaporation from a 2/1 mixture of dichloromethane/ethanol for 8, and in the case of 11, by precipitation with ethanol from its solution in chloroform. The products were obtained as metallic green solids .3-Ethyl-2-thioxothiazolidin-4-one (H-inden-1-ylidene)malononitrile  and triethylamine (0.4 mL) were added subsequently to an argon-purged solution of 5  in toluene (30 mL) and the reaction mixture was stirred at 60 \u00b0C for 3 h. The solvent was removed afterwards under reduced pressure and the crude product was purified by silica gel column chromatography (elution system: pentane/CH2Cl2/Et3N = 4/5/0.04). Further purification by crystallization from a 2/1 mixture chloroform/ethanol afforded a metallic green solid .2-. 1H-NMR  \u03b4 (ppm): 0.89\u20130.93 , 1.33\u20131.38 , 1.45\u20131.55 , 1.80\u20131.92 , 4.07 , 4.32 , 6.19 . 13C-NMR  \u03b4 (ppm): 14.20, 14.21, 22.75, 25.75, 25.82, 29.22, 30.13, 31.69, 70.76, 72.60, 97.46, 118.22, 127.60, 128.01, 144.86, 150.54. HRMS (ESI): m/z calcd for [C36H54O4S4 + H]+: 679.2978; found: 679.2966.3,3\u2019,6,6\u2019-tetrakis(hexyloxy)-2,2\u2019-bithienothiophene-5-carbaldehyde 5: orange solid; mp: 105\u2013106 \u00b0C; 58 mg, 0.08 mmol, 66%; Rf = 0.7 (pentane/CH2Cl2/Et3N = 2/1/0.02). 1H-NMR  \u03b4 (ppm): 0.90\u20130.93 , 1.33\u20131.37 , 1.45\u20131.56 , 1.81\u20131.94 , 4.08 , 4.32 , 4.42 , 4.54 , 6.26 , 10.00 . 13C-NMR  \u03b4 (ppm): 14.15, 14.20, 14.22, 22.70, 22.73, 22.79, 25.61, 25.68, 25.80, 25.86, 29.17, 29.96, 30.13, 30.15, 31.56, 31.65, 31.68, 31.72, 70.91, 72.70, 72.82, 73.07, 98.82, 115.96, 122.61, 124.29, 126.52, 126.59, 130.05, 135.90, 144.48, 146.61, 150.48, 157.96, 181.29. HRMS (ESI): m/z calcd for [C37H54O5S4 + H]+: 707.2927; found: 707.2957.3,3\u2019,6,6\u2019-tetrakis(hexyloxy)-2,2\u2019-bithienothiophene-5,5\u2019-dicarbaldehyde 6: red solid; mp: 192\u2013193 \u00b0C; 140 mg, 0.19 mmol, 86%; Rf = 0.5 (pentane/CH2Cl2/Et3N = 2/1/0.02). 1H-NMR  \u03b4 (ppm): 0.90\u20130.94 , 1.35\u20131.39 , 1.48\u20131.56 , 1.84\u20131.93 , 4.42 , 4.55 , 10.03 . 13C-NMR  \u03b4 (ppm): 14.15, 14.21, 22.70, 22.77, 25.60, 25.78, 29.96, 30.16, 31.56, 31.68, 72.93, 73.27, 123.56, 123.73, 126.29, 134.39, 146.16, 157.62, 181.53. HRMS (ESI): m/z calcd for [C38H54O6S4 + H]+: 735.2876; found: 735.2906.3,3\u2019,6,6\u2019-tetrakis(hexyloxy)-2,2\u2019-bithienothiophen-5-yl)methylene)malononitrile 7: metallic green solid; mp: 189\u2013190 \u00b0C; 44 mg, 0.06 mmol, 82%; 1H-NMR  \u03b4 (ppm): 0.90\u20130.95 , 1.35\u20131.40 , 1.47\u20131.56 , 1.81\u20131.94 , 4.09 , 4.32 , 4.48 , 4.58 , 6.31 , 7.90 . 13C-NMR  \u03b4 (ppm): 14.17, 14.20, 14.23, 22.70, 22.71, 22.74, 22.80, 25.65, 25.68, 25.80, 25.88, 29.18, 29.91, 30.09, 30.15, 31.57, 31.68, 31.72, 68.51, 70.98, 72.81, 73.20, 73.34, 99.70, 115.11, 115.19, 116.36, 116.48, 122.64, 126.12, 129.86, 131.15, 137.91, 144.18, 146.41, 147.67, 150.46, 157.94. HRMS (APCI): m/z calcd for [C40H54N2O4S4 + H]+: 755.3039; found: 755.3036.2--2,2\u2019-bithienothiophen-5-yl)methylene)-2-thioxothiazolidin-4-one 8: metallic green solid; mp: 138\u2013139 \u00b0C; 40 mg, 0.05 mmol, 55%; Rf = 0.65 (pentane/CH2Cl2/Et3N = 2/1/0.02). 1H-NMR  \u03b4 (ppm): 0.91\u20130.96 , 1.25 , 1.36\u20131.41 , 1.47\u20131.52 , 1.55\u20131.62 , 1.81\u20131.95 , 4.07 , 4.14 , 4.35 , 4.42 , 4.52 , 6.28 , 7.99 . 13C-NMR  \u03b4 (ppm): 12.56, 14.36, 14.38, 14.42, 14.45, 23.15, 23.17, 23.19, 23.21, 26.06, 26.17, 26.21, 26.30, 29.65, 30.46, 30.57, 30.60, 32.07, 32.14, 32.16, 32.17, 40.34, 71.42, 73.24, 73.43, 73.56, 99.37, 116.00, 116.29, 119.12, 123.90, 125.28, 125.55, 127.00, 130.24, 134.54, 144.71, 146.97, 150.81, 154.44, 167.49, 192.57. HRMS (APCI): m/z calcd for [C42H59NO5S6 + H]+: 850.2790; found: 850.2819.(Z)-2--2,2\u2019-bithienothiophen-5-yl)methylene)-2,3-dihydro-1H-inden-1-ylidene)malononitrile 9: metallic green solid; mp: >360 \u00b0C; 25 mg, 0.03 mmol, 57%; Rf = 0.7 (pentane/CH2Cl2/Et3N = 4/5/0.04). 1H-NMR  \u03b4 (ppm): 0.92\u20130.97 , 1.38\u20131.43 , 1.47\u20131.54 , 1.59\u20131.64 , 1.85 , 1.91 , 1.93\u20131.99 , 4.04 , 4.43 , 4.48 , 4.66 , 6.21 , 7.45 , 7.50 , 7.73 , 8.37 , 9.00 . 13C-NMR  \u03b4 (ppm): 14.22, 14.25, 14.27, 14.28, 22.77, 22.82, 25.72, 25.85, 25.86, 25.93, 29.94, 29.86, 30.14, 30.26, 31.69, 31.73, 31.81, 31.85, 65.62, 70.88, 72.89, 73.07, 73.77, 99.80, 115.43, 115.63, 116.19, 117.51, 120.53, 122.55, 122.85, 124.29, 125.78, 131.17, 131.75, 133.04, 133.17, 133.85, 136.64, 140.01, 142.63, 144.75, 148.02, 150.37, 160.49, 161.33, 188.87. Elemental analysis: calcd. for C49H58N2O5S4: C 66.63, H 6.62, N 3.17, S 14.52; found: C 66.87, H 6.48, N 3.11, S 14.36.bis(methan-1-yl-1-ylidene)dimalononitrile 10: metallic green solid; mp: 311\u2013312 \u00b0C; 65 mg, 0.08 mmol, 96%; 1H-NMR  \u03b4 (ppm): 0.90\u20130.95 , 1.33\u20131.40 , 1.48\u20131.52 , 1.56\u20131.59 , 1.87\u20131.93 , 4.47 , 4.59 , 8.00 . 13C-NMR  \u03b4 (ppm): 14.15, 14.19, 22.67, 22.75, 25.62, 25.82, 29.93, 30.13, 31.54, 31.70, 71.35, 73.47, 73.58, 114.56, 115.58, 117.87, 125.33, 125.36, 135.55, 146.65, 146.77, 157.53. HRMS (APCI): m/z calcd for [C44H54N4O4S4 + H]+: 831.3101; found: 831.3090.2,2\u2019--2,2\u2019-bithienothiophene-5,5\u2019-diyl)bis(methan-1-yl-1-ylidene)bis(3-ethyl-2-thioxothiazolidin-4-one) 11: metallic green solid; mp: 249\u2013250 \u00b0C; 135 mg, 0.132 mmol, 75%; Rf = 0.75 (toluene/CH2Cl2 = 2/0.2). 1H-NMR  \u03b4 (ppm): 0.92\u20130.96 , 1.28 , 1.37\u20131.42 , 1.52 , 1.61 , 1.87 , 1.94 , 4.17 , 4.42 , 4.49 , 8.03 . 13C-NMR  \u03b4 (ppm): 12.46, 14.19, 14.27, 22.72, 22.81, 25.66, 25.89, 30.08, 30.22, 31.64, 31.79, 40.05, 73.06, 73.15, 116.90, 120.09, 122.20, 123.42, 126.80, 132.70, 145.88, 153.61, 167.27, 191.89. HRMS (ESI): m/z calcd for C48H64N2O6S8: 1020.2525; found: 1020.2581.(5b]thienothiophene donor block and various electron-withdrawing groups as acceptors was synthesized and characterized. An in-depth study of the terminal electron-acceptor unit\u2019s impact on the optical and electrochemical properties was carried out. It was shown that the introduction of electron-withdrawing groups in parent 2,2\u2032-bithienothiophene led to a red shift of the main absorption band due to a decrease in the HOMO-LUMO band gap. All investigated systems display emission maxima in the green region, with the A-D-A compounds exhibiting poorer photoluminescence properties compared to their D-A counterparts. Finally, theoretical studies shed light on the observed optoelectronic behaviours by revealing the type and nature of the transitions that can take place and also by showing how various electron-accepting groups tune the optical bandgaps.In conclusion, a series of symmetrical and unsymmetrical conjugated systems based on a 2,2\u2032-bi[3,2-"}
{"text": "Older adults overwhelmingly prefer to age at home and in their community, yet research shows uneven access to quality home and community-based services (HCBS), especially among diverse racial and ethnic groups. Our research in Georgia and New York employs participatory research methods, including key informant interviews and focus groups conducted in 2021 and 2022, to identify root causes of disparities in access to, quality of, and experience with HCBS for older racial and ethnic and LGBTQI+ communities. Some root causes identified include: complexity in and lack of funding, tension between equal versus equitable service provision, and meaningful community outreach, particularly in Latino communities. We then identify scalable opportunities policy and programmatic interventions to improve care and service equity for older adults in those communities."}
{"text": "Logistic regression analyses assessed the association between their perceived risk of having COVID when smoking LCCs and pandemic-related behavioral changes in CAI and CAB use . Findings showed that users with a higher perceived risk of getting COVID-19 when smoking LCCs were more likely to endorse trying to quit CAI and CAB during the pandemic. Compared to the non-Hispanic White population, the non-Hispanic Black population were less likely to endorse smoking less CAI and trying to quit CAB during the pandemic. Dual users of CAI and CAB and females were more likely to endorse smoking more CAB compared to CAB-only users and males, respectively. Tailored cessation strategies are needed for dual users, non-Hispanic Black young adults, and young women. Raising awareness about the risks of LCC use can be an effective strategy for LCC smoking cessation.We examined the smoking behaviors of U.S. young adults ages 18\u201336 regarding little cigars and cigarillos (LCCs) during the COVID-19 pandemic. Survey data were collected from a nationally representative sample of young adults between October and November 2020. Respondents who reported using LCCs with tobacco (CAI) and/or with marijuana (CAB) within the past 6 months prior to the survey ( Respondents were included in the study only if they reported having used CAI or CAB within the past 6 months or less.Based on the findings from a previous focus group study in which LCC users indicated that they most often smoke a part of LCC product occasionally,  respondeRespondents were asked individual questions about changes in their CAI and/or CAB smoking behaviors because of the COVID-19 pandemic. This was measured based on the questions, \u201cHave any of the following happened to your little cigar or cigarillo smoking [as sold/as blunt] behavior because of COVID-19?\u201d Respondents rated their level of agreement to the following items: smoked CAI/CAB more than usual, smoked CAI/CAB less than usual, tried to stop smoking CAI/CAB, and worried about smoking CAI/CAB because of the risk associated with COVID-19. Dual users responded to both CAI and CAB-specific questions. The responses were measured on a 7 point scale ranging from 1 (strongly disagree) to 7 (strongly agree).Respondents were asked about using other tobacco products, including cigarette, large cigars, hookah, and e-cigarette (with and without marijuana) in the past 6 months prior to the survey. This was measured based on the questions, \u201cHave you ever used a [cigarette/large cigars/hookah/e-cigarette (with and/or without marijuana)] at least once, in your lifetime? If yes, when was the most recent time you used a [cigarette/large cigars/hookah/e-cigarette (with and/or without marijuana)] even one or two puffs?\u201d Pictures and examples of brand names of these tobacco products were shown in the survey to help respondents accurately answer the survey questions.All analyses were weighted to the general U.S. young adult population and were performed using SAS version 9.4 . Chi-square tests were used to examine the differences in demographic characteristics among the user groups. Logistic regression was used to examine COVID-19 risk perception and subsequent behavioral changes specific to CAI and CAB use, controlling for demographic-level factors  and user type . The responses for the behavioral changes in LCC use during the COVID-19 pandemic were categorized as: those who \u201cagreed\u201d , \u201cdisagreed\u201d , and \u201cneither agree nor disagree\u201d  to experiencing changes in LCC use behaviors. Those who neither agreed nor disagreed were excluded from the regression analysis.p < 0.01).Among 399 past 6 month LCC users in our sample, 72 were CAI-only users (14.5%), 160 were CAB-only users (38.0%), and 167 were dual users (47.4%) . The meaPast 6 month CAI-only users were 68.4% male, 86.8% heterosexual, 42.4% Hispanic, and 73.9% had some college experience. Past 6 month CAB-only users were 56.4% female, 70.4% heterosexual, 43.7% non-Hispanic White, and 63.4% had some college experience. Past 6 month dual users were 63.6% male, 79.9% heterosexual, 35.7% non-Hispanic White, and 57.6% had high school or less education.p < 0.01).Consistent with previous studies ,25, polyp < 0.01) between CAB-only and dual users regarding worrying about smoking CAB, trying to quit CAB, and smoking less CAB during the pandemic.Overall, respondents did not report experiencing substantial changes in CAI and CAB use during the first several months of the COVID-19 pandemic . Among CCompared to the non-Hispanic White population, the non-Hispanic Black population were more likely to endorse worrying about CAI use because of the risk associated with COVID-19 , controlling for other sociodemographic characteristics and risk perceptions . Those wCompared to CAB-only users, dual users were less likely to endorse worrying about CAB use because of the risk related to COVID-19  and smoking less CAB during the pandemic  after controlling for other characteristics. They were also more likely to endorse smoking more CAB  than CAB-only users. Those with greater perceived risk of having COVID-19 if using LCCs were more likely to endorse worrying about CAB  and trying to quit CAB  during the pandemic. The non-Hispanic Black population were less likely to endorse trying to quit CAB  compared to non-Hispanic White population. Moreover, compared to males, females were more likely to endorse smoking more CAB  during the pandemic.The current study examined factors that are associated with the reported behavioral changes in LCC use during the first several months of the COVID-19 pandemic among young adult users, specified by the modality of use , LCC use with marijuana as blunts (CAB) and user group status . Descriptive analysis of our sample indicated that dual use of CAI and CAB was the most prevalent (47.4%), compared to exclusive CAB (38.0%) and CAI use (14.5%). Such a finding is in line with emerging studies that reported increased sales of cannabis in some U.S. states  and incrRespondents in our sample did not report drastic changes in LCC use during the pandemic. Still, we observed racial and ethnic differences in CAI and CAB use behaviors. Compared to the non-Hispanic White population, the non-Hispanic Black population were less likely to endorse trying to quit CAB and smoking less CAI during the pandemic, although they were more likely to endorse worrying about smoking CAI because of risk related to COVID-19. The heightened concern about smoking CAI may be due to the disproportionate impact of COVID-19 on communities of color . Yet, thIn addition, females were less likely to endorse smoking less CAI and more likely to endorse smoking more CAB than males during the pandemic. One likely explanation for the observed racial/ethical and gender differences is that pandemic-induced stressors, such as financial concerns, death of loved ones, feeling isolated ,32, and We also found that user type  had an impact on COVID-related behavioral changes in LCC use. Compared to CAB-only users, dual users were more likely to endorse smoking more CAB and less likely to endorse smoking less CAB during the pandemic. Because dual users use multiple tobacco products and consume both tobacco and marijuana, they may experience greater nicotine dependence ,37,38 thOur findings showed that users with a higher perceived risk of having COVID-19 when smoking LCCs were more likely to endorse trying to quit both CAI and CAB during the pandemic. Nevertheless, the perceived risk of having COVID-19 when smoking LCCs was low in our sample. This finding builds upon previous research that reported cigar smokers, including LCC users, tend to underestimate its health risks compared to cigarettes ,39,40. MPreliminary research shows that tobacco use is associated with more severe symptoms and progression of COVID-19 ,42. In aThis study has a few limitations. The behavioral changes in LCC use during the COVID-19 pandemic were self-reported and were not directly measured using biological measures. There is also a potential recall bias in reporting past LCC use. Moreover, while this study is representative of the young adult population in the U.S., it is important to note the limited sample size included in the logistic regression analysis, particularly for the CAI-only users.We examined young adult LCC users\u2019 perceived risk of COVID-19 and their behavioral changes in LCC use in response to the COVID-19 pandemic. Our findings showed that young adult LCC users, particularly the non-Hispanic Black population, dual users, and females, are at higher risk of increased LCC use during the pandemic. Smoking behaviors formed during the first few waves of the COVID-19 pandemic, as well as during the repeated outbreaks caused by new variants and subvariants of the virus, will continue to affect the health of LCC users. Furthermore, because the Food and Drug Administration (FDA) is considering stricter regulations on tobacco products, such as a JUUL e-cigarette ban and reducing allowable nicotine levels in cigarettes, it is possible that users of these tobacco products switch to using LCCs. Therefore, our findings may inform the development of tobacco prevention and cessation interventions. Programs seeking to reduce LCC use must have focused efforts to reduce all modalities of LCC use, including LCC use with tobacco (CAI) and with marijuana as blunts (CAB), among vulnerable sub-populations of young adult LCC users."}
{"text": "Background: Myocardial infarction (MI), characterized by reduced blood flow to the heart, is a coronary artery disorder with the highest morbidity and mortality among cardiovascular diseases. Consequently, there is an urgent need to identify effective drugs to treat MI. Rhizoma Corydalis (RC) is the dry tuber of Corydalis yanhusuo W.T. Wang, and is extensively applied in treating MI clinically in China. Its underlying pharmacological mechanism remains unknown. This study aims to clarify the molecular mechanism of RC on MI by utilizing network pharmacology and experimental verification.Methods: Based on network pharmacology, the potential targets of the RC ingredients and MI-related targets were collected from the databases. Furthermore, core targets of RC on MI were identified by the protein-protein interaction (PPI) network and analyzed with Gene Ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Molecular docking was used to validate the binding affinity between the core targets and the bioactive components. Oxygen-glucose deprivation (OGD) was performed on H9c2 cells to mimic MI in vitro. A Cell Counting Kit-8 assay was used to assess the cardioprotective effect of the active ingredient against OGD. Western blot analysis and RT-qPCR were used to measure the cell apoptosis and inflammation level of H9c2 cells.Results: The network pharmacology obtained 60 bioactive components of RC, 431 potential targets, and 1131 MI-related targets. In total, 126 core targets were screened according to topological analysis. KEGG results showed that RC was closely related to the phosphatidylinositol 3-kinase (PI3K)/Protein kinase B  signaling pathway. The experimental validation data showed that tetrahydropalmatine (THP) pretreatment preserved cell viability after OGD exposure. THP suppressed cardiomyocyte apoptosis and inflammation induced by OGD, while LY294002 blocked the inhibition effect of THP on OGD-induced H9c2 cell injury. Moreover, the molecular docking results indicated that THP had the strongest binding affinity with Akt over berberine, coptisine, palmatine, and quercetin.Conclusion: THP, the active ingredient of RC, can suppress OGD-induced H9c2 cell injury by activating the PI3K/Akt pathway, which in turn provides a scientific basis for a novel strategy for MI therapy and RC application. Myocardial infarction (MI), characterized by myocardial necrosis, is a major cause of morbidity and mortality worldwide, resulting in an estimated 7.4 million deaths per year . The ackR. Corydalis (RC) is the dried tuber of C. yanhusuo W.T. Wang, which belongs to the Papaveraceae family. RC was first recorded by Shennong Herbal Classic and believed to be able to activate blood, move \u201cQi\u201d  and alleviate painful conditions  Database , the EncHomo sapiens. The UniProtKB database was used to unify and standardize the targets  network of RC potential targets together with MI-related targets , and topp < 0.05, and the species was limited to H. sapiens.To explore the biological processes of core targets, Gene Ontology (GO) biological function and KEGG pathway enrichment analyses were performed with the online tool DAVID Bioinformatics Resources 6.8 . The scrThe crystal structure of core targets (macromolecules) was sought in the Protein Data Bank, and the structure (in SDF format) of the ligand (component) was retrieved from the PubChem database. We then changed the format of the molecules to pdb using OpenBabel software, and utilized AutoDockTools (version 1.5.6) for molecular docking. PyMOL software was then employed to display the docking model. All websites of the databases used in this study are listed in Tetrahydropalmatine   was purchased from Shanghai Yuanye Biotechnology Co., Ltd. . The THP powder was dissolved in dimethyl sulfoxide . Diazoxide (01131863) was provided by Adamas-beta , and LY294002 (S1105) was provided by Selleck .6 cells and then maintained in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were grown at 37\u00b0C in humidified 5% CO2. Before the subsequent experiments, a serum-free conditioned medium was used for cell culture.H9c2 cells were plated into a dish (35\u00a0mm) at a density of 1 \u00d7 10A Cell Counting Kit-8 (CCK-8) assay kit  was used to examine cell activity. Briefly, H9c2 cells were seeded into 96-well plates. After treatment, the cells were incubated with 10\u00a0\u03bcl CCK-8 solution per well for 2\u00a0h at 37\u00b0C, and after incubation, the absorbance value at 450\u00a0nm was measured using a microplate reader .Western blot assays were performed as we previously reported . PrimaryAfter overnight incubation, appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were added and incubated at room temperature for 1\u00a0h. The protein level signals were visualized using ECL-enhanced chemiluminescence , and the band intensities were quantified by ImageJ software (Bio-Rad).