H2 lymphocytes, increasing evidence indicates that group 2 innate lymphoid cells (ILC2) play an important role in the production of the Type 2 cytokines interleukin (IL)-5 and IL-13, which likely amplifies the recruitment of eosinophils from the blood to the airways. In that regard, recent asthma treatments have been focusing on blocking Type 2 cytokines, notably IL-4, IL-5, and IL-13. These treatments mainly result in decreased blood or sputum eosinophil counts as well as decreased asthma symptoms. This supports that therapies blocking eosinophil recruitment and activation are valuable tools in the management of asthma and its severity. Herein, we review the mechanisms involved in eosinophil and ILC2 recruitment to the airways, with an emphasis on eotaxins, other chemokines as well as their receptors. We also discuss the involvement of other chemoattractants, notably the bioactive lipids 5-oxo-eicosatetraenoic acid, prostaglandin D2, and 2-arachidonoyl-glycerol. Given that eosinophil biology differs between human and mice, we also highlight and discuss their responsiveness toward the different eosinophil chemoattractants.Asthma is an inflammatory disease usually characterized by increased Type 2 cytokines and by an infiltration of eosinophils to the airways. While the production of Type 2 cytokines has been associated with T Asthma is a respiratory disease characterized by inflammation and hyperresponsiveness of the airways and roughly affects 300 million people worldwide . EosinopH2 lymphocytes participate in the release of Type 2 cytokines, group 2 innate lymphoid cells (ILC2) are being increasingly recognized as a significant source of Type 2 cytokines as well -4, IL-5, and IL-13 . As such as well , 10. Ast as well –25. ThisThe extensive investigation of how eosinophils were recruited really began in the 1970s. Complement component 5a (C5a) has been known to induce guinea pig eosinophil migration since 1970 –29, and 4, and N-formylmethionyl-leucyl-phenylalanine (fMLP). Numerous reports indicate that PAF induces the migration of eosinophils , leukotriene (LT) Binophils , 35–41. igration , 42, 43.igration , 44, 45 igration , 46.The expansion of the chemokine field in the 1990s allowed the characterization of additional eosinophil chemoattractants. CCL5  was the first chemokine documented as a human eosinophil chemoattractant in 1992  and was The discovery of eotaxins was a substantial leap forward in understanding how eosinophils were selectively recruited into the tissues. CCL11 (eotaxin-1) was first discovered by Jose et al. in guinea pigs , 62. Twoin cellulo (4 was the first cysteinyl leukotriene (CysLTs) to be defined as a direct chemoattractant of human eosinophils  was identified as a potent chemoattractant of eosinophils in 1996 , 73. To  cellulo , 65, 66.inophils  but induinophils –78. It w to LTD4 .2 selectively induced the migration of eosinophils, Th2 lymphocytes cells, and basophils  was shown to induce the recruitment of eosinophils , 80, 81.asophils , and incf asthma . Howeverinophils , 84–88. igration . Finallyigration ; this efd GM-CSF , 90, 91.2 receptors, the expression of chemoattractant receptors do not change, at the mRNA level, in human eosinophils isolated from the blood of healthy subjects, mild and severe eosinophilic asthmatics, as defined in Ref. , more asthma exacerbations, and increased sputum eosinophil counts , induced sputum, blood, and bronchial biopsies –115. Morl counts , 121. Itl counts , but notl counts . CCL11 ll counts , 125. Thl counts . CCL24 el counts , but is l counts . As for l counts , 126, busubjects , 127. Fisubjects , 129.Studies on CCL3, CCL7, CCL8, CCL13, and CXCL12 in relation with asthma are limited. Among the latter, CCL13 is better associated with eosinophils and asthma. Its expression is higher in BAL, bronchial biopsies, induced sputum, and plasma samples from asthmatics than from healthy controls , 131. On1 receptor blockade usually decreases eosinophil counts, although it is not clear whether this is a direct or indirect effect  improves lung function and the quality of life of asthmatics compared to placebo (Other soluble mediators might also participate in the recruitment of eosinophils in asthma. In that regard, CysLTt effect –144. LTBn asthma –147, thethmatics . As for subjects –152, butsubjects , 154. Of placebo , 156. Fi placebo , 158, an placebo .As for PAF, 5-KETE, fMLP, and 2-AG, their association with asthma is not well documented and this requires further investigations. For example, we have no idea to