\u2212\u0394\u0394Ct method.The H9c2 cells were lysed with Accurate Biology RNAex Pro Reagent  to extract total RNA. Then 2\u00a0\u03bcg of RNA was reversely transcribed to cDNA with the Evo M-MLV RT Kit . The mRNA levels were determined with a SYBR Green qPCR kit  by QuantStudio 5 Real-Time PCR instrument . Rat-specific primers for BCL2 Associated X (Bax), B cell lymphoma-2 (Bcl-2), p53, interleukin-6 (IL-6), interleukin-1 beta (IL-1\u03b2), tumor necrosis factor-\u03b1 (TNF-\u03b1), and \u03b2-actin were synthesized by Sangon Biotech (Shanghai) . All semA one Step TdT-Mediated dUTP Nick-End Labeling (TUNEL) Apoptosis Assay Kit  was used to measure cell apoptosis. H9c2 cells were washed with phosphate buffered saline (PBS) three times and fixed with 4% paraformaldehyde for 10\u00a0min. After washing with PBS three times, H9c2 cells were incubated with 50\u00a0\u03bcl of TUNEL detection solution at 37\u00b0C for 1\u00a0h in the dark and then incubated with 4\u2032,6-diamidino-2-phenylindole (DAPI) for 10\u00a0min to visualize nuclei. The green spectrum and blue spectrum were used to detect TUNEL-positive cells and nuclei, respectively. Finally, the cells were photographed by confocal scanning microscopy . TUNEL-positive cells were quantified and analyzed from four different views. Data were expressed as the percentage of the number of TUNEL-positive nuclei in the total number of nuclei detected by DAPI staining. ImageJ software was used to calculate the number of TUNEL positive cells.p < 0.05 was considered statistically significant.The data in this study are presented as mean \u00b1 SD from three independent replicates. One-way analysis of variance (ANOVA) with the Bonferroni post-hoc test was performed among the multiple groups. In all cases, To collect the components of RC, we searched three databases, and in all, 60 bioactive components were collected by filtering the TCMSP, ETCM, and TCMID databases by the limitations of OB \u2265 30% and DL \u2265 0.18. The active ingredients mainly included berberine, sitosterol, tetrahydropalmatine, corydaline, and quercetin. The information about the components is listed in p < 0.05 and absolute fold change of 1.5 or greater. After deleting duplicates, 1,131 targets were obtained in total.To facilitate statistics, we set screening conditions for MI targets derived from corresponding databases. After taking the third quartile value, a relevance score \u2265 3.66 was set as the threshold for targets from the GeneCards database, and a score \u2265 0.1 was set as the threshold for those from the DisGeNET database. While searching GEO datasets, we selected the reference series GSE48060 to obtain the differentially expressed genes between normal cardiac function controls and first-time MI patients . The filWe used the Cytoscape plugin BisoGenet to construct a PPI network of RC potential targets   and MI-rp = 0.000000000020993, 31 targets/981), negative regulation of transcription from RNA polymerase II promoter , transcription, DNA-templated , viral process , negative regulation of apoptotic process , negative regulation of transcription, DNA-templated , cell-cell adhesion , positive regulation of transcription, DNA-templated , negative regulation of gene expression, epigenetic  and cellular protein metabolic process .To identify the biological function of the core targets, GO and KEGG enrichment analyses of the 126 core targets were performed using the DAVID online tool. Ultimately, a total of 126 targets were analyzed by GO, while 105 targets were enriched in KEGG pathways. The top 10 results of biological process analysis, cellular component analysis, and molecular function analysis of GO function and the top 10 major KEGG enriched pathways are shown in p = 0.000000000000000, 94 targets/5,415), nucleoplasm , cytoplasm , cytosol , membrane , extracellular exosome , protein complex , nucleolus , cell-cell adherens junction  and focal adhesion .The top 10 cellular components were nucleus , poly(A) RNA binding , ubiquitin protein ligase binding , enzyme binding , ATP binding , DNA binding , cadherin binding involved in cell-cell adhesion , protein heterodimerization activity , identical protein binding  and transcription factor binding .The major molecular functions were protein binding . As As the KEGG analysis of the core target results showed, among the 10 pathways, we found that the 126 core targets were primarily involved in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B  signaling pathway  significantly inhibited the overproduction of these inflammatory biomarkers compared to the OGD-induced group . CollectThe results from KEGG enrichment showed that RC and THP were closely related to the PI3K/Akt signaling pathway against MI and that most components could regulate Akt. Therefore, to confirm whether THP exhibits a protective effect via the regulation of Akt, the phosphorylation level and protein expression of Akt were detected. As shown in The PI3K/Akt signaling pathway is significant in defending against myocardial infarction damage . AccordiMI is a severe disease with high motility that places a heavy burden on individuals and society. The current mechanisms of MI include cell death, mitochondrial dysfunction, inflammatory response, oxidative stress, and ATP depletion . In addiThe chemical components of RC can be classified as alkaloids  . In thisMoreover, we obtained a PPI network by merging the active component target PPI of RC and the potential target PPI of MI . Then, ain vivo  The internal absorption and utilization of RC is a complicated process, meaning the function of RC may not simply accumulate the effects of multiple compounds. 2) The predicted targets are greatly affected by the quality of the databases and the algorithm. 3) The anti-apoptotic effect and the exact mechanism of THP against MI should be further demonstrated with animal models and AKT knockout animals.In conclusion, the present study aimed to investigate the effect and mechanism of RC against MI via network pharmacology analysis, experimental validation and molecular docking studies. We identified 60 bioactive components with 431 potential targets and 1,131 MI-related targets through network pharmacology analysis. Then, 126 core targets were screened by PPI network topology analysis. The KEGG enrichment results and the experimental validation results suggested that RC achieves the protective effect against MI via the PI3K/Akt signaling pathway. The molecular mechanisms by which RC inhibits MI were associated with a high binding affinity between THP and AKT. In light of these findings, understanding the function and mechanism of RC can provide novel insight for therapeutic strategies to treat MI."}
{"text": "A full cell consists of the cobalt sulfide@reduced graphene oxide nanocomposite anode and a commercial LiCoO2 cathode, and is able to hold a high capacity of 574.7 mA h g\u22121 at 200 mA g\u22121 after 200 cycles. The reduced graphene oxide plays a key role in enhancing the electrical conductivity of the electrode materials; and it effectively prevents the cobalt sulfide nanoparticles from dropping off the electrode and buffers the volume variation during the discharge\u2013charge process. The cobalt sulfide@reduced graphene oxide nanocomposites present great potential to be a promising anode material for lithium-ion batteries.Cobalt sulfide@reduced graphene oxide composites were prepared through a simple solvothermal method. The cobalt sulfide@reduced graphene oxide composites are composed of cobalt sulfide nanoparticles uniformly attached on both sides of reduced graphene oxide. Some favorable electrochemical performances in specific capacity, cycling performance, and rate capability are achieved using the porous nanocomposites as an anode for lithium-ion batteries. In a half-cell, it exhibits a high specific capacity of 1253.9 mA h g Cobalt sulfide@reduced graphene oxide nanocomposites obtained through a dipping and hydrothermal process, exhibit ascendant lithium-ion storage properties. The present commercial anode, graphite with a low theoretical capacity of 372 mA h g\u22121, greatly hinders the application fields of LIBs.4\u20136 Thus, the search for an anode with a high capacity, high rate capability and good cycling stability becomes urgent for the extensive applications of LIBs.Nowadays, lithium-ion batteries (LIBs), as convenient and effective devices for the storage and conversion of energy, have extended their practical application in alternative energy sources, and hybrid electric vehicles, as well as other electrical equipment.x) such as FeSx,7 NiSx,8,9 MnSx,10 MoS2,11,12 SnS2,13 CuS,14,15 ZnS16 have been fabricated and served as anode materials for LIBs due to their high theoretical capacities, low cost and abundant raw materials. Cobalt sulfide (CoSx) has been considered as an alternative anode material because of its high theoretical specific capacity of 590 mA h g\u22121 and superior electrochemical performance.17 Unfortunately, the pure CoS anode suffers from the low rate capability and a poor cycling stability, which were caused by the low electric conductivity, huge-structural change, and easy pulverization of the CoS anode material.17\u201320 Therefore, the tremendous efforts have been paid to improving the conductivity of anode materials and the diffusion of lithium ions/electrons.Recently, various transition metal sulfides  anchored on reduced graphene oxide (rGO) via combination of hydrothermal with sulfidation process. The as prepared CoS NFs\u2013rGO electrodes delivered the discharge a capacity of 939 mA h g\u22121 after the 100th cycle at 100 mA g\u22121 with coulombic efficiency above 98%.30 Zhu et al. prepared CoSx/rGO nanocomposite via a facile hydrothermal method with CoSx NPs uniformly embedded in rGO. When utilized in LIBs, the composite demonstrated a high discharge capacity of 796 mA h g\u22121 after 50 cycles at 100 mA g\u22121.31 Lu et al. reported Cox1\u2212S hollow spheres formed by in situ growth on reduced graphene oxide layers. When evaluated as an anode material for LIBs, it delivers a specific capacity of 969.8 mA h g\u22121 with a high coulombic efficiency of 96.49% after 90 cycles at 50 mA g\u22121.32 Compared with the pure CoS anodes, improvements in the specific capacity, cycling life and rate performance were achieved. Nevertheless, most graphene-based nanocomposites were obtained through a simple hydrothermal method, mixing the reactant with graphene oxide in a Teflon-lined autoclave. The resulting active materials obtained by this method cannot fully anchor on the surface of the rGO and often fall off easily, leading to a poor electrochemical property.25,27,32,33 Furthermore, the loading amount of CoS on rGO would also greatly affect the Li-storage performances of the nanocomposites. To seek for the desirable electrochemical behaviors for LIBs, it is important to develop a CoS@rGO nanocomposite with various CoS loadings, in which all CoS could anchor on the surface of rGO densely and tightly.To address the above problems, many attentions have been paid to the design of the CoS-based composites with nanostructures. The nanostructured CoS composites with an improved structural stability and electronic conductivity could be fabricated by incorporation of the CoS nanoparticles (NPs) with various carbonaceous materials such as amorphous carbon,via an electrostatic reaction to form CoS NPs uniformly distributing on the surfaces of the rGO. Employed as an anode, the CoS@rGO nanocomposites show outstanding electrochemical behaviors for LIBs.In this study, CoS@rGO nanocomposites with a 3D nanostructure were synthesized through a simple hydrothermal method. The composites consist of numerous CoS NPs and the rGO. The functional groups on the surfaces of rGO capture the Co ions All chemical reagents in this study were at the analytical level. The preparation of graphene oxide and 3D rGO is described in the ESI.\u20203CSNH2 and various amount of CoCl2\u00b76H2O  were dissolved in 14 mL of DMF/water mixture solvent (1\u2009:\u20091 v/v). The mixed precursor solution was stirred to form a transparent solution at room temperature using a magnetic stirrer. Then, a columnar rGO was soaked in the solution for 2 days. After that, the solution system was transferred into a Teflon-lined stainless steel autoclave and maintained at 200 \u00b0C for 24 h. When it cooled down to room temperature, the product was taken out and rinsed for several times with deionized water and absolute ethanol, respectively. At last, the product was vacuum-dried at 60 \u00b0C for 10 h. The amounts of CoCl2\u00b76H2O for the preparation of CoS@rGO-1, CoS@rGO-2 and CoS@rGO-3 were 0.4, 0.5 and 0.6 g, respectively.Synthesis of CoS@rGO 0.8 g of CHThe as-prepared samples were characterized using X-ray diffraction , transmission electron microscopy , field emission scanning electron microscopy  with energy dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS) (ESCALAB 250), Brunauer\u2013Emmett\u2013Teller (BET) nitrogen adsorption\u2013desorption (Nova 2000E), and Raman spectroscopy . A thermogravimetric analysis (Setaram Labsys Evo SDT Q600) was conducted to determine the rGO content of the composites.6 mixed with ethylene carbonate (EC) and diethyl carbonate (DEC), with a volume ratio of 1\u2009:\u20091. The mass loading of the electrode and the active mass are ca. 1.08 and 0.76 mg, respectively. The assembly process of the cells was carried out in a glove box ) filled with Ar gas, with oxygen and water concentrations below 0.1 ppm and 1 ppm, respectively. For a full-cell, a commercial LiCoO2 was used as the counter/reference electrode using a polypropylene separator (Celgard 2400). The commercial LiCoO2 mixed with a binder polyvinylidene fluoride (PVDF) and carbon black (mass ratio of 8\u2009:\u20091\u2009:\u20091) in N-methyl-2-pyrrolidone (NMP) solvent to form a slurry. The uniform slurry was then cast onto the surface of an aluminum foil and dried at 80 \u00b0C for 12 h. The mass loading of the electrode and the active mass are ca. 10.01 and 8.01 mg, respectively. The ratio of cathode and anode active materials is ca. 10.54. For a half-cell, a Neware battery tester  with a voltage ranging from 0.01\u20133.0 V was used to monitor the discharge/charge behavior. For the full-cell, the voltage range was 1.5\u20133.9 V. An electrochemical workstation  was used to record the cyclic voltammetry (CV) performance at a scanning rate of 0.1 mV s\u22121. The data of the electrochemical impedance spectrum (EIS) was obtained from 0.01 Hz to 100 kHz.For a half-cell, the electrochemical tests of the as-prepared CoS@rGO nanocomposites were conducted in a CR2032 coin-kind cell using a polypropylene separator (Celgard 2400), and the lithium plate acted as a counter/reference electrode. The active materials mixed with a sodium carboxymethyl cellulose binder, styrene butadiene rubber, and carbon black (mass ratio of 7\u2009:\u20091.5\u2009:\u20090.5\u2009:\u20091) in water to form a slurry. The uniform slurry was then cast onto the surface of a copper foil and dried at 80 \u00b0C for 12 h. The electrolyte consisted of 1 M LiPF34,35 No other obvious diffraction peaks are found in The crystal structure of the CoS@rGO samples were determined by XRD as shown in The FE-SEM images of the samples  are presented in More detailed information on the morphology and structure of CoS@rGO-1 could be obtained using TEM and high-resolution TEM (HRTEM). The TEM images of the CoS@rGO-1 composite are presented in ca. 1347 and 1588 cm\u22121, which can be respectively ascribed to the D and G bands of rGO. Furthermore, 2D and D + G peaks not shown here should locate at approximately 2694 and 2935 cm\u22121, respectively.36 The formation of the D and G bands results from the disorder and defects in the graphene layers, and sp2-bonded carbon atoms in the hexagonal lattice, respectively. For carbon-related materials, the intensity ratio (ID/IG) of the D and G bands represents the defects and degree of graphitization.37ID/IG of the CoS@rGO-1, CoS@rGO-2, CoS@rGO-3, and GO are 1.12, 1.16, 1.22, and 0.917, respectively. This clearly indicates that most of the oxygen-containing functional groups on graphene oxide have been reduced in the CoS@rGO composites.36 Furthermore, the increase of the intensity ratio (ID/IG) may also be due to due to the widely separated rGO layers promoted by the intercalation of CoS NPs.38Raman spectroscopy was applied to investigate the structure of carbon materials in details. And Raman spectra of the CoS@rGO and GO samples are displayed in 2+.39 The peaks at 778.5 and 793.7 eV are the Co 2p3/2 and 2p1/2 of cobalt sulfides.40 The binding energy at 780.6 and 796.3 eV are consistent with Co 2p3/2 and 2p1/2 with the splitting value over 15 eV demonstrating the coexistence of Co3+ and Co2+.41,42 The higher state of Co3+ would provide extra capacity for LIBs.43 The S 2p spectrum as shown in 32,44\u201347 In addition,  PBM data was replaced with SVG by xgml2pxml:<glyph-data id=\"z.dbd\" format=\"PBM\" resolution=\"300\" x-size=\"8\" y-size=\"10\" xml:space=\"preserve\">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform=\"translate scale\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>C bonds. Also, two weak peaks at 288.5 eV and 286.3 eV are detected, coming from the O\u2013CO and C\u2013O/C\u2013S bonds, respectively.48 Obviously, the intensity of the C\u2013C/CC bonds are much stronger than that of O\u2013CO and C\u2013O/C\u2013S bonds. This result proves that the overwhelming majority of GO were reduced and transformed into the rGO via the hydrothermal process. The presence of C\u2013S bond further confirms the formation of tight bonding between the C atoms and S atoms, which demonstrates that the CoS particles tightly attached on the surface of the rGO.25XPS analysis was used to determine the valence state and elemental composition in the CoS@rGO nanocomposites. From the survey spectrum shown in 49 The third weight loss from 410 to 650 \u00b0C is mainly due to the decomposition by oxidation of CoS into Co3O4.50 As we know that the TGA data cannot be so clearly separated to rGO and CoS formation. The carbon contents within the CoS@rGO-1, CoS@rGO-2 and CoS@rGO-3 samples are approximately 4.5 \u00b1 0.3 wt%, 3.6 \u00b1 0.2 wt% and 3.1 \u00b1 0.2 wt%, respectively.The content of rGO in the CoS@rGO nanocomposite was calculated based on the TGA results, which were performed at the temperature range of room temperature to 650 \u00b0C in air, as shown in Fig. S4 (see ESI for detail).2 g\u22121 for the CoS@rGO-1, CoS@rGO-2 and CoS@rGO-3 samples, respectively, in terms of a Brunauer\u2013Emmett\u2013Teller (BET) model. Obviously, the specific surface area of the samples slightly decreases as the load of the CoS NPs increases. The insets of the figures show the distributions of the pore sizes ranging from 1 to 22 nm. The pore sizes for the CoS@rGO-1, CoS@rGO-2 and CoS@rGO-3 respectively average 2.4, 2.1 and 1.9 nm. The as prepared CoS@rGO nanocomposites with high specific surface area and sandwich structure would present some exceptional performances in the application of LIBs.For the investigation of the porosity texture of the CoS@rGO nanocomposites, the nitrogen adsorption\u2013desorption isotherms and pore size distributions (inset) were performed in Fig. S5.xLi+ + xe\u2212 \u2192 LixCoS.19,33,51 Another slight peak at \u223c1.20 V corresponds to the reduction reaction: (2 \u2212 x)Li+ + LixCoS + (2 \u2212 x)e\u2212 \u2192 Co + Li2S.19,33,51 In the next two cycles, two peaks positively shift to \u223c1.70 V and \u223c1.29 V, respectively. The weak broad peak at ca. 0.41 V is assigned to the formation of the solid electrolyte interface (SEI) film which disappear in the following two cycles. The prominent peak at \u223c0.11 V is attributed to the insertion of lithium into the rGO nanosheets and the lattice of carbon: yC + xLi+ + xe\u2212 \u2192 LixCy.52,53 In the anodic process, two apparent peaks at \u223c2.08 and \u223c2.40 V can be identified as the reverse reaction to form LixCoS and CoS, respectively. The CV curves are overlapped well in the second and third cycles indicating the good electrochemical reversibility of the electrode.The electrochemical properties of the electrodes were studied using cyclic voltammograms (CV) tests. 33 The obvious plateau at \u223c0.12 V is ascribed to the lithium ion insertion of carbon.) In the charge process, two plateaus at \u223c2.0 and \u223c2.4 V are consistent with the anodic peaks in CV curves. In the first discharge and charge curves, the CoS@rGO-2 composite delivers specific capacities of 2262.0 and 1521.9 mA h g\u22121, respectively. And the coulombic efficiency is calculated as 67.3%, which mainly due to the SEI layer formed on the surface of the electrode. Besides, in the first lithiation process, some lithium ions were consumed because of the defects in the nanocomposite.54 In the second and third cycles, the specific discharge capacities of the CoS@rGO-2 composites declined to 1613.4 and 1589.2 mA h g\u22121, respectively. The coulombic efficiency of the second cycle increased to 97.8%, and it maintained ca. 97.9% in the third cycle, proving the good electrochemical cycling reversibility.The discharge and charge profiles the CoS@rGO-2 nanocomposites are shown in \u22121 at the current densities of 0.1, 0.2, 0.5, and 1.0 A g\u22121, respectively. The CoS@rGO-2 electrode delivered a high capacity of 668.5 mA h g\u22121 even at 2.0 A g\u22121. For the CoS@rGO-1, CoS@rGO-2 and CoS@rGO-3 electrodes, the specific capacities could respectively reach to 1305.8, 1462.6 and 1335.1 mA h g\u22121, when the current density returns to 0.1 A g\u22121. Furthermore, the CoS@rGO nanocomposite electrodes present a much better rate capacity than those of cobalt sulfide-based composites such as CoS NFs\u2013rGO,30 CoSx/rGO nanocomposite31 and Cox1\u2212S hollow spheres/rGO.32The rate performance of the CoS@rGO-1, CoS@rGO-2, CoS@rGO-3 were presented in \u22121. Observed from \u22121 with the coulombic efficiency of 99.2%, 98.9% and 99.9%, respectively. The theoretical capacity of CoS@rGO-2 can be calculated to be \u223c596.6 mA h g\u22121 (CoS contribution (capacityCoS = 590 \u00d7 96.4%) + rGO contribution (capacityrGO = 774 \u00d7 3.6%)). The capacity of the CoS@rGO-2 electrode is much higher than the theoretical value, which may be attributed to an excellent synergistic effect between CoS NPs and rGO.48 In comparison with the CoS-based electrodes recent reported, the CoS@rGO nanocomposite electrodes exhibit superior electrochemical performance than those of CoS NPs,18 lantern-like CoS,55 CoS/CNTs hybrid,22 CoS nanosheets/rGO foams,25 cobalt sulfides/rGO composite,27,28 CoS NFs\u2013rGO,30 CoSx/rGO nanocomposite,31 Cox1\u2212S hollow spheres/rGO,32 CoS2/rGO composites,38 and other CoS/rGO composites,33,56,57 which have been listed in Table S1 (ESI).\u22121) among three nanocomposite electrodes after 100 cycles. And the suitable loading of active materials (CoS NPs) in the nanocomposites could account for the highest discharge capacity of the CoS@rGO-2 electrode. Since the theory capacity of CoS is higher than that of the rGO, the capacity of the CoS@rGO increases with the increase of the amount of the CoS within the composites. As a result, the specific capacity of the CoS@rGO-2 is higher than that of CoS@rGO-1. However, when there are too many CoS NPs anchored on the rGO, they could easily detached from rGO during the charge\u2013discharge process, resulting a poor capacity.58 The detachment of the CoS NPs from the rGO can be observed from the SEM and TEM images (Fig. S7\u22121. Therefore, the specific capacity of the CoS@rGO-3 is lower than that of CoS@rGO-2.The cycling performance of the CoS@rGO composites was investigated when a high current density was set as 0.5 A gs Fig. S7 of the CRs. The resistance and constant phase element for the SEI film can be respectively expressed by Rf and CPE1. And for electrode/electrolyte interface, the charge-transfer resistance and constant phase element can be represented by Rct and CPE2, respectively. In addition, the Warburg impedance produced in the process of the lithium-diffusion can be referred as Wo.59 The Rct of the CoS@rGO-1, CoS@rGO-2 and CoS@rGO-3 electrodes before cycling test are 80.7, 56.6 and 61.3 \u03a9, respectively, which can be revealed by the fitting results. After 100 cycles, Rct of these electrodes respectively decreases to 53.0, 20.1 and 40.5 \u03a9. This may be due to the obvious pulverization of CoS NPs on the surface of rGO during the cycling process. The pulverization of CoS NPs can be observed from the TEM image (\u22121. Among these CoS@rGO nanocomposite electrodes before cycling test and after 100 cycles, the Rct of CoS@rGO-2 electrode is the smallest, for CoS NPs anchored on the rGO in the composites contribute to the small Rct value. Moreover, the Rct of the CoS@rGO-3 electrode is higher than that of the CoS@rGO-2 electrode, which may be due to the slight detachment of active material CoS from the surface of rGO. The slight detachment of the CoS NPs marked with an arrow can be observed from the SEM image (\u22121. The straight line in the low-frequency region is associated with Warburg behaviour where the lithium-ion diffusion occurs in the nanostructure. This clearly indicates that the unique porous structure is quite advantageous for improving the speed of charge transfer at the interface, and reducing the overall resistance of the battery, thus leading to excellent electrochemical performances.The Nyquist plots of the CoS@rGO nanocomposite electrodes before cycling test and after 100 cycles were presented in EM image  of the CEM image  of the C\u22121 for 10 or 100 cycles. Fig. S83S4, cubic Co9S8, orthorhombic CoSO4, and rhombohedral carbon in the final electrode material. This is consistent with the result of the EDS analysis after cycling. Moreover, Fig. S6\u22121. This clearly shows the presence of Co, S and C in the composites after 100 cycles. A small part of S has been oxidized to some higher valence states, and after the cycles, the composite consists of S2\u2212, S22\u2212, SO32\u2212, and SO42\u2212.The morphology and composition of the CoS@rGO nanocomposites were investigated by SEM, TEM, EDS, and XRD after the composite electrode was tested at 0.5 A g2 and the CoS@rGO-2 nanocomposites which respectively serve as a cathode and an anode. The capacity of the LiCoO2 was 1.2 times than that of the anode in design. When a current density and potential range was respectively set as 0.2 A g\u22121 and 1.5\u20133.9 V, the charge/discharge profiles were presented in \u22121, and the coulombic efficiency is \u223c50.2%. The formation of SEI layer on the surfaces of the electrodes may account for the major loss in capacity. In the second and third cycles, the coulombic efficiency was up to 97.1% and 97.5%, respectively. This suggests that there is efficient electron transport as well as smooth insertion and extraction of Li+. Moreover, the cycling performance of the full battery was exhibited in \u22121 at 0.2 A g\u22121 when the battery cycled 200 times, which indicates outstanding cycling stability for the full battery.A full cell consists of commercial LiCoO\u22121 at 500 mA g\u22121 after 100 cycles. For a full-cell, the CoS@rGO-2 nanocomposite anode exhibited a capacity of 574.7 mA h g\u22121 at 200 mA g\u22121 after 200 cycles. In addition, in contrast to other CoS-based composite anodes reported, the as prepared nanocomposite anodes present the superior rate performance. And the CoS@rGO-2 nanocomposite delivers specific capacities of 892.1 and 668.5 mA h g\u22121, at respective 1000 mA g\u22121 and 2000 mA g\u22121. The unique nanostructure of the electrode materials accounts for the superior electrochemical behaviors. Furthermore, the rGO embedded in the nanocomposites can effectively buffer the volume variation, mitigate the aggregation of CoS particles, and prevent the CoS particles from dropping off the electrode during the cycling process. These notable electrochemical characteristics indicate promise of the nanocomposites as anode materials in LIBs.The CoS@rGO nanocomposites were synthesized through a facile solvothermal method. Some favorable electrochemical performances in specific capacity, cycling performance and rate capability are achieved when the nanocomposites were used as anode materials for LIBs. For a half-cell, the CoS@rGO-2 nanocomposite anode exhibited a capacity of 1253.9 mA h gThere are no conflicts to declare.RA-010-D0RA01351J-s001"}
{"text": "However, during the insertion/de-insertion process, Mn3O4 suffers from particle aggregation, poor conductivity, and low-rate capability, which, in turn, limits its practical application. To overcome these obstacles, we have successfully prepared Mn3O4 nanoparticles distributed on the nitrogen (N)-doped and nitrogen, sulphur -doped reduced graphene oxide (rGO) aerogels, respectively. The highly crystalline Mn3O4 nanoparticles, with an average size of 15\u201320 nm, are homogeneously dispersed on both sides of the N-rGO and N,S-rGO aerogels. The results indicate that the N-rGO and N,S-rGO aerogels could provide an efficient ion transport channel for electrolyte ion stability in the Mn3O4 electrode. The Mn3O4/N- and Mn3O4/N,S-doped rGO aerogels exhibit outstanding electrochemical performances, with a reversible specific capacity of 374 and 281 mAh g\u22121, respectively, after 100 cycles, with Coulombic efficiency of almost 99%. The interconnected structure of heteroatom-doped rGO with Mn3O4 nanoparticles is believed to facilitate fast ion diffusion and electron transfer by lowering the energy barrier, which favours the complete utilisation of the active material and improvement of the structure\u2019s stability.Owing to their high theoretical capacity, transition-metal oxides have received a considerable amount of attention as potential anode materials in sodium-ion (Na-ion) batteries. Among them, Mn SHE) for Na-ion, most examined electrodes, especially the anode, are not ideal hosts for Na-ion insertion .,53.3O4, aerogels . Both Mn3O4 agglomerated in a size of 0.5\u20131.1 \u00b5m, which is an aggregate of individual Mn3O4 nanoparticles, anchored uniformly on the porous N-rGO . A typical crumpled structure of rGO and interconnected and cross-linked random rGO layers construct a 3D framework with open-pore structures ). The HRTEM images  plane of the tetragonal phase of the Mn3O4 nanoparticles. These results suggested that the Mn3O4 nanoparticles have a strong connection and network with the heteroatom-doped rGO aerogels, which is in accordance with the SEM images.Further characterisation of the morphology and structure of the Mnructures a,b. The M images e,f show \u22121, respectively, were observed in the heteroatom-doped rGO aerogels. The G band reflected the radial C-C stretching of ordered sp2 -linked carbon atoms, whereas the D band indicated the defects or irregularities on the graphene edges [ID/IG) for the N-rGO aerogel was 0.95, whereas it was 0.98 for the N,S-rGO aerogel. After the nitrogen and sulphur doping, defects were introduced to the rGO aerogel layers [ID/IG ratios of the Mn3O4/N-rGO and Mn3O4/N,S-rGO aerogels increased to 0.98 and 1.00, respectively. Two peaks at 658 and 369 cm\u22121 were observed in the Mn3O4/N-rGO and Mn3O4/N,S rGO aerogels, respectively [2+ ions and thus demonstrated that Mn3O4 is successfully attached to the rGO layer [\u22121 were associated with the second-order two-phonon mode 2D band. It is worth noting that the Raman spectrum associated with Mn3O4 in the Mn3O4/N-rGO and Mn3O4/N,S-rGO aerogels was shifted to a low wavenumber in comparison with the pristine Mn3O4, indicating the electronic coupling between Mn3O4 and heteroatom rGO aerogel.The disordered degree of the heteroatom-doped rGO aerogels was characterised via Raman spectroscopy and is presented in ne edges ,59. Furtl layers ,61, wherectively ,63. ThesGO layer ,65. Addi3O4/N-rGO and Mn3O4/N,S-rGO aerogels to further support the presence of heteroatom in the rGO aerogel, as well as the Mn3O4 nanoparticles in the nanocomposites. The peak positioned at 1730, 1363, and 1215 cm\u22121 corresponded to the stretching vibration C=O of carboxylic groups, O-H deformation, and C-O stretching vibration from epoxy groups, respectively, indicating that GO was successfully converted into rGO [\u22121 associated with the absorption band of C=S and S-H stretching vibration, respectively, was observed in the N,S-rGO and Mn3O4/N,S-rGO aerogels and, thus, confirmed the presence of S atom on the surface of N,S-rGO aerogel samples [\u22121 could be assigned to the characteristic band of nitrogen doping. For the Mn3O4 nanoparticles, the strong peaks at 528 and 621 cm\u22121 were attributed to the Mn-O stretching of the tetrahedral and octahedral sites in Mn3O4 [3O4 nanoparticles were successfully integrated into the heteroatom rGO layers.into rGO ,67. The  samples . In addi3O4/N,S-rGO and Mn3O4/N-rGO aerogels, respectively. From the survey scan XPS spectra, the presence of nitrogen  for the Mn3O4/N-rGO aerogel. Thus, it further confirmed the presence of N and S-atoms in the nanocomposites [3/2, S p1/2, and SOx, respectively, indicating the existence of Mn-S [3O4/N-rGO and Mn3O4/N,S-rGO aerogels is presented in 3/2 and Mn 2p1/2, respectively, indicating the existence of the Mn species, implying the possible presence of Mn3O4 in the nanocomposites [3/2 and Mn 2p1/2 were 12.2 and 11.4 eV for the Mn3O4/N,S-rGO and Mn3O4/N-rGO aerogels, respectively, and were in accordance with other earlier reports [3O4 nanoparticles have formed in the Mn3O4/N-rGO and Mn3O4/N,S-rGO aerogels.The XPS technique was used to obtain further insight into the chemical states of elements on the surface of the samples. nitrogen a and nitnitrogen b is notiFor O 1s c,d, the For O 1s e,f couldmposites . Figure mposites . As can  of Mn-S ,71. The mposites . The spl reports , which f3O4 to MnO as well as the formation of Na2O, which is attributed to the decomposition of the electrolyte according to Equation (1)To evaluate the sodium storage performances of the samples, the CV and galvanostatic charge/discharge testing have been conducted in a half-cell within the potential range of 0.01\u20133.00 V. As can be seen from The peaks at 0.45 to 0.01 V could be attributed to the reduction of MnO to metallic Mn (Equation (2)) and solid electrolyte interphase (SEI) layer formation on the electrode surface of the electroactive material.3O4/heteroatom rGO aerogel was reversible during the insertion/de-insertion process of Na+ ions [+ extraction into the graphitic carbon layers, which was the inverse process of Na+ intercalation. When compared with Li-ion batteries, the CV peaks in Na-ion batteries were broader and weaker. This may be due to the larger radius of Na+ than Li+ and the slower Na+ intercalation between graphitic carbon layers [3O4/N-rGO aerogel .During subsequent cycles, the CV curves nearly overlapped, indicating that the MnNa+ ions . The tinn layers ,76. Cont aerogel b, the Mn aerogel c exhibit3O4 and the Mn3O4/N-rGO and Mn3O4/N,S-rGO aerogels electrodes for selected cycles at a current density of 0.1 A g\u22121. The subsequent CV curves are different from the first sodiation, and the discharge plateau for the Mn3O4/N-rGO and Mn3O4/N,S-rGO aerogels is much longer than the pristine one, indicating that more Na-ions can be inserted into these nanocomposites [mposites . Further3O4, Mn3O4/N-rGO aerogel and Mn3O4/N,S-rGO aerogel were measured to be 522, 1950, and 884 mAh g\u22121, respectively. In the second cycle, the discharge capacities were 336, 470, and 425 mAh g\u22121 for Mn3O4, Mn3O4/N-rGO aerogel and Mn3O4/N,S-rGO aerogel, respectively. The irreversible capacity loss was mainly due to the irreversible formation of the SEI layer. Interestingly, the Mn3O4/N-rGO aerogel maintained its discharge capacity at 374 mAh g\u22121 after 100 cycles with an 80% retention rate. Contrarily, the Mn3O4/N,S-rGO aerogel and Mn3O4 exhibited much lower discharge capacities of 281 mAh g\u22121 (68% retention rate) and 185 mAh g\u22121 (55% retention rate) after 100 cycles. For the few initial cycles, the porous structure of rGO aerogel promoted the formation of excessive SEI layers, resulting in a lower initial Coulombic efficiency [3O4/N,S-rGO aerogel electrodes demonstrated much lower discharge capacity, presumably because of the large atomic radius of S, as well as the large crystallite size than that of Mn3O4/N-rGO aerogel electrodes.The cycling stability of all electrodes is presented in ficiency . Overall3O4/N-rGO and Mn3O4/N,S-rGO aerogel electrodes exhibited remarkable rate performance, as presented in 3O4/N-rGO aerogel electrode exhibited discharge capacities of 203, 166, 145, 135, 128, and 156 mAh g\u22121 at various current densities of 0.2, 0.4, 0.6, 0.8, 1.0, and returning to 0.2 A g\u22121. The minimal drop in capacities with increased current densities indicated a higher degree of reversible Na-ion insertion/de-insertion owing to their expanded interlayer spacing [3O4/heteroatom-doped rGO aerogels may be due, in part, to the 3D porous structure, which may minimise the electron and ion transport path.In addition to their high discharge capacity and good cycling stability, the Mn spacing . The imp3O4/N-rGO aerogel and Mn3O4/N,S-rGO aerogel were improved, which benefitted from the synergistic effect of the heteroatom doping and porous structure of the rGO, as well as the small particle sizes of Mn3O4. The rGO sheets as well as the porous structure in the conductive network of the Mn3O4/heteroatom rGO aerogels provided an efficient electron and ion transport path, thereby decreasing the internal resistance to enhance the reaction kinetics and resulting in a high specific capacity and rate capability [3O4 nanoparticles anchored on rGO can prevent the restacking of the rGO layers, preserve their high active surface area and maintain the channels for Na-ion diffusion, which is advantageous for increasing the Na storage within the nanocomposites [3O4/heteroatom-doped rGO aerogels. This strategy can be used as one of the approaches for mitigating the large volume change and poor electrical conductivity, which is associated with bare transition-metal oxide anodes.The specific capacity and cyclability of the Mnpability ,82,83. Ppability . Meanwhimposites . Doping mposites . The incmposites ,87,88, t3O4/heteroatom-doped rGO aerogels have been successfully synthesised via a hydrothermal route, followed by a freeze-drying process using NH3 and L-cysteine as nitrogen and nitrogen\u2013sulphur sources, respectively. The aerogel structure built well-interconnected heteroatom-doped rGO layers, and the Mn3O4 nanoparticles distributed on the rGO layers prevented the graphene layers from restacking again. The 3D structure provides a large active surface area and eases electron diffusion and Na-ion transportation. Both the N- and N,S-doped rGO aerogels with Mn3O4 exhibited high specific capacity, excellent cycling stability, and rate capability than the pristine Mn3O4. The heteroatom-doped rGO aerogel acts as a robust structure to accommodate the volume expansion of Mn3O4 nanoparticles and enables reversible Na-ion insertion/de-insertion. Our work demonstrates that N- and N,S-doped rGO aerogels can efficiently improve the Na storage capacity of Mn3O4 and offer a useful strategy for synthesising high-yield anode materials. Considering the simple step of the preparation process and the excellent cycling stability of the samples, the Mn3O4/heteroatom-doped rGO aerogel can be considered a potential candidate and provide an opportunity to explore these materials for the next generation of Na-ion batteries.In summary, the Mn"}