which extent 2-AG and 5-KETE levels are modulated in asthma and its severity.1, and more asthma exacerbations (As underscored with the data from the allergen challenges presented in the previous section, it is not possible to pinpoint one chemoattractant explaining the recruitment of human eosinophils. They rather indicate that they collaborate together and that they might be involved at different times during the asthmatic response. In addition, it is possible that the mediators responsible for eosinophil recruitment might also change as the disease worsens. For example, CCL11 and/or CCL26 levels are greater in induced sputum from severe or moderate asthmatics than from mild asthmatics or healthy controls , 160. Inrbations . In contrbations , 121. Forbations . As for rbations , raising2 and DP2/CRTH2 levels are increased in asthma severity in BAL (2/CRTH2 antagonist OC000459 improves FEV1 and the quality of life of subjects with eosinophilic uncontrolled asthma and steroid-free subjects with moderate persistent asthma (In asthma, CysLTs levels in induced sputum are increased in moderate asthmatics compared to severe asthmatics and healthy controls . Also, sy in BAL , 152, ant asthma , 156. Fit asthma .The potential and/or documented roles of multiple chemoattractant involved in eosinophil recruitment in asthma underscore the need to revisit this concept and to establish when and how those actors are involved. The development of experimental asthma models with mice, rats, or guinea pigs has been very helpful to broaden our knowledge about asthma pathogenesis and to identify some eosinophil and ILC2 chemoattractants in allergic asthma. However, eosinophils and their functional responses are very different between species . In that2/CRTH2 and ST2, the IL-33 receptor (H2 cytokines IL-5, IL-13 and, to a lesser extent, IL-4. Of note, the number of ILC2 correlate with sputum eosinophils in allergic asthma (First identified in 2010, ILC2 are defined as lymphoid cells lacking specific lymphocytes lineage markers and the expression of the DPreceptor –218. Thec asthma . This suc asthma , 221, es2 and CysLTs are defined as potent chemoattractants of ILC2. Indeed, PGD2 is almost five times more potent than IL-33 (2-induced migration is greater in ILC2 from allergic subjects compared to healthy subjects (2/CRTH2 or treated with a DP2/CRTH2 antagonist have lower ILC2 levels in the lungs after intranasal administration of PGD2 (1 and its expression is increased in atopic subjects (in vitro, LTE4 ≫ LTD4 > LTC4 ≈ IL-33, indicating that perhaps another CysLT receptor might be involved in this process (Since IL-25, IL-33, and TSLP are potent activators of ILC2, their ability to induce the migration of ILC2 was first evaluated. IL-33 and TSLP induce a weak migration of human ILC2 , 223. Hoan IL-33 , 224, an of PGD2 . As for subjects , 227. In2, and the CysLTs have been identified as chemoattractants of ILC2, some studies reported that human ILC2 express the chemokine receptor CCR4 and mouse ILC2 express the LTB4 receptor BLT1 (Although only IL-33, TSLP, PGDtor BLT1 , 227. Futor BLT1 . Other sex vivo and their recruitment in vivo. This underscores that targeting eosinophil recruitment as a therapeutic approach in asthma might not be readily successful, as suggested with the attempt at blocking the eotaxin receptor CCR3 (This review highlights that many chemokines and soluble mediators are very good to excellent at inducing the migration of eosinophils tor CCR3 . Additiotor CCR3 ]. Anothetor CCR3 , raisingAll the authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metal nanoclusters, with dimensions between atomic and nanoparticles, have attracted a great deal of attention due to their significantly unusual properties. Water-soluble palladium nanoclusters (Pd NCs) with blue-green fluorescence were synthesized by a water bath heating method, with methionine as a stabilizer and ascorbic acid as a reducing agent. We investigated the optimal synthesis conditions, stability, and pH response of the obtained products in detail. The synthesized materials were characterized by ultraviolet-absorption spectroscopy, fluorescence spectroscopy, high-resolution transmission electron microscopy, and atomic force microscopy. These experimental results showed that the Pd NCs had a small size of ~1.91 nm, with a uniform size distribution. Additionally, the Pd NCs emitted blue-green fluorescence under ultraviolet light with a quantum yield of 5.47%. Notably, both stabilizers and reducing agents used in this