{"text": "Sodium carboxymethyl cellulose (CMC) is firstly applied as the binder for ZnS based anodes and shows a more advantageous binding effect than PVDF. To simplify the synthesis procedure, l-cysteine is added as the sulfur source for ZnS and simultaneously as the reducing agent for rGO. The average diameter of ZnS nanoparticles is measured to be 13.4 nm, and they uniformly disperse on the rGO sheets without any obvious aggregation. As anode materials, the CMC bound ZnS\u2013rGO nanocomposites can maintain a high discharge capacity of 705 mA h g\u22121 at a current density of 500 mA g\u22121 for 150 cycles. The significantly improved electrochemical performance mainly derives from the combined effects of the small and uniformly dispersed ZnS nanoparticles, the high conductivity and structural flexibility of rGO and the strong binding ability of CMC.ZnS nanoparticles are  in situ grown on reduced graphene oxides (rGO) via a simplified one-step hydrothermal method.ZnS nanoparticles are  Among the vastly investigated anode materials, ZnS is a promising candidate as it possesses a high theoretical capacity of 962 mA h g\u22121.3 The naturally abundant zinc and sulfur are cheap and environmental friendly, which makes ZnS anodes even more advantageous. But the practical application of ZnS based anode materials is still hindered by their low rate capability and severe capacity decay which are caused by their low conductivity and huge volume expansion during cycling. Thus it is imperative to find effective methods to solve these problems.The ever growing demand for green and high-efficiency energy storage devices has greatly promoted the development of the lithium-ion batteries (LIBs) industry. However, the power and energy density of current commercial LIBs still cannot satisfy the high requirements of next generation batteries which are expected to be applied in electric vehicles.4\u20136 And among the various carbon based materials, reduced graphene oxide (rGO) is an ideal performance-enhancing additive because of its high electrical conductivity, large surface area and excellent structural flexibility.7\u20139 Actually, the advantages of the above two strategies can be combined through the fabrication of composite materials which contain rGO nanosheets and uniformly dispersed ZnS nanoparticles. But synthesizing such ZnS\u2013rGO nanocomposites with desired particle size and morphology is not an easy task because zero-dimensional nanoparticles often suffer from severe aggregation problems. Aggregated particles will block the penetration of electrolyte, decrease the ion transport efficiency and lower the utilization of active materials.10\u201312 Moreover, rGO is usually obtained by the reduction of graphene oxide (GO), which means the need for extra reducing agent (like toxic hydrazine) or additional thermal annealing process.13,14 And how to guarantee the firm adhesion of ZnS nanoparticles on the rGO sheets is another problem that should be considered. Physically adsorbed particles can be easily peeled off from rGO, which will result in the loss of cell capacity. Considering these issues, it is obvious that green and simplified method is still urgently needed to fabricate stable ZnS\u2013rGO nanocomposites while ensuring the uniform dispersion and firm adhesion of ZnS particles.For ZnS anode materials, reducing the particle size to the nanometer scale and compositing ZnS with carbon materials are two adoptable strategies to improve their long-term cycling stability and rate performance. Nanosized materials, especially nanoparticles and nanodots, are quite advantageous as their shortened ion and electron transport path will greatly facilitate the lithium storage process at high current rate.15,16 So PVDF also seems not to be a good choice for ZnS since alloying and conversion reactions are both existed in its charge\u2013discharge process. Compared with PVDF, sodium carboxymethyl cellulose (CMC) is probably more suitable for ZnS as it can offer a homogeneous 3D networking around the active materials, endowing the electrode with stronger ability to accommodate mechanical stress.17 The successful application of CMC in Si and metal oxides anodes further demonstrates its advantage.18\u201320 However, to the best of our knowledge, whether CMC binder has a positive effect on ZnS anodes has not yet been reported. Figuring this out would be meaningful as it may provide an easy and scalable way to improve the cycling performance of ZnS.In addition to optimizing ZnS anodes from the structural and materials preparation aspects, choosing suitable binder for ZnS is also of great importance. Traditional PVDF binder has been proved to be inappropriate for anode materials based on alloying or conversion mechanism because it is difficult for PVDF to withstand the huge volume expansion that is involved.in situ grow ZnS nanoparticles on rGO and firstly use CMC as the binder for ZnS based anodes. In the synthesis procedure, l-cysteine is added as both the sulfur source for ZnS and simultaneously as the reducing agent for rGO. No extra annealing treatment or reagent is needed to complete the reduction process. The introduced rGO has proved itself to be a desirable matrix as it can improve the dispersity of ZnS and enhance the conductivity and structural stability of the as-prepared composite materials. The binding effect of CMC has been compared with traditional PVDF binder. With the synergistic effects of the flexible rGO nanosheets, firmly attached ZnS nanoparticles and the advantageous CMC binder, the ZnS\u2013rGO nanocomposites exhibit a much enhanced electrochemical performance.Based on the above considerations, herein we develop a simplified one-step hydrothermal route to 3COO)2\u00b72H2O, l-cysteine were used as received without further purification. GO was prepared by the oxidation of natural flake graphite powder by a modified Hummers method.21 Typically, 3 mmol Zn(CH3COO)2\u00b72H2O was dissolved into 50 ml deionized water under magnetic stirring for 10 minutes. Then 10 ml of 2.0 mg ml\u22121 GO aqueous suspension was added and treated by ultrasonication for 30 minutes. Subsequently, 4.5 mmol l-cysteine was dissolved into the above solution. After being vigorously stirred for 20 minutes, the resulting mixture was transferred into a Teflon-lined stainless steel autoclave and heated to 180 \u00b0C for 12 h. The product was collected by centrifugation and washed thoroughly by deionized water and absolute ethanol. After being dried in air at 60 \u00b0C for 12 h, the ZnS\u2013rGO nanocomposites (denoted as ZSG) were obtained. As control samples, pure ZnS (denoted as ZS) and rGO were also prepared using the same method without the addition of GO suspension or Zn(CH3COO)2\u00b72H2O.Analytical-grade Zn with Cu K\u03b1 radiation (\u03bb = 1.5406 \u00c5). ESCALAB 250 spectrometer with Al K\u03b1 X-ray source was used to collect the X-ray photoelectron spectroscopy (XPS) data. The morphology of the as-prepared samples is observed by field emission scanning electron microscopy . Thermogravimetric analysis  was carried out under air flow (100 ml min\u22121) at a heating rate of 10 \u00b0C min\u22121. Raman spectra measurements were recorded on a Renishaw InVia Raman microscope with an excitation wavelength of 633 nm. TEM and HRTEM images were obtained by a FEI-TECNAI G2 F20/America microscope.The crystallographic structure and phase purity of as-prepared samples were characterized by X-ray diffraction , ethyl methyl carbonate (EMC) and dimethyl carbonate (DMC) were mixed in a volume ratio of 1\u2009:\u20091\u2009:\u20091, and 1 M LiPF6 was dissolved in the mixture, which was used as the electrolyte. A LAND CT2001A battery instrument was applied to perform galvanostatic charge\u2013discharge tests at various current densities in 0.01\u20133.0 V. The cyclic voltammetry (CV) tests were conducted in 0.01\u20133.0 V at a scan rate of 0.1 mV s\u22121 on a CHI650D electrochemical workstation. Electrochemical impedance spectra (EIS) measurements were also recorded on the CHI650D electrochemical workstation with an AC signal amplitude of 5 mV from 0.01 Hz to 100 kHz.The working electrode is prepared by mixing active materials, acetylene black and polyvinylidene fluoride (PVDF) or sodium carboxymethyl cellulose (CMC) to form a homogenous slurry. The weight ratio of the active materials, conducting agent and binder is 7\u2009:\u20092\u2009:\u20091. The slurry was uniformly pasted on a copper foil and then dried at 70 \u00b0C for 12 h in a vacuum oven. The electrode materials prepared with PVDF is denoted as ZS\u2013PVDF, ZSG\u2013PVDF and rGO\u2013PVDF. Similarly, electrode using CMC as the binder is denoted as ZS\u2013CMC, ZSG\u2013CMC and rGO\u2013CMC. CR2025-type half-coin cells were assembled in an argon filled glove box with Hl-cysteine, Raman spectroscopy measurements were carried out on ZSG and pristine GO powder. The Raman spectra displays disorder-induced D band (\u223c1350 cm\u22121) and tangential G band (\u223c1590 cm\u22121) in ID/IG value of ZnS\u2013rGO is 1.14, and the value for GO is 1.03. The increased ID/IG value indicates the successful reduction of GO as it can decrease the average size of the sp2 domains.22,23 And among all the reagents, only l-cysteine possesses the ability to reduce GO.24 Thus it is obvious that l-cysteine, with its strong reducibility and excessive amount, has successfully reduced GO to rGO.The crystal structure and phase purity of the as-prepared products were characterized by XRD . All the1/2 and Zn 2p3/2 peaks are found at 1044.5 eV and 1021.4 eV in the high-resolution spectra of Zn 2p in 25 For the S 2p spectra in 1/2 and S 2p3/2 spin orbit peaks of ZnS. For ZSG, the successful reduction of GO can also be proved by the change of C 1 s XPS spectra. Four peaks are detected in the C 1 s spectra of unreduced GO in  PBM data was replaced with SVG by xgml2pxml:<glyph-data id=\"z.dbd\" format=\"PBM\" resolution=\"300\" x-size=\"8\" y-size=\"10\" xml:space=\"preserve\">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform=\"translate scale\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>C bond. The binding energy of the CC bond is slightly deviated from the usually reported value (\u223c284.6 eV).26 This might be caused by the modification of the electronic structure of GO by some electronic acceptor molecules. As reported by Mullen, the electronic donor and acceptor molecules within the functional groups on graphene can modify its electronic structure, which can lead to the upshift/downshift of the carbon peaks in the XPS spectra.27 Moreover, the defective carbon structures on GO also possess the ability to reduce the binding energy of CC bond and cause the downshift of the XPS peak.28 Considering the complex electronic and defective structures of GO, further investigation is still needed to figure out the detailed mechanism. The other three peaks at 285.8, 286.3 and 287.5 eV are typical C\u2013O, CO and O\u2013CO bonds in GO.29 In contrast, the peak intensity of these oxygen-containing bonds decreases dramatically in the C 1 s spectra of ZSG  and differential scanning calorimetry (DSC) were conducted in air to evaluate the rGO content in the composites (X-ray photoelectron spectroscopy (XPS) analysis was carried out to further verify the reduction of GO and investigate the oxidation state of the component elements in ZSG. As shown in a of ZSG , indicatmposites  and S1\u2020.2 adsorption\u2013desorption measurement. Type IV isotherm with a distinct hysteresis loop in relative pressure of 0.45 < P/P0 < 1.00 is observed in 2 g\u22121, respectively, proving that the introduction of rGO has increased the surface area of the composites. Large surface area is beneficial as it can increase the contact area between the electrode and electrolyte and provide more active sites for electrochemical reactions.31 It is apparent that the ZSG sample, with the in situ formed ZnS nanoparticles and the wrinkled rGO nanosheets, possesses a more advantageous nanostructure than the ZS sample.32The TEM images in \u22121 in 0.01\u20133 V. During the first cathodic scan, the broad and weak reduction peak below 0.8 V is related with the reduction of ZnS to metallic zinc and the formation of Li2S.33 Besides, the Li\u2013Zn alloying process and formation of SEI film also take place in this voltage range.34 During the first anodic scan, three small peaks are observed in 0.01\u20130.7 V, which can be assigned to the multi-step dealloying process of Li\u2013Zn alloys. The oxidation peak at \u223c1.4 V corresponds to the back conversion of metallic zinc to ZnS. The corresponding reversible reactions are summarized as follows:To investigate the electrochemical performance and compare the binding effect of CMC and PVDF, ZS and ZSG electrode materials are prepared with both the two sorts of binders, and they are denoted as ZS\u2013PVDF, ZSG\u2013PVDF, ZS\u2013CMC and ZSG\u2013CMC, respectively. As shown in + into the nanocomposites. Such phenomenon is also reported in other metal oxides with small particle size.35\u201337 The irreversible formation of SEI film has led to the change in the curve shape of the second cathodic scan. The peaks corresponding to the reduction of ZnS has right shifted to voltage above 0.8 V. The CV curves coincide well from the second cycle onwards, indicating the good reversibility of ZSG. As for the CV peaks of rGO, the insertion of Li+ should appear around 0.5 V and the extraction of Li+ from rGO happens in a wide voltage range from 0.05 to 3 V.13 However, the overlap of the reaction voltage with ZnS and the low content of rGO in the nanocomposites has made the peaks corresponding to rGO invisible in \u22121 in 0.01\u20133 V. The slope ranging from 0.01\u20130.7 V in the first discharge curve and the plateau around 1.4 V in the first charge curve are consistent with the above CV results. During the first cycle, the ZnS\u2013rGO nanocomposites deliver an initial discharge and charge capacity of 1184 mA h g\u22121 and 625 mA h g\u22121, exhibiting a relatively low coulombic efficiency of 55.1%. The large capacity loss is mainly due to the formation of the solid electrolyte interphase (SEI) layer.38 In the 2nd, 10th and 30th cycle, the coulombic efficiency has increased to 80.9%, 96.2% and 98.7%, demonstrating the high reversibility of the electrode materials.Interestingly, in the first scanning cycle, one tiny cathodic peak at \u223c1.6 V and two anodic peaks at \u223c1.8 V and \u223c2.3 V are observed and they gradually disappear in subsequent cycles. This can be ascribed to the insertion and extraction of a small amount of Li\u22121 is displayed in \u22121 and 1334 mA h g\u22121. Then the capacity of ZS\u2013PVDF gradually decreases and drops to 337 mA h g\u22121 in the 35th cycle while the ZSG\u2013PVDF sample can maintain a stable capacity of 496 mA h g\u22121. This suggests that the incorporation of rGO with ZnS has effectively helped to buffer the volume expansion, making the electrode more stable during charge\u2013discharge cycles. When CMC is used as the binder, the cycling performance of ZSG is further improved. It can deliver an initial discharge capacity of 1184 mA h g\u22121 and maintain at 645 mA h g\u22121 after 35 cycles, retaining 54.5% of its initial capacity, which is much higher than that of ZSG\u2013PVDF (37.2%). The improved capacity retention may mainly derive from the stronger binding ability of CMC than PVDF.18 During cycling, the carboxylic groups in CMC can form hydrogen binding with ZnS nanoparticles, alleviating pulverization and therefore increase the utilization rate of the active materials. Though the content of rGO in the nanocomposites is low and its contribution to the capacity of the electrode is limited, investigating the performance of pure rGO anodes is necessary and would be conducive to the understanding of the electrochemical properties of ZSG. The cycling performance of rGO\u2013PVDF and rGO\u2013CMC is shown in Fig. S2a.\u22121 and maintained at 574 mA h g\u22121 after 35 cycles at 100 mA g\u22121, while the rGO\u2013PVDF can also retain a stable capacity of 443 mA h g\u22121.The cycling performance of ZS\u2013PVDF, ZSG\u2013PVDF, ZS\u2013CMC and ZSG\u2013CMC at 100 mA g\u22121 to 2 A g\u22121. ZS\u2013PVDF can only retain 52 mA h g\u22121 and ZSG\u2013PVDF can hold a capacity of 174 mA h g\u22121 at 2 A g\u22121. The improved rate performance of ZSG\u2013PVDF mainly derives from the good conductivity of rGO and the uniformly dispersed nanosized ZnS particles. On the one hand, the problem of low conductivity of ZnS will be ameliorated after the introduction of rGO. On the other hand, the small particle size and uniform dispersion of ZnS will shorten the diffusion length of ions and electrons, offer larger surface area for reactions and facilitate the penetration of electrolyte. Interestingly, ZS\u2013CMC shows a much inferior capacity than ZSG\u2013PVDF at low current density, but the capacity gap narrows with the increase of current and even becomes comparable at 2 A g\u22121. The low-density PVDF binder possess a porous structure. Such structure can guarantee a good immersion of active materials in the electrolyte. That is why the PVDF bound ZSG shows comparable electrochemical performance at low current density. However, according to Winter et al., the porous nature of PVDF will result in a low electronic conductivity, thus weakening the electrode performance under high current rate.39 In comparison, the relatively compact CMC binder is not faced with this problem. Its performance under high rates is only limited by the Li diffusion coefficient within the electrode materials. Moreover, there are also reports showing that CMC could modify the Li diffusion coefficient and accelerate the ion transport, thus leading to a more advantageous rate capability.40 ZSG\u2013CMC still displays the highest rate performance with capacities of 379 mA h g\u22121 at 1 A g\u22121 and 325 mA h g\u22121 at 2 A g\u22121. Moreover, its capacity can recover to 640 mA h g\u22121 after the current is changed back to 100 mA g\u22121. For pure rGO  which reflects the diffusion of lithium ions in electrode appears as the slope long line in the low frequency region. It can be clearly seen that the diameter of the curve of ZSG\u2013CMC is smaller than the other three samples, indicating that the nanocomposites possess a lower charge transfer resistance and higher conductivity. This is further verified by the fitting results. The inset of Rct value for ZS\u2013PVDF, ZSG\u2013PVDF, ZS\u2013CMC and ZSG\u2013CMC are 388.7 \u03a9, 59.9 \u03a9, 61.7 \u03a9 and 47.1 \u03a9, respectively. Lower charge transfer resistance means a higher transport efficiency of Li+ ions through the interface of the electrode and electrolyte, which is conducive to the enhancement of rate capability of the electrode materials.47To gain more information about the nanocomposites, electrochemical impedance spectroscopy (EIS) measurements were performed after the cells were tested at 500 mA gThe excellent electrochemical performance of ZSG\u2013CMC can be ascribed to the following reasons. Firstly, the high structural flexibility and conductivity of rGO can help to alleviate the huge volume expansion and increase ion transport efficiency of the composite materials. Secondly, the small particle size and uniform dispersion of ZnS can offer larger surface area for reactions and facilitate the penetration of electrolyte, guaranteeing a high utilization of active materials. Thirdly, the conversion reaction mechanism and small size of ZnS can trigger a capacity increase phenomenon through the formation of gel-like films and pseudo-capacitive behavior. Finally, the CMC binder can provide a strong binding effect to alleviate pulverization and to some extent modify the Li diffusion coefficient to further improve the rate capability. These advantages have combined to enhance the overall cycling stability and rate capability of the ZSG\u2013CMC electrode.via a simplified one-step hydrothermal route and CMC is firstly used as the binder for the ZnS based anode. With an average diameter of 13.4 nm, ZnS nanoparticles are uniformly anchored on rGO nanosheets. l-cysteine plays an indispensible role during the whole fabrication process as it not only serves as the sulfur source for ZnS but also acts as the reducing agent for rGO. The incorporation of rGO and ZnS has successfully alleviated the severe aggregation of ZnS nanoparticles. Besides, rGO has also endowed the composites with higher electronic conductivity and structural flexibility. The CMC binder can provide a strong binding effect to further stabilize the electrode, thus guaranteeing a higher capacity retention. Owing to these beneficial features, the CMC binded ZnS\u2013rGO nanocomposites can retain a high discharge capacity of 705 mA h g\u22121 at 500 mA g\u22121 for 150 cycles. The excellent electrochemical performance fully demonstrates that the ZnS\u2013rGO nanocomposites are quite promising as high-performance anode materials for next generation Li-ion batteries. And the idea of adding l-cysteine as the sulfur source and reducing agent to simplify the synthesis procedure can be extended to fabricate other metal sulfides\u2013rGO composites.In summary, ZnS\u2013rGO nanocomposites are synthesized There are no conflicts to declare.RA-008-C8RA00470F-s001"}