synthesis method are nutrients for humans, non-toxic, and harmless. This method could be viewed as a biologically friendly and green way of preparing fluorescent metal nanoclusters. The as-prepared fluorescent Pd NCs also possessed excellent fluorescence detection ability and were very sensitive to low concentrations of hemoglobin, with a linear response in the range of 0.25–3.5 μM and a detection limit of 50 nM. Metal nanoclusters (NCs), are an important kind of fluorescent nanomaterial, which have recently received considerable attention ,2. NCs c4 to reduce HAuCl4, with a luminescence quantum yield exceeding 40% [To date, the rational design, controllable synthesis, and applications of ultra-small NCs have drawn considerable interest ,16,17. Tding 40% . Huang eding 40% . Cao et ding 40% . Tanaka ding 40% .2 is not easily soluble in water, and reduction of palladium is a relatively slow process. Nano-sized Pd plays an important role in different types of chemical reactions and exhibits different characteristics from those of nanoparticles, such as gold and silver. Pd nanoparticles can catalyze C–C cross-couplings [N,N-dimethylformamide (DMF) solution [Research on fluorescent gold, silver, copper, and platinum metal nanoclusters has matured, and regulation of the emission spectrum and quantum efficiency can be achieved to a certain extent . Howeverouplings , olefin ouplings , and CO ouplings . The proouplings ,30,31. Asolution ,33. AlthInspired by this method, we synthesized Pd NCs using methionine as a stabilizer, ascorbic acid as reducing agent, and ammonium tetrachloropalladate(II) as metal precursor via water bath heating . Bio-mol4)2PdCl4, 98%) and dl-methionine  were obtained from Adamas-Beta . Ascorbic acid , human serum albumin (HSA), ribonuclease A (RNA), cytochrome C, transferrin, bovine serum albumin (BSA), lysozyme, and hemoglobin were obtained from Solarbio . All competitive metal ions and glucose used for selectivity testing were acquired from Sinopharm Chemical Reagent Co. Ltd. . All reagents were used as received without further purification. Ultrapure water (resistivity: 18.2 MΩ⋅cm) was obtained from a Millipore purification system.Ammonium tetrachloropalladate (II) . Fluorescence spectra were performed on a Hitachi F-7000 fluorescence spectrometer . High-resolution transmission electron microscopy (HR-TEM) data were obtained on a FEI Tecnai G2 F20 S-TWIN transmission electron microscopy instrument operating at 200 kV . Time-resolved luminescence intensity decays were recorded on a Horiba JY Fluorolog-3 molecule fluorometer , and samples were excited by a 395-nm laser light source. Atomic force microscopy (AFM) data were obtained on a Bruker MultiMode8 .3 v/v) and rinsed with ultrapure water prior to use. In a typical procedure, 12 mL of (NH4)2PdCl4 aqueous solution (2.5 mM) was mixed with 24 mL of methionine solution (0.1 M), and 3.6 mL of NaOH (0.6 M) for 30 min, and then 9 mL of l-ascorbic acid (0.14 M) was added into the solution at 60 °C. Within 5.5 h, yellow solutions of Pd NCs were obtained. Afterwards, the reaction solution was centrifuged at 8000 rpm for 10 min to remove large particles and dialyzed with water via a dialysis membrane (1000 Da) to remove the free ions and ligand. The resulting solution was stored in the dark at 4 °C for use. All glassware used in the following procedures was thoroughly cleaned with freshly prepared aqua regia  to measure the change of fluorescence intensity. The fluorescence spectra were recorded at room temperature with excitation at 420 nm; both the excitation and emission slit widths were 5 nm.To evaluate the sensitivity toward hemoglobin, 100 µL of hemoglobin solution of various concentrations was added into 1.9 mL of the prepared Pd NCs solutions and the mixtures were incubated at room temperature before spectral measurements. To check the selectivity of the Pd NCs, a series of competitive molecules at 5 µM were tested, including HSA, RNA, cytochrome C, transferrin, BSA, lysozyme, glucose, K4)2PdCl4 was first reacted with the ligand (methionine) in aqueous solution under alkaline conditions to form a low valence metal-stabilizer complex and then further reduced under the action of the reducing agent ascorbic acid to generate methionine-coated zero-valent palladium metal nanoclusters. These results showed that the amount of ligand methionine, the concentration of sodium hydroxide, the concentration of reductant ascorbic acid, and the reaction temperature and time affected the formation of Pd NCs. The reaction conditions of the Pd NCs synthesis were optimized. As shown in In the process of synthesizing Pd NCs, the aqueous solution of the metal precursor (NH4)2PdCl4, and reducing agent (ascorbic acid). The synthesized Pd NCs exhibited a clear absorption band centered at 400 nm. In contrast, pure methionine, (NH4)2PdCl4, and ascorbic acid did not show any notable absorption.Photoluminescence is a typical property of small-size palladium clusters because of the formation of discrete energy levels. As shown in the inset of 4)2PdCl4 nor pure methionine emit any fluorescence under the same conditions, the luminescence can be attributed to the synthesized Pd NCs , which is comparable to the yields reported for other metal nanoclusters stabilized by amino acids and higher than those reported for methionine-stabilized fluorescent Au NCs .To further characterize the morphology and the size distribution of the Pd NCs, the synthesized Pd NCs were characterized by high-resolution transmission electron microscopy (HRTEM) and atomic force microscopy (AFM). The results of HRTEM a showed The environmental stability of fluorescent nanoclusters is an important factor for practical applications. The effects of pH and ionic strength on the stability of the NCs were investigated. As shown in 2 of 0.9915  is a protein presenting in higher organisms, which is responsible for carrying oxygen ,40. It tf 0.9915 b. The def 0.9915 c. Furtheetection d.4)2PdCl4, which has good water solubility, as a metal precursor. The prepared Pd NCs were well dispersed in water with a uniform particle size distribution, strong blue-green fluorescence, and good biocompatibility. We found that the synthesized Pd NCs were sensitive to hemoglobin and could be successfully applied to detection of hemoglobin. Owing to the inherently excellent biocompatibility and detection performance, our fluorescent Pd NCs show promise for various applications in biosensing.In summary, we present a simple, non-toxic, harmless, environmentally friendly method for the synthesis of fluorescent Pd NCs. The method uses amino acids with a high biocompatibility as protective agents and (NH


A viable virus aerosol sampler (VIVAS) effectively collected viable influenza A and B viruses from air inside a student health care center during an influenza outbreak. The viruses had “drifted” genes, showcasing the usefulness of the VIVAS for air sampling and noninvasive surveillance of viruses in circulation. The dynamics and significance of aerosol transmission of human influenza viruses remain controversial , 2. A maThe quantity of viruses collected in air samplers is typically low and below the detection limit of PCR-based methods. To improve the chances of virus detection, virus isolation was attempted by inoculation of aliquots of the collection medium onto a variety of cell lines. Remarkably, mixed virus-induced cytopathic effects (CPE) were observed in various cell lines, indicating the isolation of different types of viruses on two of the test days (only human metapneumovirus was isolated on the third attempt).Typical influenza virus (IFV) CPE, including the formation of focal enlarged granular cells, followed by nonspecific cell deterioration and detachment of the swollen cells from the growth surface, were observed in Madin-Darby canine kidney (MDCK) cell monolayer cultures. To quickly screen for the presence of influenza A and B virions, the spent medium of MDCK cell cultures that exhibited typical IFV-induced CPE was tested using a commercial solid-phase enzyme-linked immunosorbent assay (ELISA)  , 5. ViraSequencing revealed that the pdmH1N1 viruses belonged to hemagglutinin (HA) subclade 6B.1 –10, the –We conclude that the VIVAS could be useful for passive noninvasive virus surveillance applications.KX398060, KX398061, KX398062, and KX398063; IFV pdmH1N1 NA gene, KX398064, KX398065, KX398066, and KX398067; IFV pdmH1N1 M2 and M1 genes, KX398068, KX398069, KX398070, and KX398071; IFV H3N2 HA gene, KX398081, KX398082, and KX398083; IFV H3N2 NA gene, KX398084, KX398085, and KX398086; IFV H3N2 M2 and M1 genes, KX398087, KX398088, and KX398089; IFV B HA gene, KX398072, KX398073, and KX398074; IFV B NB and NA genes, KX398075, KX398076, and KX398077; and IFV B M1 and BM2 genes, KX398078, KX398079, and KX398080.The sequences of this study have been deposited in the GenBank database under the following accession numbers: IFV pdmH1N1 HA gene,