{"text": "Nfkb, Trem2 , GFAP (astrocyte marker), Il1b, and S100a8 in DSS-treated mice. While the expression of Nfkb, Trem2, and GFAP showed a peak on day 10, the S100a8 expression was high on days 10 and 17 and subsided on day 42. Interestingly, expression of Bdnf remained elevated in the times assessed . Together, these results demonstrated that DSS-induced colitis could induce prolonged neuroinflammation and impaired contextual fear memory.Accumulating evidence has shown that intestinal inflammations in inflammatory bowel disease (IBD) also drive pathological responses in organs outside the intestine, including the brain. Previous studies using the dextran sodium sulfate (DSS)-induced colitis model have shown that colonic inflammation contributes to the development of anxiety- and depression-related behaviors; however, little is known about whether memory function is affected. Here, we subjected male and female C57BL/6J mice to DSS-induced colitis for 6 days, followed by Pavlovian conditioned fear (CF) tests 15 days after the start of inflammation, when local colonic inflammation has receded. The contextual and cued CF tests were used to assess associative fear memory. We found that DSS-induced colitis led to significant impairment in contextual fear memory in both male and female mice; on the other hand, auditory cued fear memories were comparable between control and DSS-treated mice. There were marked signs of astrogliosis in the hippocampal regions 17 days (D17) after colitis induction. Furthermore, molecular characterization of hippocampi showed marked but transient increases in the expression of inflammatory genes The online version contains supplementary material available at 10.1186/s13041-022-00961-4. The prevalence of inflammatory bowel disease (IBD), a chronic inflammatory condition of the gastrointestinal tract, continues to rise . IBDs, iTo model IBD in rodents, dextran sulfate sodium (DSS)-induced colitis has been widely used, which elicits intestinal pathologies similar to human ulcerative colitis . PreviouIn the present study, we subjected male and female C57BL/6J mice to DSS-induced colitis for 6 days, followed by Pavlovian conditioned fear (CF) tests 15 days after the start of colitis induction  . Some male mice showed cued fear response below the 5% threshold and were excluded from data analysis, while all female mice responded. Overall, freezing behaviors for auditory cues were comparable between control and DSS-treated mice  and recovered gradually after cessation of DSS Fig.\u00a0b. FemaleNfkb, Trem2 , Gfap, and Il1b were significantly increased on day 10 and decreased thereafter to basal levels by day 42, except for Il1b  peaked on day 17 and subsided to basal level on day 42 (Fig.\u00a0Bdnf remained elevated compared to control (Fig.\u00a0It has been shown that contextual information is encoded by neurons in the hippocampus and conveyed directly to the amygdala, which generates conditioned fear responses , 11. Nex colitis . InteresIn conclusion, our study showed that contextual memory function was negatively impacted by an episode of colitis, with prolonged neuroinflammation in the hippocampal regions. Further work is required to determine mechanistically the interactions between innate neuroinflammatory response and neurons encoding specific inputs to hippocampal-amygdala neurocircuit to affect contextual fear memory . Of noteAdditional file 1. Experimental Methods."}
{"text": "Crocodilians are an order of ancient reptiles that thrive in pathogen-rich environments. The ability to inhabit these harsh environments is indicative of a resilient innate immune system. Defensins, a family of cysteine-rich cationic host defence peptides, are a major component of the innate immune systems of all plant and animal species, however crocodilian defensins are poorly characterised. We now show that the saltwater crocodile defensin CpoBD13 harbors potent antifungal activity that is mediated by a pH-dependent membrane-targeting action. CpoBD13 binds the phospholipid phosphatidic acid (PA) to form a large helical oligomeric complex, with specific histidine residues mediating PA binding. The utilisation of histidine residues for PA engagement allows CpoBD13 to exhibit differential activity at a range of environmental pH values, where CpoBD13 is optimally active in an acidic environment. Defensins are a class of host defence peptides that contribute to the immune system of eukaryotes. Here, the authors report the structure of saltwater crocodile defensin CpoBD13 and the mechanism of pH-dependent antifungal activity. During this time, these animals have evolved to thrive in microbe-laden environments. Yet despite regularly receiving wounds and even losing limbs in territorial disputes and interspecies conflicts, the development of infection is rare2. This unique ability to ward off pathogens before they can invade systemically is indicative of a formidable innate immune system1. A major constituent of an organism\u2019s innate immune system are defensins; a family of cysteine-rich cationic host defence peptides (CHDPs) that harbor a broad range of activities, including direct microbicidal properties and inflammatory response signalling4. Defensins are grouped into two major superfamilies based on their intramolecular disulphide bonds; cis-defensins  and trans-defensins (expressed by vertebrates and invertebrates)4. Within vertebrates, the trans-defensins are further subdivided into three sub-families termed the \u03b1-, \u03b2- and \u03b8-defensins, with their distinguishing feature being the cysteine-pairing order of their disulphide bonds. While \u03b1- and \u03b8-defensins are unique to mammals and old-world primates respectively, \u03b2-defensins are the most abundant and expressed by all vertebrates4.Crocodilians are an order of ancient reptiles that have existed in their modern-day forms for approximately 83 million years5, fungi6, viruses7 as well as tumourigenic cells8. This broad range of activities is primarily attributed to the destabilisation of the target cell plasma membrane, leading to death by lysis9. This mechanism of action was first characterised in plant defensins that were found to directly target and bind negatively charged phospholipids. Target phospholipids are typically members of the phosphatidylinositol phosphate (PIP) family and interact electrostatically with cationic residues of the defensin 11. Engagement of a target lipid by plant defensins triggers the formation of oligomeric defensin\u2013lipid complexes which place torsional stress on the membrane until the point of lysis. Recently, this ability to target PIPs has been shown to be conserved in \u03b2-defensins, specifically human \u03b2-defensin 2 (HBD-2), which was able to kill the human pathogenic fungus Candida albicans through the binding of phosphatidylinositol 4,5-bisphosphate P2)6. While plant and human defensins have been studied in detail11, the defensins of other phyla, including reptiles, are poorly characterised.\u03b2-defensins effectively inhibit and/or kill a wide range of pathogens including bacteriaCrocodylus porosus \u03b2-defensin 13 (CpoBD13). Our findings reveal that despite limited primary sequence identity, the three-dimensional structure of CpoBD13 bears a striking resemblance to HBD-2, suggesting strong evolutionary conservation of the \u03b2-defensin structure between humans and reptiles. Furthermore, we have also determined the structure of CpoBD13 bound to phosphatidic acid (PA), which identifies a continuous helical oligomer and reveals in atomic detail how trans-defensins are able to form large oligomeric membrane attack complexes. Importantly, we show that CpoBD13 permeabilises fungal cell membranes in a pH-dependent manner as a result of histidine-mediated binding of PA, revealing a mechanism to regulate defensin antimicrobial potency.We now report the functional and structural characterisation of C. porosus open reading frames acquired from the NCBI nucleotide database for potential defensin genes. CpoBD13, the homologue of avian \u03b2-defensin-13 and Alligator sinensis \u03b2-defensin-13, was selected for further characterisation based on its unusual amino acid sequence that featured a high proportion of histidine residues  to pH +8 (at pH 5.5), we surmised that changes in the net charge of CpoBD13 may impact its functionality. To assess if changes in the environmental pH modify the membrane-permeabilising activity of CpoBD13, we repeated the PI uptake assays in buffered media , the net charge of CpoBD13 is significantly altered by the pH of its surrounding environment Fig.\u00a0. As the dia Fig.\u00a0. At pH 5C. albicans with fluorescently labelled CpoBD13 and observed the process by confocal microscopy -tagged CpoBD13 in lipid overlay assays on PIP Strips Fig.\u00a0 bound oncis-defensins and adopt large intricate protein:phospholipid oligomeric complexes. In contrast, whilst trans-defensins (such as those found in humans) have been shown to bind phospholipids, the atomic detail of putative large oligomeric defensin:phospholipid complexes remain to be determined11. Consequently, we subjected CpoBD13 to chemical crosslinking analysis in the presence and absence of PA 5, clear dimers are observed at 0 and 23\u2009\u00b5M PA concentrations. At 115\u2009\u00b5M PA, multiple higher order bands are clearly observable, indicative of multimeric complexes up to the size of pentamers. To visualise the morphology of these higher order complexes, samples of CpoBD13, PA only and CpoBD13:PA were examined by transmission electron microscopy (TEM)  Fig.\u00a0. CpoBD13To understand the structural basis of CpoBD13 activity, we determined its atomic structure using X-ray crystallography. A single copy of CpoBD13 was found in the asymmetric unit, with clear and continuous density observed for the entire protein chain. CpoBD13 adopts a fold comprising the family-defining structural elements observed in mammalian \u03b2-defensins comprising a single \u03b1-helix and a three-stranded \u03b2-sheet, which are connected via three disulphide bonds. A Dali analysis of CpoBD13 revealed that human \u03b2-defensin-2 (HBD-2) is the closest structural homologue in the Protein\u00a0Data\u00a0Bank\u00a0(PDB), with a rmsd of 0.8\u2009\u00c5 (Z-score 6.2) over 40\u2009C\u03b1 atoms, and a peptide sequence identity of 26% .An overlay of the two defensin structures Fig.\u00a0 revealedIn contrast, the type I site Fig.\u00a0 featuresTo further understand the role of PA interactions for CpoBD13 activity, we performed structure-guided mutagenesis on key residues involved in the proposed type I (R9 and H21) and type II (R17 and H35) PA-binding sites and performed fungal growth inhibition assays using our panel of CpoBD13 mutants Fig.\u00a0. We also50 of ~6\u2009\u00b5M) and CpoBD13 (H21A) (IC50 could not be determined) both showed significant reductions in fungal growth inhibition activity. This pattern in activity was reflected in PI uptake assays with CpoBD13 (H21A) being the least effective at permeabilising C. albicans cell membranes followed by CpoBD13 (R17A)  compared to the wild type and only minor changes in activities of CpoBD13 (R9A) and CpoBD13 (H33A) Fig.\u00a0, whereas7A) Fig.\u00a0. In cont7A) Fig.\u00a0. Results. CpoBD13\u00a0(H21A) was the least effective at binding PA at pH 5.5, whereas the remaining histidine mutants showed no significant differences to the wild type.We then examined PA binding of our panel of mutants in liposome pulldown assays Fig.\u00a0. Both arFig.\u00a0. CpoBD13To establish if the differences in PA binding are reflected in the lytic activity of the mutants, we again treated PA-enriched liposomes and monitored calcein-release Fig.\u00a0. The resAs single amino acid substitutions of the PA-binding residues elicited mild changes in the antifungal activity of CpoBD13 (excluding H21A), we generated a range of compound mutations to further investigate the importance of PA interactions on the defensin\u2019s ability to kill fungal cells. In total, five compound mutant defensins were recombinantly expressed to investigate two aspects of the defensin\u2019s activities Fig.\u00a0, the pH C. albicans  had greater membranolytic activity and was able to permeabilise ~75% of the fungal cells at 50\u2009\u00b5M. Furthermore, when the pH was adjusted to pH 7.5, neither mutant showed a significant difference in activity when compared to pH 5.5, indicating that the antifungal activities of the compound histidine mutants were not regulated by the surrounding pH.As single amino acid substitutions had little effect on the pH sensitivity of CpoBD13, we assessed the ability of the double histidine substitution mutants CpoBD13 (H21A/H35A) and CpoBD13 (H21R/H35R) to permeabilise fungal cell membranes at pH 5.5 and 7.5 Fig.\u00a0. At pH 510. While reptilian defensin genes have been identified from the genomes of species such as the Komodo dragon12, Chinese turtle13 and recently members of the crocodilian family15, the same depth of structural and mechanistic insight has not been obtained in comparison to their human and plant counterparts.The structure\u2013function relationships of human and plant defensins have been well documented. These CHDPs target and destabilise the plasma membranes of a broad range of microbial organisms and, in certain cases, tumourigenic cells by interacting with negatively charged phospholipidsIn this study, we characterised the antifungal activity of the saltwater crocodile \u03b2-defensin CpoBD13 and discovered that the ability to bind anionic phospholipids, specifically phosphatidic acid, has been evolutionarily conserved by reptilians. By solving the first three-dimensional crystal structure of a reptilian defensin in complex with PA, we identified that the binding of PA is mediated by key histidine residues that regulate the membranolytic activity of the peptide in a manner dependent on the environmental pH. To our knowledge, this phenomenon has not been observed for any characterised defensin.C. albicans, which is mediated via accumulation at the cell membrane prior to cell lysis  in a pH-dependent manner21. For CpoBD13, this in-built pH-sensitivity could be a mechanism that allows the peptide to be stored or transported in a low-activity state within the crocodile when maintained in a neutral pH to help prevent harm to the host. However, once CpoBD13 reached an acidic pH at the site of an infection (e.g. Candida blastophores favour acidic pH in humans22), the peptide could be activated and clear target pathogens.The antifungal activity of CpoBD13 is regulated by the environmental pH and has optimal activity at acidic pH. Its ability to permeabilise fungal membranes improves as the net charge of the peptide increases. While this is the first defensin determined to be pH-sensitive based on direct histidine-mediated lipid binding, the ability for pH to modulate membranolytic activity has been previously observed23. Our crystal structure of CpoBD13 bound to PA  tag (YPYDVPDYA) was codon-optimised for expression in the methylotrophic yeast Pichia pastoris, synthesised by Bioneer Pacific , subcloned into the pPIC9 vector for recombinant protein expression in P. pastoris, and subsequently purified by cation exchange chromatography, as previously described25. CpoBD13 point mutations  were generated using the QuikChange II Site-Directed Mutagenesis Kit  as per the manufacturer\u2019s instructions. CpoBD13 compound mutants were synthesised by GenScript Biotech  directly into the pPIC9 vector.The open reading frame encoding CpoBD13 was identified from the NCBI nucleotide database as accession XM_019537183.1. The DNA sequence encoding the mature defensin domain of CpoBD13 (nucleotides 142\u2013255), as determined using the web-based software SignalPC. albicans ATCC 10231 cells  were pelleted , washed once in 0.5\u00d7 potato dextrose broth  and finally resuspended in the same media. Cells were seeded onto a 96-well plate  and treated with an equal volume of varying concentrations of defensin in 0.5\u00d7\u00a0potato dextrose broth. After incubating the samples , the plates were shaken to distribute the cells and growth was determined by performing a 9-well scan (600\u2009nm) using a Spectramax M5e microplate reader . For assays testing defensin salt tolerance, C. albicans cells were instead resuspended in 1x potato dextrose broth and treated with an equal volume of CpoBD13 + NaCl solution at their intended concentrations. Propidium iodide (PI) uptake assays were performed by preparing the cells as per the standard growth inhibition assay however they were instead seeded onto a U-bottom 96-well plate . After incubating the cells with an equal volume of defensin in 0.5\u00d7\u00a0potato dextrose broth\u00a0at a range of concentrations , PI (diluted in ice-cold PBS) was added to the samples  before analysis by flow cytometry using a BD FACSCanto II with FACSDiva 8.0.1 (BD Biosciences). For PI uptake experiments performed at a specific pH, the same method was followed with the exception that cells were resuspended in 1\u00d7 potato dextrose broth while defensin titrations were diluted in 40\u2009mM MES buffer at the intended pH. An example PI uptake gating strategy is presented in Supplementary Fig.\u00a0For fungal growth assays, overnight cultures of 2.Human epithelial cervical cancer , prostate cancer  and histiocytic lymphoma  cells were obtained from the American Type Culture Collection  and cultured in RPMI-1640 medium  supplemented with 5\u201310% (v/v) foetal bovine serum (FBS), 100\u2009U/mL penicillin and 100\u2009U/mL streptomycin . Adult human dermal fibroblasts  cells were obtained from Lonza  and cultured in Dulbecco\u2019s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS, 100\u2009U/mL penicillin and 100\u2009U/mL streptomycin. Human umbilical vein epithelial  cells were obtained from Lonza and cultured in EBM-2 medium supplemented with EGM-2 SingleQuots (Lonza). All cell lines were maintained at 37\u2009\u00b0C in a humidified incubator with 5% CO8. For propidium iodide uptake assays in serum-free medium, U937 cells  were washed once in serum-free RPMI-1640, pelleted again (as previous centrifugation) and resuspended in serum-free media. 