Pulsatillae Radix (PR) is widely used to treat amoebic dysentery and renowned for its anti-inflammatory effects. This study aimed to confirm evidence regarding the potential therapeutic effect of PR on Crohn's disease using a system network level based in silico approach. Study results showed that the compounds in PR are highly connected to Crohn's disease related pathways, biological processes, and organs, and these findings were confirmed by compound-target network, target-pathway network, and gene ontology analysis. Most compounds in PR have been reported to possess anti-inflammatory, anticancer, and antioxidant effects, and we found that these compounds interact with multiple targets in a synergetic way. Furthermore, the mRNA expressions of genes targeted by PR are elevated significantly in immunity-related organ tissues, small intestine, and colon. Our results suggest that the anti-inflammatory and repair and immune system enhancing effects of PR might have therapeutic impact on Crohn's disease.In East Asian traditional medicine, Inflammatory bowel disease (IBD) may be categorized clinically as Crohn's disease or ulcerative colitis . Crohn'sAccording to a statistical report issued by the Health Insurance Review and Assessment Service in Korea in 2016, the number of Crohn's disease patients increased from 13,920 in 2011 to 18,332 in 2015, an average annual increase of 7.1%. Furthermore, over the same period, total medical cost has increased by 19.4% annually, and more than half of patients are in 20s or 30s. A systematic review about the economic and quality-of-life burden of Crohn's disease reported that, in the USA, Germany, France, UK, Italy, and Spain, in all countries combined, Crohn's medical costs totaled €30 billion annually and that patient quality of life was substantially diminished by the physical, emotional, and social effects of the disease  A (MAOA), dual oxidase 2 (DUOX2), and others are associated with “oxidation-reduction process.”Bcl-2-like protein 1 (BCL2L1), interleukin-6 (IL6), mitogen-activated protein kinase 8 (MAPK8), and cellular tumor antigen p53 (TP53) are associated with both the “positive regulation and negative regulation of apoptotic process.”To summarize, it is likely that the therapeutic effect of PR in Crohn's disease is due to its anti-inflammatory and repair process and immune system enhancing effects.To visualize more conveniently the multitargeted effects of PR, network analysis was used to investigate its actions within the context of the whole human genome , 53. As The C-T network showed 415 interactions between the 182 targets and 32 active compounds of PR. Ursolic acid  had the highest number of interactions with targets, followed by beta-sitosterol  and isorhamnetin , and, thus, these results demonstrated that single molecules can target multiple receptors . LikewisκB p65 subunit  demonstrate higher affinitive connections with pathways. Betweenness centrality and degree centrality results were similar; there was only little difference. “Pathways in cancer” (betweenness = 0.19) had the highest betweenness among the targets, which concurred with degree centrality, and this was followed by “neuroactive ligand-receptor interaction” (betweenness = 0.15) and “PI3K-Akt signaling pathway” (betweenness = 0.10). Regarding highest betweenness targets, PIK3CA (betweenness = 0.10), PIK3CG (betweenness = 0.10), and prostaglandin E2 receptor EP3 subtype  showed most affinitive connections with pathways.In addition, 40 pathways related to Crohn's disease were extracted to construct the T-P network. According to degree centrality, “pathways in cancer” (degree = 49) had the highest number of connections with the targets, followed by the “PI3K-Akt signaling pathway” (degree = 34) and “hepatitis B” (degree = 33). In the same manner, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform , phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform , and nuclear factor NF-The tissue mRNA expression profiles of target genes at the organ level were investigated to identify effects of PR on Crohn's disease. No mRNA expression information of three of genes, muscarinic acetylcholine receptor M1 (CHRM1), G-protein coupled receptor TGR-1 (NMUR2), and taste receptor type 2 member 31 (TAS2R31), was found in the BioGPS. In total, the mRNA expression profiles of 179 of 182 genes were analyzed (Supplementary Table S2). 