4\u00d7104 cells were seeded and incubated with a range of CpoBD13 concentrations (diluted in serum-free medium) for 30\u2009min at 37\u00b0C. PI (diluted in ice-cold PBS) was added to a final concentration of 1\u2009\u00b5g/mL before analysis by flow cytometry using a BD FACSCanto II with FACSDiva 8.0.1 (BD Biosciences).Cell viability assays were performed using MTT/MTS as previously describedLyophilised CpoBD13 (2\u2009mg) was resuspended in 900\u2009\u00b5L of conjugation buffer  followed by the addition of 0.71\u2009mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 mg N-hydroxysuccinimide . After incubating at RT for 15\u2009min, the pH of the reaction was adjusted to between 7\u20138 by the addition of 20\u00d7 PBS. BODIPY FL EDA (Thermo Fisher Scientific) was resuspended in dimethylsulfoxide to a concentration of 20\u2009mg/mL before it was subsequently added to the reaction mixture . After incubating the reaction for 150\u2009min, salts and unbound BODIPY were removed by using a PD-10 desalting column  as per the manufacturer\u2019s instructions.2. C. albicans ATCC 10231 cells suspended in 0.5\u00d7 potato dextrose broth were immobilised onto the surface of glass chamber wells pre-coated with 0.01% poly-L-lysine  and were treated with CpoBD13-BODIPY (20\u2009\u00b5M) immediately prior to imaging. For microscopy experiments containing PI, the dye was added to the cell media to give a final concentration of 1\u2009\u00b5g/mL.Live cell imaging was performed on a Zeiss LSM 800 confocal microscope using a 63\u00d7 oil immersion objective at 30\u2009\u00b0C in 5% COPIP Strips  were initially incubated in PBS/3% BSA for 60\u2009min. The blocked strips were then incubated with CpoBD13-HA (1\u2009\u00b5g/mL) diluted in PBS with 1% BSA for 60\u2009min followed by three 10\u2009min washes with PBS/0.1% Tween-20 at RT. Bound CpoBD13-HA was probed for using rabbit anti-HA tag antibody  diluted to 1:4000 in PBS/1% BSA for 120\u2009min at RT. The PIP Strips were washed before being incubated with HRP-conjugated donkey anti-rabbit Ig  diluted to 1:5000 in PBS/1% BSA for 60\u2009min at RT. Following a final round of washing, antibody reactivity was detected by chemiluminescence using the ECL Prime Western Blotting System (Cytiva) as per the manufacturer\u2019s instructions.5, Thermo Fisher Scientific) in the aforementioned buffer solution. After a 30\u2009min incubation, samples were subjected to SDS-PAGE analysis and colloidal Coomassie staining. Crosslinking experiments with CpoBD13 mutants were performed using the same method however only a single PA concentration (0.46\u2009mM) was assessed.CpoBD13  was incubated with an equivalent volume of 0, 0.092 or 0.46\u2009mM 08:00 phosphatidic acid (PA)  in 20\u2009mM HEPES pH 7.1 for 30\u2009min before the addition of equal parts 20\u2009mM PEGylated bis(sulfosuccinimidyl) suberate (BS(PEG)6. For liposome pulldowns performed at pH 5.5, the same protocol was used with minor variations. The desiccated lipid films were resuspended in 20\u2009mM MES pH 7.4 and were washed in the same buffer during the centrifugation process. After the final wash step, the liposome pellets were resuspended in 20\u2009mM MES pH 5.5 and incubated with CpoBD13 or mutants thereof .Liposomes were prepared using natural chicken egg L-\u03b1-phophatidylcholine and L-\u03b1-phosphatidic acid dissolved in chloroform (Avanti Polar Lipids), and liposome pulldowns (at pH 7.4) and calcein-encapsulated liposome lysis assays were\u00a0performed as previously describedSamples were prepared by mixing 1\u2009mg/mL CpoBD13 with 4\u2009mM PA or PC at a 9:1 ratio . For samples of CpoBD13, PA or PC only, protein/lipid was diluted in water to give the same concentration as the CpoBD13 + lipid sample. Samples (10\u2009\u00b5L) were allowed to settle onto 400-mesh copper grids before excess liquid was removed with blotting paper. Each sample was negative stained twice with 2% uranyl acetate, allowed to air-dry and imaged using a JEOL JEM-2010 transmission electron microscope . For experiments in reducing conditions, CpoBD13 and PA were mixed in the presence of 100\u2009mM dithiothreitol (DTT) prior to negative staining.2HPO4/KH2PO4, 0.1\u2009M sodium HEPES pH 7.5 using a protein:lipid molar ratio of 1:1.2 at a concentration of 14\u2009mg/mL. Diffraction data were collected on the MX2 beamline at the Australian Synchrotron. All diffraction data were integrated using Dials26 or Xia227 version 3.2.1. and scaled using AIMLESS version 0.7.429. The structure of CpoBD13 was solved by molecular replacement with PHASER30 using the structure of HBD-2 (PDB ID 6CS9)6 as a search model. The structure of CpoBD13:PA was solved by molecular replacement with PHASER30 using the previously determined structure of CpoBD13. Final models were manually built using Coot31 and refined using PHENIX version 1.18.2_387432. All data collection and refinement statistics are summarised in Supplementary Table\u00a0Crystals of CpoBD13 were grown at 20\u00b0C in 0.1\u2009M trisodium citrate pH 5.5, 20% PEG 3000 using protein at a concentration of 20\u2009mg/mL. Crystals of CpoBD13:PA were grown at 20\u00b0C in 0.8\u2009M NaAll data are presented as the mean\u2009\u00b1\u2009SEM of three independent experiments. Where stated, statistical analyses of these experiments (including two-way ANOVA and unpaired t-tests) were performed using Prism 9.1 .Further information on research design is available in the\u00a0Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary"}
{"text": "The appropriate design of studies is essential for their quality, successful execution, and interpretation. Randomized controlled trials (RCTs) are considered the \u201cgold standard\u201d for intervention studies. However, the most recent large-scale , long-term RCTs involving vitamin D3 did not provide any statistically significant primary results. This may be because they are designed similarly to RCTs of a therapeutic drug but not of a nutritional compound and that only a limited set of parameters per individual were determined. We propose an alternative concept using the segregation of study participants into different groups of responsiveness to vitamin D3 supplementation and in parallel measuring a larger set of genome-wide parameters over multiple time points. This is in accordance with recently developed mechanistic modeling approaches that do not require a large number of study participants, as in the case of statistical modeling of the results of a RCT. Our experience is based on the vitamin D intervention trials VitDmet, VitDbol, and VitDHiD, which allowed us to distinguish the study participants into high, mid, and low vitamin D responders. In particular, investigating the vulnerable group of low vitamin D responders will provide future studies with more conclusive results both on the clinical and molecular benefits of vitamin D3 supplementation. In conclusion, our approach suggests a paradigm shift towards detailed investigations of transcriptome and epigenome-wide parameters of a limited set of individuals, who, due to a longitudinal design, can act as their own controls.Vitamin D intervention studies are designed to evaluate the impact of the micronutrient vitamin D However, over many thousands of years hunter\u2013gatherer populations adapted genetically to reduced UV-B exposure, such as a less active DHCR7 (7-dehydrocholesterol reductase) enzyme [3 [SLC24A5 (solute carrier family 24 member 5) and SLC45A2 of the solute carrier family [3-rich food like fatty fish to their diet [3 became a real vitamin. For example, in England in the 19th century, rickets was a very common disorder in children [Vitamin Dsed skin ,3, the nsed skin . Above a) enzyme . Reducednzyme [3 ,7. In par family  involvedr family . In addieir diet . Althougeir diet ,13, justchildren ,15. Thischildren .3 (25(OH)D3) [3 [3 is considered insufficient [Mycobacterium tuberculosis [The vitamin D status of an individual is defined as the serum concentration of the most stable and abundant vitamin D metabolite, 25-hydroxyvitamin D5(OH)D3) . There iH)D3) [3 . As a reH)D3) [3 . This sufficient , becausefficient , and a lfficient ,23. Whilfficient , multiplfficient  and inflfficient  as well fficient ,29 or thrculosis ,31, stilrculosis , as wellrculosis . Howeverrculosis . To eval3, 1,25-dihydroxyvitamin D3 2D3), which acts as a high-affinity ligand to the transcription factor VDR (vitamin D receptor) [VDR-expressing tissues and cell types [3 intervention studies can be considered as nutrigenomic experiments, in which the action of vitamin D3 and its endogenously produced metabolites are investigated under human in vivo conditions. This had already been demonstrated by studying vitamin D-triggered changes in chromatin accessibility [The physiological effects of vitamin D are mediated by the biologically most active form of vitamin Deceptor) . In thisll types . Thus, all types , (ii) asll types , (iii) mll types , and  are balanced. This clearly distinguishes RCTs from observational studies. However, the design of RCTs requires a larger number of participants. Finally, when the RCTs are finished, they are unblinded and mostly analyzed in comparison of the groups to which the individuals had been assigned, i.e., treatment versus placebo.RCTs aim to measure the effectiveness of interventions or treatments, such as whether vitamin D3 supplementation have been published  with more than 5000 adults over 3.3 years concerning the prevention of cardiovascular events and mortality [3 (1600 or 3200 IU) per day over 5 years for investigation of the primary outcomes of cardiovascular disease and invasive cancer [3 (4000 IU) over 2.5 years, for a possible conversion of prediabetes to type 2 diabetes (T2D) [3 dose of 50 \u00b5g (2000 IU) alone or in combination with \u03c9-3 (1 g/d) and a strength-training exercise program over 3 years improves the health conditions of elderly, such as the likelihood of falls [Within the past 6 years, the results of a few large-scale RCTs involving vitamin Dublished . The lard cancer . The ViDortality . In the e cancer . The stues (T2D) . Finallyof falls .3 in serum, i.e., hardly any study participants were vitamin D deficient. This is not representative of the world population, of which some 7% are severely vitamin D deficient and 33% have an insufficient vitamin D status [3 supplementation clearly increased the vitamin D status of the participants to sufficiency. However, primary analysis of the data showed a trend in the expected direction, but did not provide any statistically significant indication that vitamin D3 supplementation, either daily or monthly, was beneficial for the expected clinical outcome of the respective trials, i.e., the studies had null results [The average vitamin D status of a total of 38,054 individuals in these five studies was, at baseline, more than 50 nM 25(OH)DD status . Moreove results .3 supplementation did not improve their clinical status (only 12.7% of participants in VITAL and 9.1% in FIND were vitamin D deficient). In the classical RCT, the entrance criterion into a study should be low vitamin D3 status to observe a stronger effect of supplementation, but for ethical reasons, long-term RCTs with vitamin-D3-deficient individuals as a placebo group (control) cannot be performed. Additionally, for some of the individuals, daily supplementation with only 40 \u00b5g (FIND) or 50 \u00b5g (VITAL and DO-HEALTH) vitamin D3 may have been insufficient. Furthermore, the RCTs were designed for outcomes such as cancer, cardiovascular disease, and T2D that do not reflect the primary physiological role of vitamin D, which is the control of calcium homeostasis and the modulation of immunity [3 supplementation reduced the risk of autoimmune diseases by 22%, i.e., for a directly immune-related outcome a significant effect could be observed [3 supplementation on cancer mortality [3.There are several explanations for this unexpected result. The main point may be that the study participants were recruited an excessively high basal vitamin D status, i.e., many of them were already vitamin D sufficient and further vitamin Dimmunity . For exaortality , while Vwww.internationalgenome.org, accessed on 5 July 2023) have described the variability of the human genome [\u201cBig biology\u201d projects such as 1000 Genomes , CYP2R1 , CYP24A1 (rs17216707), and GC  contribute to basal serum levels of 25(OH)D3, but each of them only with a small OR [DHCR7 gene, increases the 7-dehydrocholesterol concentrations in the skin, and leads to a more efficient synthesis of vitamin D3 [3. Similarly, the frequency of other SNVs related to genes mediating vitamin D signaling, such as VDR (rs2228570 (known as FokI polymorphism), rs1544410 (Bsm1), rs731236 (Taq1)), and the VDR target genes CD14 (rs2569190) and CARD9  is increased in European populations [3 serum levels [The 20/80% contribution of genetics versus environment/epigenetics also applies to vitamin D status. GWAS indicated that SNVs in the regions of the genes small OR ,55. Moretamin D3 . Today\u2019s3 over 5 months of a Finnish winter , i.e., it follows the treatment protocol of the FIND trial. The study aimed at preventing the onset of type 2 diabetes; blood samples were collected at the beginning and end of the intervention. In contrast, in VitDbol 35 young healthy subjects were exposed only once to a vitamin D3 bolus  and samples were taken at days 0, 1, 2, and 30. Importantly, the analysis of both VitDmet and VitDbol differed from other vitamin D intervention studies by relating the changes of vitamin D-triggered parameters, such as vitamin D target gene expression in PBMCs , which had been isolated at the end and beginning of the study, to the ratio of the vitamin D status at the respective time points  ,59,60,61e points A. Accord3 supplementation. For example, the study BEST-D had a similar three-arm design as VitDmet, also focused on the elderly, and measured the expression of cytokines [3 supplementation [The method of data analysis of these smaller-scale studies is essential for obtaining significant effects of vitamin Dytokines . Howeverentation  2D3). In addition, this study included two different approaches, which were a traditional cohort analysis built on single repeats of 25 individuals and a personalized analysis based on testing a limited number of selected participants in triplicate [50-value of 1,25(OH)2D3 stimulation [The vitamin D intervention study VitDHiD (NCT03537027) followed the VitDbol approach in design, but studied 25 participants on the level of their transcriptome  Table 2Table 2. iplicate . This lemulation . In humaVKORC1 (vitamin K epoxide reductase complex subunit 1) and CYP2C9 [The molecular basis of the vitamin D response index needs to be explored further. In analogy to the anti-coagulant drug warfarin, as the interindividual difference in the response to it is determined by SNVs in the genes d CYP2C9 , the vit3 supplementation, can be used [In general, the transcriptome-wide analysis has an advantage in that, in contrast to the set of 24 vitamin D target genes, which had been selected in the context of the VitDmet study ,59,60,61 be used . This si3 production. High vitamin D responders better tolerate these conditions and should suffer less frequently from autoimmune diseases [3 supplementation doses than suggested by population-based recommendations and guidelines that may serve primarily mid vitamin D responders. However, daily vitamin D3 supplementation should not be higher than 4000 IU (100 \u00b5g) in order to prevent overdose in high vitamin D responders. The most appropriate supplementation may be 1 \u00b5g (40 IU)/kg body mass to account for obese individuals.A low vitamin D status most likely occurs in the winter season, in particular in the about 15% of the world\u2019s population living above a latitude of 38\u00b0 N ,72, i.e.diseases , infectidiseases , and/or diseases  because It is possible that due to the evolutionary adaptation of the European populations to changing environmental conditions, such as the northern migration after the end of the ice age, there are more high vitamin D responders in Northern Europe than in Southern Europe. Since populations in Nordic countries have a higher rate of ancestry from Caucasian pastoralists  and Siberian hunter\u2013gatherers than those from the South ,77, inte3 supplementation than mid or high responders, which already may be saturated with a lower vitamin D status. Accordingly, RCTs performed exclusively with accurately adjusted dosage on low vitamin D3 responders would provide more conclusive results. However, ethical considerations do not allow the use of low vitamin D responders as a control group with no or insufficient vitamin D3 supplementation. This prevents the design of classical RCTs with low vitamin D responders as well as studies with vitamin-D-deficient persons. In this respect, a longitudinal design of vitamin D intervention studies, such as in VitDmet, VitDbol, or VitDHiD, where each participant is serving as his/her own control, are more appropriate. Since in these studies each participant is investigated individually, they are also referred to as N = 1 studies  , Wikipat3, for which no clean zero controls exist, long-term RCTs with vitamin-D3-deficient individuals cannot be performed for ethical reasons and alternative study designs, such as N = 1 approaches, may be more appropriate. Finnish studies such as VitDbol and VitDHiD were designed to use a vitamin D3 bolus (3 intervention study in Saudi Arabia [3 bolus should not be used over longer periods in order to prevent hypercalcemia and tissue calcification [3 supplementation is recommended [The most recent large-scale, long-term RCTs involving vitamin D did not provide any significant primary results. This is largely because they were designed similarly to RCTs of therapeutical compounds, i.e., drugs that do not naturally occur in the human body. Thus, for endogenous molecules, such as vitamin DD3 bolus , i.e., ti Arabia ,86. Howefication . Thus, dommended .3 supplementation may serve as a trait that identifies members of the general population who have a significantly higher susceptibility to multiple types of diseases, such as autoimmune diseases, cardiovascular disorders, T2D, and cancer. In contrast, high responsiveness may reflect a high immune resilience, i.e., an appropriate response of the immune system to various health challenges [3 supplementation.A low responsiveness to vitamin Dallenges . In this"}
{"text": "Trisomy 18 syndrome (T18) is the second most common autosomal trisomy and has a high risk of fetal loss and stillbirth. Aggressive surgical treatments for the respiratory, cardiac, or digestive systems of patients with T18 were previously futile, while the results of recent studies are controversial. Over the past decade, there have been approximately 300,000 to 400,000 births annually in the Republic of Korea; however, there have been no nationwide studies on T18. This nationwide retrospective cohort study aimed to determine the prevalence of T18 in Korea and its prognosis according to the presence of congenital heart disease and relevant interventions.This study utilized NHIS-registered data between 2008 and 2017. A child was defined as having T18 if the ICD-10 revision code Q91.0\u20133 was reported. Subgroup analysis was performed for children with congenital heart diseases, and survival rates were compared based on the history of cardiac surgical or catheter interventions. The primary outcomes in this study were the survival rate during the first hospitalization period and the 1-year survival rate.Of the children born between 2008 and 2017, 193 were diagnosed with T18. Of these, 86 died, with a median survival of 127 days. The 1-year survival rate for children with T18 was 63.2%. The survival rate in the first admission of children with T18 who did and did not have congenital heart disease was 58.3% and 94.1%, respectively. Children with heart disease who underwent surgical or catheter intervention had a longer survival time than those who did not.We suggest these data could be used in ante- and postnatal counseling. Ethical concerns about the prolonged survival of children with T18 remain; however, the potential benefits of interventions for congenital heart disease in this population need further study.The online version contains supplementary material available at 10.1186/s12887-023-04056-4. Trisomy 18 syndrome (T18) is the second most common autosomal trisomy and has a high risk of fetal loss and stillbirth. The prevalence in live births is estimated as 1/6,000\u20131/8,000. However, the actual prevalence may be higher  because of the high risk of fetal death and pregnancy termination after prenatal diagnosis . Recent In contrast, some reports have indicated that the survival rate of children with T18 may increase while receiving active treatment. Data of vital statistics from Japan demonstrated that more surgical interventions provided during 1995\u20132016 increased the median survival time of children with T18 from 28 to 54 days . A multiIn 2019, the Constitutional Court in the Republic of Korea (Korea) determined the anti-abortion law unconstitutional officially. Until then, abortion due to fetal disease was illegal . The govNHIS data containing demographics, healthcare utilization, and diagnoses based on the International Classification of Diseases, 10th revision (ICD-10), were used in this study. Several studies utilizing the Korean NHIS cohorts have been conducted, and more recently, studies on the long-term outcomes of neonates using the NHIS database have been published \u201312. ThisA child was defined as having T18 if the ICD-10 codes  were reported during an inpatient admission or visit to outpatient clinics. Follow-up observations of these infants were conducted until December 31, 2018. The type of insurance  and the NHIS premium depending on income levels were employed as proxies for measures of income level. The lowest income category was designated as those receiving medical aid. In addition, the NHIS group was split into four groups: category 1, <\u200925% premium; category 2, 25\u201350% premium; category 3, 50\u201375% premium; and category 4, >\u200925% premium. We reviewed and ranked all ICD-10 codes associated with congenital malformation, deformation, and chromosomal abnormalities (Q codes). A child was defined as having a history of congenital heart disease (CHD) if diagnosed with Q20\u201326. Heart anomalies that usually regress spontaneously, such as atrial septal defect (ASD) (Q21.1) and patent ductus arteriosus (PDA) (Q25.0), were excluded from CHD codes. Among CHDs, the annual number of children who underwent surgical or catheter intervention (intervention) for CHDs was counted using health insurance claim codes (eTable 1 in Supplement 1). Additionally, the number of children with T18 who had tracheotomy or gastrostomy was counted. The mortality rate was analyzed in two categories. We examined mortality during the first hospitalization period and before 1 year after birth.The study employed Pearson\u2019s chi-square analysis to compare the mortality rates between groups according to the history of cardiac intervention. Kaplan-Meier survival analysis and log-rank tests were utilized to describe long-term survival and compare groups, respectively. In this study, we used a two-sided test with a significance level of p\u2009<\u20090.05 to determine statistical significance. All analyses were performed using SAS .The NHIS database included 193 children diagnosed with T18 who were born between 2008 and 2017. Annually, 12\u201325 children are diagnosed with T18  \u2019, which includes ASD and ventricular septal defect; \u2018congenital malformations of great arteries (Q25)\u2019, which subsume PDA; \u2018congenital deformities of feet (Q66)\u2019; and \u2018other congenital malformations of the brain (Q04)\u2019 (eTable 2 in Supplement 1). Except for PDA and ASD, the ventricular septal defect was the most common CHD, followed by coarctation of the aorta and double outflow of the right ventricle (eTable 3 in Supplement 1). Consequently, 108 (56.0%) children with T18 were diagnosed with CHDs, and 47 (43.5%) received cardiac intervention. In 2008 and 2009, few children with T18 underwent heart intervention; however, 23.1\u201377.8% of children with T18 and CHD underwent intervention from 2010. Only one or two infants underwent a tracheotomy yearly; however, approximately more infants underwent surgery in 2014 and 2017 .P\u2009=\u20090.028) (Table\u00a0P\u2009<\u20090.001)  Table\u00a0, with si01) Fig.\u00a0.This nationwide population-based study was the largest on intervention and outcomes of children with T18 in Korea. The median survival time of mortality cases was 127 days and the 1-year survival rate was 63.2%, with no evident change in the survival rate for 10 years. Among 108 children with CHD, 47 (43.5%) underwent cardiac intervention, and their survival days were longer than those who did not undergo the intervention.In this study, 193 children were diagnosed with T18 over 10 years , a lower number than in previous studies conducted in other countries. Approximately 1.5 per 10,000 live births in the US were affected by T18, and multinational research reported that the mean prevalence of T18 among live births was 1.07  per 10,000 births , 6. The The median survival time (25\u201375 percentiles) of mortality cases was 127  days in our study, which was longer than those in previous studies with similar study periods. A recent study from Japan has reported that the median survival time of children with T18 who were born between 2006 and 2016 was 54 (1\u2013196) days . In anotOur study included 108 children with T18 and CHD. In previous studies, there has been debate about the association between CHD and the survival of T18 . HoweverSevere developmental delay, long-term dependence on medical equipment, and survival have been considered in deciding on a treatment policy for T18 patients. However, several studies on the quality of life of children with T18 have further indicated that considering the family\u2019s perspective and knowledge of natural history when formulating policies for T18 treatment is crucial. Parents of patients with T18 or trisomy 13 syndrome patients reported that their child was happy and enriched their family despite their children\u2019s severe conditions . In anotThis study had some limitations. First, in this study, the observation period varied from 1 year to 11 years due to the children\u2019s different birth years, and ended in December 2018. Although we attempted to mitigate this issue by using a survival curve, there were still limitations to this approach. We acknowledge the limitations of our analysis due to the absence of information on the number of patients at risk at each time point and censoring marks. We had access to this information at the time of analysis, but unfortunately overlooked it and are now unable to retrieve the data. Thus, our presented survival curve may not provide a comprehensive view of the survival outcomes, and readers should interpret our results with caution. Second, we did not have patient information on T18 mosaicism. Patients with T18 mosaicism revealed an extremely variable phenotype; they occupy a small portion of the disease group and may not affect the study\u2019s result . In addiIn this study, fewer children with T18 were reported in the NHIS database in Korea than in other countries; however, many of them received active treatments and had longer survival rates. Recently, advance care planning has been implemented, and the laws regarding abortion have been amended in Korea. Due to these social and legal changes, more ante- and postnatal consultation on T18 treatment are expected, and patient-centered care will be more warranted. Therefore, we expect these data to help determine the disease prognosis and find the best interests for children with T18.Below is the link to the electronic supplementary material.Supplementary Material 1"}
{"text": "Studies have revealed that the language network of Broca\u2019s area and Wernicke\u2019s area is modulated by factors such as disease, gender, aging, and handedness. However, how occupational factors modulate the language network remains unclear.In this study, taking professional seafarers as an example, we explored the resting-state functional connectivity (RSFC) of the language network with seeds .The results showed seafarers had weakened RSFC of Broca\u2019s area with the left superior/middle frontal gyrus and left precentral gyrus, and enhanced RSFC of Wernicke\u2019s area with the cingulate and precuneus. Further, seafarers had a less right-lateralized RSFC with Broca\u2019s area in the left inferior frontal gyrus, while the controls showed a left-lateralized RSFC pattern in Broca\u2019s area and a right-lateralized one in Wernicke\u2019s area. Moreover, seafarers displayed stronger RSFC with the left seeds of Broca\u2019s area and Wernicke\u2019s area.These findings suggest that years of working experience significantly modulates the RSFC of language networks and their lateralization, providing rich insights into language networks and occupational neuroplasticity. The human brain controls language , movemenResting-state functional magnetic resonance imaging (rsfMRI) is widely used to map the physiology and behavior of the healthy/diseased brain . FurtherIn this paper, taking seafarers as an example, two important sub-functions of language, namely, the language network (study 1) and the lateralization of the language network (study 2), were investigated to explore the association between the occupational factor and language by using the method of resting state functional connectivity (RSFC) . The anaSince seafarers have been engaged in repetitive technical work for a long time we recruited twenty male professional seafarers  from a shipping company in Shanghai, China. All of them had more than 10 years of experience in navigation. For non-seafarers, 20 Chinese male participants , were recruited from land-based jobs  at university or secondary school campuses. All the subjects in the non-seafarer group had no maritime professional training, maritime navigational skills, or long-term experience on the sea. All subjects signed the informed consent form and were considered to have normal functions of language and communication. Also, no history of mental health conditions or neurological diseases were reported. The blood-oxygen-level-dependent imaging (BOLD) rsfMRI data for each participant was scanned at the Shanghai Key Laboratory of Magnetic Resonance. All participants were informed about the purpose of the study and signed a written consent form according to the procedures approved by the IRB of East China Normal University (ECNU). The specific parameters were listed as follows: GE 3.0 Tesla using a gradient echo EPI, a total of 36 slices covering the whole brain area, 160 time points, TR (time of repetition) = 2 s, matrix size = 64 \u00d7 64, in-plane resolution = 3.75 mm \u00d7 3.75 mm, and slice thickness = 4 mm. The detailed information related to the dataset can also be found in 1 and the Resting-State fMRI Data Analysis Toolkit (REST).2 The preprocessing steps for the Resting-State fMRI data of each subject were as follows: (1) slice timing; (2) realignment; (3) normalization by EPI template (resampling voxel size = 3 mm*3 mm*3 mm); (4) spatial smoothing using a Gaussian kernel with FWHM = 6 mm; (5) nuisance regression including covariates such as six head motion parameters, whole brain mean signal, white matter signal, and cerebrospinal fluid signal; (6) band-pass temporal filtering (0.01\u20130.1 Hz); and (7) scrubbing volumes with sudden head motion, i.e., a threshold of frame-wise displacement (FD) was set to 0.05, and we removed one volume before and two volumes after the motion spike.All data preprocessing was performed using the Data Processing Assistant for RS-fMRI software package (DPARSF)  which isThe RSFC method generates a high-precision functional connection diagram of a complex brain system by interpreting the relevant patterns of low-frequency fluctuations in the blood oxygen level signals, which can be used to identify language-related functional tissues. In this paper, the Broca and Wernicke in the left side of brain were selected as the region of interest (ROI) (lBro and lWer), with MNI coordinates  and  as the center of the seed points  with a rt-test results  is defined by following formula (2)  :zFCL is a whole brain functional connection diagram based on the left seed points , respectively, and zFCflipped R is a left-right flipped functional connection diagram based on the right seed points (rBro and rWer), respectively. Similarly, as in previous studies  and the rBro and rWer and the right hemisphere (RH). Also, contralateral asymmetry was established on the right side of the AI map, indicating the differences between the lBro or lWer and the RH and the rBro and rWer and the LH. Further, a one-sample t-test was conducted to reveal regions which show significant hemispheric asymmetry based on individual AI maps. Moreover, the two-sample t-test was applied to analyze the differences of language lateralization between seafarers and the control participants. All RSFC maps and AI maps were established with the test criteria of p 0.005 and cluster size >200 voxels (corresponding to corrected pFWE 0.05).where  studies , ipsilatt-test analysis of the linguistic functional connectivity patterns from the seafarer and non-seafarer groups revealed that: the negative functional connectivity of Broca\u2019s region appeared weaker in the seafarer group than the non-seafarer group, especially in the left superior/middle frontal gyrus and left precentral gyrus .A profile of the lateralization for each subject was obtained based on the RSFC map in terms of Broca\u2019s area and Wernicke\u2019s area. REST software was used to conduct a one-sample According to t-test analysis on the AI maps of the two core language regions for the two groups. The statistical results are shown in AI maps corresponding to the Broca\u2019s region, the seafarer group elicited greater functional asymmetry in the paracentral and precuneus (BA7 and BA31). For the AI maps corresponding to the Wernicke\u2019s area, the lateralization difference between the seafarer group and the non-seafarer group was mainly reflected in the left frontal gyrus and bilateral precuneus.In order to further quantify the differences in language lateralization between the seafarer group and the non-seafarer group, we performed a two-sample Previous studies have investigated whole-brain language networks using the RSFC method . SulpiziIn this study, we selected the special occupation group of seafarers as the research object, and compared the functional language network seafarers with non-seafarers. Regarding the seafarers, the functional connectivity related to functional language networks showed a negative connection with Broca\u2019s area, though strongly left-lateralized, including in the left SFG/MFG (BA 6) and the precentral gyrus (BA 6 and 4), which may be involved due to the extended length of time spent in a relatively closed environment and the lack of spoken interaction. AI asymmetry in seafarers and non-seafarers. We found slightly rightward lateralization in the Broca\u2019s seed of seafarers with left IFG, and almost leftward asymmetric distribution in the non-seafarers . The patients/participants provided their written informed consent to participate in this study.HW and DP: conceptualization, methodology, validation, formal analysis, and writing\u2014original draft. HY: conceptualization, methodology, validation, formal analysis, writing\u2014original draft, and funding acquisition. YY: investigation and writing\u2014review and editing. WZ: investigation, writing\u2014review and editing, and data curation. CC and NW: conceptualization, resources, writing\u2014review and editing, supervision, funding acquisition, and project administration. All authors contributed to the article and approved the submitted version."}
{"text": "Renal cell carcinoma (RCC) is the most common type of kidney cancer. The therapeutic strategies are based on surgery and/or specific therapies able to inhibit growth factors that have been shown to promote the growth and spread of tumors. Currently, there is no established biomarker which helps in early diagnosis and in better disease monitoring with a high sensitivity. Much information could be provided by body fluids, especially blood liquid biopsy (LB), that are increasingly interesting to researchers. LB is a non- or minimally invasive procedure that could allow clinicians to monitor cancer evolution, also thanks to the presence of small vesicles known as extracellular vesicles (EVs) secreted by tumor cells and containing useful information. In particular, growing interest is focused on small RNA molecules (miRNAs) that are involved in tumor growth and could represent potential diagnostic, prognostic, and predictive biomarkers in RCC, as we summarize in this review.Renal cell carcinoma (RCC) is the second most common cancer of the urinary system. The current therapeutic strategies are based on partial or total nephrectomy and/or targeted therapies based on immune checkpoint inhibitors to which patients are often refractory. Preventive and screening strategies do not exist and the few available biomarkers for RCC are characterized by a lack of sensitivity, outlining the need for novel noninvasive and sensitive biomarkers for early diagnosis and better disease monitoring. Blood liquid biopsy (LB) is a non- or minimally invasive procedure for a more representative view of tumor heterogeneity than a tissue biopsy, potentially allowing the real-time monitoring of cancer evolution. Growing interest is focused on the extracellular vesicles (EVs) secreted by either healthy or tumoral cells and recovered in a variety of biological matrices, blood included. EVs are involved in cell-to-cell crosstalk transferring their mRNAs, microRNAs (miRNAs), and protein content. In particular, transferred miRNAs may regulate tumorigenesis and proliferation also impacting resistance to apoptosis, thus representing potential useful biomarkers. Here, we present the latest efforts in the identification of circulating miRNAs in blood samples, focusing on the potential use of EV-derived miRNAs as RCC diagnostic and prognostic markers. Renal cell carcinoma (RCC) is the second most lethal malignancy in the urological system  and reprThe incidence of the disease varies widely throughout the world and appears to be highest in more developed countries. White people have a lower risk of RCC than Native, African, and Hispanic Americans, probably due to the disparities in diets, lifestyles, socioeconomic conditions, education, and economical possibility for healthcare. Risk factors connected to RCC could be distinguished as (a) unmodifiable, such as age, race, and gender, and (b) modifiable, that include diet, alcohol, occupational exposure, and drugs . The peaUnmodifiable and modifiable risk factors are often related: RCC is doubly diagnosed in men where some modifiable risks such as hypertension, tobacco smoking, and obesity are more frequently observed. RCC originates from the kidney cortex, in particular from the epithelial cells of renal tubules ,4, and iRCC is a chemo- and radio-resistant neoplasia. The current therapeutic strategies are based on the surgical approach of radical and/or partial nephrectomy that remains the only effective therapy for clinically localized RCC . TraditiThose approaches target the peculiar hypervascularity of RCC and trigger immune cell infiltration by immune checkpoint inhibitors (ICIs), with or without antiangiogenic tyrosine kinase inhibitor (TKI)-based combinations . Thus, tICIs can stimulate the host immune response against cancer through PD1 inhibitors (Nivolumab and Pembrolizumab) or anticytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors (Ipilimumab). The first group can block the interaction between programmed cell death protein 1 (PD1) and programmed death-ligand 1 (PD-L1) able to downregulate the host T cells and, therefore, the immune response . Many stHowever, the response and tolerance to these combination treatments are not always satisfying, probably due to the lack of recognized molecular targets, an intratumoral heterogeneity, and different RCC molecular subtypes. The therapeutic strategy is based on the clinical prognostic score  and Memorial Sloan Kettering Cancer Center (MSKK) criteria), drug toxicities, patients\u2019 comorbidities, and preference. In fact, nowadays the International mRCC Database Consortium risk model remains the only prospectively known predictive biomarker in mRCC .Although targeted therapies represent the standard treatment options for RCC, nearly all patients treated with targeted drugs will eventually experience disease progression. Unfortunately, a significant number of RCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilization nor clinical benefits, probably due to