158 Of the genes displayed above average mRNA expressions in 17 relevant organ tissues, namely, in BDCA4+ dendritic cells, bone marrow, CD14+ monocytes, CD19+ B cells, CD33+ myeloid cells, CD34+ hematopoietic stem cells, CD4+ T cells, CD56+ NK cells, CD8+ T cells, colon, colorectal adenocarcinoma, liver, lymph nodes, lymphoblasts, small intestine, smooth muscle, and thymus. In addition, these 17 organ tissues, retina, prefrontal cortex, pineal, amygdala, cardiac myocyte, heart, whole blood, and other tissues, were also associated with relatively high mRNA expressions. Networks of the tissue mRNA expressions of 158 target genes and PR compounds are shown in In detail, among 84 organ tissues, CD33+ myeloid showed the most overexpressed mRNA expression, 104 genes of 158 target genes were overexpressed in CD33+ myeloid, followed by 97 genes in lymphoblasts, 95 in each of smooth muscle and CD34+ hematopoietic stem cells, 91 genes in liver and CD56+ NK cell, 84 in bone marrow and colorectal adenocarcinoma, 82 in BDCA4+ dendritic cells, 75 in CD14+ monocytes, 73 in small intestine, 72 in CD4+ T cells, 70 in CD19+ B cells, 67 in colon, 38 in CD8+ T cells, 23 in thymus, and 22 in lymph nodes. It is evident that most genes were overexpressed in several organs at the same time.Additionally, intestinal alkaline sphingomyelinase (ENPP7), DNA polymerase catalytic subunit (POLG), and carbonic anhydrase 13 (CA13) recorded beyond average mRNA expressions in all 17 organs. Furthermore, more than 146 (92%) of 158 target genes were overexpressed in two or more organ tissues, suggesting that these organs and compounds in PR are closely related. Furthermore, because the above 17 organs are highly related to immunity, our study results indicate that the therapeutic effects of PR on Crohn's disease are due to its targeting and activating the immune system.The other 21 target genes, such as acetylcholinesterase (ACHE), pancreatic alpha-amylase (AMY2A), and muscarinic acetylcholine receptor M3 (CHRM3), did not show above average mRNA expression in the 17 organs.Comparison data between normal tissue and Crohn's disease patients' mRNA expression pattern from Gene Expression Omnibus (GEO) was collected. We employed GEOquery and limma R packages of GEO2R tool to identify highly expressed genes in 6 datasets. Dataset accession numbers are as follows: GSE24287, GSE60083, GSE6731, GSE36807, GSE68570, and GSE72780. To sum up, 86 normal samples and 149 Crohn's disease samples were analyzed in each dataset. GEO2R presented the top 250 highly expressed genes in Crohn's disease group compared to the control group and we deleted overlaps, so, in the end, 1182 genes were sorted out.We found out that there were 23 common genes  between In order to investigate further the effect of PR in Crohn's disease, we performed pathway enrichment analysis . Using tα, is mediated by NF-κB, which is a key regulator of inflammation [κB p65 subunit (RELA); and scoparone (C25) and ursolic acid (C30) target NF-κB inhibitor alpha (CHUK), which suggests that these compounds affect NF-κB activity. Cernuoside (C12) targets IL-2. Beta-sitosterol (C8) targets transforming growth factor beta-1 (TGFB1) and transcription factor AP-1 (JUN), and ursolic acid (C30) also targets transcription factor AP-1 (JUN) and signal transducer and activator of transcription 3 (STAT3). Furthermore, NF-κB and AP-1 in combination are highly related to the initial inflammatory response and to the development of acquired immunity [The synthesis of inflammatory cytokines, such as IL-1, IL-6 and TNF-ammation , 56. We immunity . Moreoveimmunity .In this study, a network pharmacology method with active compounds filtration, multiple drug target prediction, gene ontology, network analysis, relevant organ location network, and pathway enrichment analysis were employed to determine the targets of PR in relation to Crohn's disease. Our study shows that PR is highly connected to the pathways, biological processes, and organs of Crohn's disease. A pharmacological approach was used to identify the actions of PR at the systems network level.In this study, pathway mapping result showed that the target genes of PR overlap more with Crohn's disease than with ulcerative colitis. Experimental study also suggested that the markers of both diseases are different from each other , 60. TheIn addition, IBD is usually referred to as an autoimmune disorder , whereas Pulsatillae Radix , Phellodendri Cortex (Hwangbaek), Coptidis Rhizoma (Hwangryeon), and Citrus reticulata (Jinpi) herb combination  should be explored in the future.In the present study, we focused on the use of PR as a potential therapy for Crohn's disease. However, herb pairs and combinations are more commonly prescribed and are regarded to be more effective and safer . In