the intratumoral heterogeneity and molecular subtypes ,16.Therefore, the identification of new promising biomarkers in RCC is an urgent clinical need that leads clinicians and researchers to investigate the role of cell components as microRNAs (miRNAs) secreted by tumor cells and packaged into extracellular vesicles (EVs) released in biological fluids .For decades, surgical tissue biopsy has firmly been considered the gold standard for solid tumor diagnosis; however, it may become a diagnostic obstacle for its invasiveness, unrepeatability, and especially its peculiarity to immortalize that moment without taking into account the tumor heterogeneity and dynamism . The molOver the past decade, liquid biopsy (LB) has revolutionized the field of clinical oncology, providing ease in tumor sampling and continuous tumor monitoring .Thus, LB is gaining relevance in the clinical scenario of cancer for early diagnosis and treatment stratification, as well as residual disease detection and recurrence monitoring . In particular, overall survival in mRCC patients remains low despite the development of novel targeted therapeutic approaches. LBs could provide an attractive and noninvasive method to determine the risk of recurrence or metastatic dissemination during follow-up and, thus, improve the clinical outcomes and quality of life of RCC patients . SeveralThe identification of biomarkers in LB could enable the continuous and real-time collection of RCC patients\u2019 information for diagnosis, prognosis assessment, and treatment response monitoring .Many studies have attempted to use LB as a routine method for the clinical diagnosis of RCC and for the prediction of patients\u2019 grades, stages, and survival to identify patients at high risk for metastasis and recurrence ,27,28,29EVs and miRNAs are emerging as a platform with potentially broader and complementary applications in LB . EVs are small vesicles enclosed in a lipid bilayer, continuously released by all viable cells, in physiological and pathological conditions . AccordiEVs can transport tumor-derived material, including lipids, a wide variety of RNA , DNA, proteins, enzymes, and metabolites. They play a key role in cell-to-cell communication ; by tranThese small messengers are involved in tumorigenesis and tumor metastasis due to their widespread distribution throughout the blood and lymphatic circulation. Several studies indicate that tumor-released EVs may orchestrate tumor-progression-stimulating proliferation, angiogenesis, chemoresistance, and immune escape ,38,39. IRecently, EVs showed increasing potential in the field of urological malignancies . An incrThe recent knowledge is related to the biological role of EVs shed by RCC tumor cells in RCC progression, such as angiogenesis, immune escape, and tumor growth .Horie K et al. analyzed the hypoxic conditions in RCC that stimulate the release of tumor-cell-derived EVs. Their in vitro results, based on migration and tube formation assays, suggested the possibility that carbonic anhydrase 9 (CA9) enriched in exosomes and released from hypoxic RCC may enhance angiogenesis in the microenvironment, thereby contributing to cancer progression . In addiMoreover, specific protumorigenic roles have been attributed to EVs released by CD105+ renal CSCs, due to their cargo enriched in several proangiogenic mRNAs and miRNAs . Those cA specific role of renal-CSC-derived EVs has been additionally described for the formation of the premetastatic niche, which consists of a complex network of information exchanges. These EVs may sustain an unfavorable outcome for the tumor by enhancing tumor vascularization and by contributing to the establishment of a premetastatic niche . More reMicroRNAs (miRNAs) are small noncoding RNA molecules of 18\u201322 nucleotides capable of post-transcriptionally regulating protein translation, due to increased mRNA degradation ,38. For Over the last few years, the aberrant expression of miRNAs in cancer development has been dissected, identifying some miRNAs highly expressed in cancer cells with an oncogenic role and miRNAs acting as tumor suppressors. The deregulation of both groups of miRNAs contributes to tumor development mechanisms: oncogenic miRNAs (onco-miRNAs) are commonly overexpressed in cancerous tissues, and tumor suppressor miRNAs are conversely downregulated in tumors . Their sIn 2017, for the first time, Tian and colleagues compared the expression levels of miRNAs in plasma and in plasma-derived exosomes, showing no differences in healthy donors. However, in lung cancer patients, the onco-miRNAs were more enriched in exosomes than free in circulation, assuming an increased involvement of EVs in cancer for miRNA-based cell-to-cell communication .Cancer cells can actively load miRNAs into EVs for supporting tumor development and spread due to their peculiarity of protecting their cargo from the environment. The EV double layer allows the content to be preserved, thus improving miRNA\u2019s half-life and stability . FurtherIn RCC, many efforts have been made in the research of circulating miRNAs in biological fluids, such as serum, plasma, and urine. Because urine can be easily collected, urinary miRNAs are attractive as they may be promising biomarkers in diseases affecting the urinary tract. Several studies demonstrated their stability in urine, particularly when carried by EVs. However, urine samples and, consequently, urinary miRNA detection may be influenced by several physiological and pathological conditions which make their study very challenging ,55.Due to the ease of sampling, better specificity of detection, and higher stability, most studies focused on serum and plasma miRNAs as potential noninvasive diagnostic, prognostic, or predictive biomarkers in RCC patients .The expression profile of several miRNAs has revealed their valid potential as diagnostic biomarkers in human RCC, since they are differentially expressed in tumors when compared with their normal counterpart, but also between primary and metastatic tumors . They haAn increasing challenge in recent studies is to find potential biomarkers able to improve sensitivity to chemoradiotherapy, predicting patient\u2019s response and resistance to targeted therapy. Circulating miRNAs might also be involved in acquiring resistance to treatment in RCC. Their promising role as predictors of response to therapy may acquire considerable importance in RCC, providing clinicians with crucial information to determine the optimal treatment plan and, thus, avoiding severe side effects of ineffective overtreatment  Figure .We reviewed the studies on free or encapsulated miRNAs differentially expressed in the blood samples of RCC patients compared to normal controls, as summarized in Tusong et al. reported the overexpression of miR-21 and miR-106a in RCC patients\u2019 serum compared to healthy controls and their significant reduction a month after surgery compared with the preoperative group. This outcome suggests the potential diagnostic and predictive role of serum miR-21 and miR-106a as noninvasive biomarkers for RCC . MiR-21 Cheng et al. showed an upregulation of miR-21, miR-34a, and miR-224 and a downregulation of miR-141 in the sera of patients with ccRCC, reporting consistency in their expression profile with those of the corresponding tumor tissue samples .Interestingly, only the miR-21 expression levels in the serum of the patients were correlated with the patients\u2019 clinical stage, while miR-224 expression levels were correlated with gender. However, this study showed that miR-34a, miR-224, miR-21, and miR-141 could be considered as potential ccRCC tumor markers .A diagnostic role has been attributed to several miRNAs, such as: miR-625-3p, miR-508-3p, and miR-885-5p, dysregulated in the serum of ccRCC patients ,62; miR-The late diagnosis of RCC, primarily due to a lack of early-stage diagnosis, encouraged researchers to discover novel miRNA signatures that discriminate RCC patients from healthy controls with a high degree of accuracy. In particular, in 2015, Whang et al. focused on a 5-miRNA panel including miR-28-5p, miR-362, miR-572, miR-193a-3p, and miR-378, demonstrating their potential value as an ancillary clinical diagnostic tool to also detect early-stage RCC, for which surgery is most effective . Next toMore recently, the upregulation of miR-765 was described in the plasma of ccRCC patients after tumor resection, suggesting its tumor suppressor role and identifying the proteolipid protein 2 (PLP2) as a candidate downstream target gene . In the In contrast to other studies, Heinemann et al. did not only compare the serum of patients with ccRCC and healthy donors, but also a group of patients with benign renal tumors (BRTs). As a result, the downregulation of miR-122-5p and miR-206 in the serum of both ccRCC and BRT groups compared to controls suggested their doubtful role in diagnosis. However, they demonstrated their prognostic value, showing that high levels of miR-122-5p and miR-206 were associated with a significantly shorter period of progression-free, cancer-specific, and overall survival . Plasma The comparison of plasma of ccRCC patients with localized and metastatic disease before and after surgery suggested that EV content varies depending on the presence or absence of the disease. An increased level of miR-301a-3p and a decreased expression of miR-1293 may be potential biomarkers of metastatic disease . Two stuRCC is considered one of the most unfavorable tumor diseases due to the late diagnosis and the poor prognosis. Nowadays, specific biomarkers validated for RCC early detection are not available and the actual treatments are often unable to avoid recurrence of the disease. LB could provide an attractive and noninvasive tool to assist the research for biomarkers in RCC tumors to capture a larger amount of the molecular heterogeneity compared to tissue biopsy and give prompt information on the risk of recurrence/relapse during follow-up.Many efforts are directed towards the research for circulating miRNAs that play a role in almost all aspects of cancer biology and development. In particular, miRNAs packaged in EVs and released in blood flow could allow expanding the spectrum of potential biomarkers for future use in the diagnosis and prognosis of RCC and in the prediction of therapeutic response.However, nowadays, there are no miRNAs widely applied as biomarkers in the clinical setting, partly due to the lack of isolation and quantification standardized protocols, the heterogeneity of study cohorts, and the variety of body fluids under investigation. Several studies have analyzed plasma- and serum-derived miRNAs, without focusing on the vesicle compartment. EV-derived miRNAs appear to be more stable than free miRNAs as EVs seem to protect them from degradation by macrophages. The EVs\u2019 double-layered membrane and nanoscale size grant the miRNAs stability, thus prolonging their circulation half-life and enhancing their biological activity . After rNonetheless, beyond these technical limitations, the most exciting but challenging application will be to utilize EVs and their cargo as a clinical tool to diagnose and monitor disease. Thus, EV-derived miRNAs could provide a novel therapeutic tool in RCC clinical practice, where the lack of adequate biomarkers makes functional investigations urgently needed."}
{"text": "The mechanical environment of endothelial cells (ECs) in PAD is characterized by disturbed blood flow (d-flow) and stiff extracellular matrices. In PAD, the stiffness of arteries is due to decreased elastin function and increased collagen content. These flow and stiffness parameters are largely missing from current models of PAD. It has been previously proven that ECs exposed to d-flow or stiff substrates lead to proatherogenic pathways, but the effect of both, d-flow and stiffness, on EC phenotype has not been fully investigated. In this study, we sought to explore the effect of sex on proatherogenic pathways that could result from exposing endothelial cells to a d-flow and stiff environment. We utilized the scRNA-seq tool to analyze the gene expression of ECs exposed to the different mechanical conditions both in vitro and in vivo. We found that male ECs exposed to different mechanical stimuli presented higher expression of genes related to fibrosis and d-flow in vitro. We validated our findings in vivo by exposing murine carotid arteries to d-flow via partial carotid artery ligation. Since women have delayed onset of arterial stiffening and subsequent PAD, this work may provide a framework for some of the pathways in which biological sex interacts with sex-based differences in PAD.Peripheral arterial disease (PAD) is an age-related medical condition affecting mostly muscular arteries of the limb. It is the 3 PAD most commonly affects the older populations, with an incidence of 12%\u201320% in those older than 65 years . The onsecade 30\u20130\u2005s, but to males , 8. Prevto males . The endto males . Our resh factor , 13.in vitro and in vivo studies have been done to understand the molecular pathways that lead to atherosclerosis  were less impacted than male HUVECs when exposed to higher and lower laminar shear stress and increasing substrate stiffness since they presented no change in morphology and YAP1 nuclear translocation (in vitro response of human aortic ECs (HAECs) in the context of PAD conditions (stiff substrate and d-flow). Given the powerful independent effect of stiffness or flow on ECs, we hypothesized that scRNA-seq would reveal sex-based phenotypic differences under PAD biomechanical conditions that may contribute to sex-based differences in PAD.Despite the NIH mandate to include sex as a biological variable, sex-differences have not been adequately investigated in this space , 18. Thelocation . In para2.2.1.2) while HAECs grown in the inner circle mimicked disturbed flow . The cells were sheared for 96\u2005h utilizing an orbital shaker at 100\u2005rpm, changing media every two days. For comparison, HAECs cultured on tissue culture plastic (TCP) immediately prior to subculture conditions above were analyzed. ECs on TCP (2\u2005GPa) and static conditions are known to mimic many of the pathways seen under s-flow conditions, including THBS1  and two female (ages 46 and 53) donors  were expanded, cultured, and sheared in complete endothelial cell growth medium . HAECs of passages 4\u20136 were seeded onto modified 6-well plates functionalized with collagen-coated hydrogels  of different stiffnesses (25\u2005kPa and 100\u2005kPa) that mimic the EC environment in physiologic (25\u2005kPa) and PAD conditions (100\u2005kPa) , 21. The2.2.Following exposure to shear stress, HAECs were recovered utilizing TrypLE  and suspended in 1X PBS (Cat.# MT21040CV) with 0.04% BSA. After ensuring the proper cell concentration was collected, the samples were delivered to the Emory Integrated Genomics Core for single-cell processing and barcoding with the 10X Genomics Chromium device. NovaSeq S4 was utilized to prepare the libraries and sequence the samples. Then, the CellRanger software was utilized to demultiplex and align the sequenced data.2.3.scRNA-seq data was processed and analyzed using the Seurat package in R (RRID:SCR_016341). To remove low-quality cells or cell multiplets, standard quality control filters were applied . Cells c2.4.in situ control. D-flow has been shown to induce arterial stiffening similar to that seen in aged 80-week-old animal . The gene analysis for the in vivo validation was presented as the fold change normalized against 18S.Partial carotid artery ligation (PCL) surgery was performed on 9 to 15-week-old S129 and C57BL/6 wild type (WT) mice as previously published . The lefd animal . 2\u20133 mou2.5.p-value of <0.05.The validation data collected from quantitative PCR was analyzed utilizing GraphPad Prism. Each data point represents 2\u20133 LCAs or RCAs from mouse following 24\u2005h of PCL. The fold change gene expression of the two WT strains utilized here (S129 and C57BL/6) were pooled for plotting. A two-sample Wilcoxon-Mann\u2013Whitney test was done to compare between biological sex. Significance was set as a 3.3.1.XIST and RPS4Y1 rank among the highest in female and male, respectively. However, other non-sex-linked genes were identified. Specifically, in male HAECs, genes like VWF and BMP4 are differentially expressed, while in female HAECs, PIEZO2 was increased.To identify sex-specific differences in static conditions, HAECs on TCP and under static culture were pooled across donors. This condition mimics how ECs are typically expanded. After pooling all donors, eight distinct clusters were identified following unsupervised clustering and are shown using a UMAP plot . The clu3.2.CCN1, CCN2, COL8A1 and d-flow associated genes like THBS1 that present higher expression in male HAECs after mechanical stimuli.Motivated by the sex-specific differences shown in the static culture group, we next investigated the existence of sex-specific differences when HAECs were exposed to different magnitudes of shear stress and substrate stiffness. The relative gene expression across all shear stress and substrate conditions per donor was plotted in violin plots . Of inte3.3.in vitro scRNA-seq findings with two commonly used WT murine strains (S129 and C57BL/6). Here, EC-enriched RNA was collected after 24\u2005h of d-flow, induced by PCL in the LCA; the contralateral RCA serving as an in situ control . After finding distinct differences between male and female HAECs on TCP under static culture conditions, we considered the effect of mechanical stimuli on sex-specific differences. We found that male HAECs presented higher expression of genes related to fibrosis and d-flow. Lastly, we validated our scRNA-seq results with CCN2, TAGLN, and THBS1 , which is similar in donor number to many publications \u201328. All TAGLN, TAGLN2, MYL9, and TPM2 in male and female HAECs  to HUVECs seeded on substrates of different stiffnesses (10\u2005kPa or 100\u2005kPa), they discovered that female HUVECs remained mostly invariant to the different combinations of mechanical stimuli suggesting that females might be more resistant to changes in the mechanical environment of ECs (TGF-\u03b2) has been proposed to be important to atherosclerotic remodeling, but the data presented was all from male animals (THBS1 can activate TGF-\u03b2, this mechanism may be relatively more important in males compared to females. Biological sex-based differences are present in cardiovascular diseases such as pulmonary arterial hypertension, aortic valve stenosis, PAD, and more (Biological sex is underreported in ar field , 18. In t of ECs . Here, w animals . Since Tand more , 26, 36.and more , 38. Forand more . The effin vitro model utilized in this work also enables rigorous delineation of sex-based EC differences in a setting that can exclude hormonal interactions of in vivo studies as well as comparisons between humans and mice that have distinct hormonal patterns across all ages. Still, this in vitro platform could be used to incorporate exogenous hormone levels to test in vivo conditions. Further, our in vivo validation supports acute sex-based EC expression differences under PAD flow conditions.This work highlights significant sex-based EC phenotypic differences induced by various clinically relevant culture conditions that may have profound impact on our understanding of sex-based clinical differences. In particular, this study found that male HAECs on TCP and cultured under static conditions and HAECs exposed to OS conditions had relatively similar gene expression in markers of EndMT and mesenchymal cells. In contrast, female HAECs showed higher EndMT and mesenchymal cell markers in HAECs cultured on static conditions when compared to other shear and substrate stiffness conditions. The In conclusion, sex-based differences are seen in HAECs under PAD conditions. These EC phenotypes may contribute to sex-based clinical differences in PAD. The use of scRNA-seq is a powerful technique that will allow better understanding of ECs under complex biomechanical conditions. Future work may further aid in the discovery of EC heterogeneity, genetic differences of PAD patients, as well as sex-based donor differences under static culture and under clinically relevant conditions."}