termThrough GO analysis, we found out that targets of PR are associated with liver diseases such as hepatitis B, hepatitis C, and nonalcoholic fatty liver. In addition, the mRNA expression of 91 of 179 genes in liver was overexpressed according to the target organ location network result. Liver inflammation is as common extraintestinal symptom of Crohn's disease , and theThis study demonstrated that 73 and 67 of 158 targets of PR were highly expressed in small intestine and colon, respectively, results which were accordance with not only the organs commonly affected by Crohn's disease but also the properties of PR in terms of meridian tropism theory. However stronger evidence with another research design is required to support this result.We confirmed a multicompound and multitarget interaction through the C-T network, which showed that 88% of the active compounds were connected with more than two targets and 47% of the targets interacted with more than one compound. Although it demonstrated the synergetic network of multitarget actions, we should explore more differentiated drug action based on degree centrality and how different the drug actions are when more compounds target one gene in simultaneous way compared to the case of one compound targeting one gene.Previous studies indicate that most compounds in PR have anti-inflammatory, anticancer, and antioxidant effects. In the present study, we found that these compounds interact with multiple targets in a synergetic manner and that PR is highly connected to Crohn's disease related pathways, biological processes, and organs. C-T and T-P network results demonstrated complex multicompound and multitarget drug actions and the relations between targets and various pathways. Furthermore, target genes were found to be overexpressed in organs highly related to immunity. These findings suggest the anti-inflammatory effects of PR, and its enhancements of repair processes and immune system might be of therapeutic benefit in Crohn's disease.Supplementary Table S1 showed the ADME profiles of 57 compounds in PR, and supplementary Table S2 demonstrated the mRNA expression profiles of 179 of 182 genes.


On the other hand, Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine , surface receptor , transcription factors B lymphocyte maturation protein 1, signal transducer and activator of transcription 5, T-bet, KLF-2 signaling, and repressor miR 146a. Interestingly, miR-17–92 and FOXO1 act as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review, we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin ligase, and microRNA as positive and negative regulators for Tfh differentiation.The follicular helper T (Tfh) cells help is critical for activation of B cells, antibody class switching, and germinal center (GC) formation. The Tfh cells are characterized by the expression of CXC chemokine receptor 5 (CXCR5), ICOS, programed death 1 (PD-1), B cell lymphoma 6 (BCL-6), and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. On the one hand, Tfh cells are generated from naive CD4 Robust ormation , 3, wherormation . Moreoveormation , 12. Becormation . In caseormation , 15. Thu+ cells representing effector Tfh , surface receptor , transcription factors , ubiquitin ligase, microRNA (miRNA), and Tfr cells. The mechanistic details for positive and negative regulation of Tfh differentiation are highlighted in this review.Follicular helper T cells are characterized by expression of CXC chemokine receptor 5 (CXCR5) that is attracted toward CXCL-13 present in the B cell zone enabling these cells to enter interior of B cell follicle , 17. Thector Tfh  or memorctor Tfh –30 are actor Tfh . The Tfhctor Tfh  as exempctor Tfh . Thus, T+ T cells upon antigen presentation and co-stimulation, marks the early event of Tfh differentiation  . During  of IL-2 . A recen of IL-2 . The cytnd Cxcr5 . Therefo+ T cell to differentiate into effector T cell, it requires antigenic peptide presentation through MCH-II and CD28 costimulation by antigen-presenting cells such as dendritic cells, macrophage, or B cells along with lineage driving cytokines. Signaling from CD28 by binding with its ligand CD80 (B7-1) or CD86 (B7-2) is critical for antibody class switching and GC formation , a hallmark Tfh cell surface marker, differentiates these cells from other T cell lineages , 61. Mig+ CD45RO+ memory cells validates the importance of the ICOS in Tfh differentiation  with ICOS mutant in PI3k-binding site of IProx motif and adoptive transfer into ICOS−/− mice and then LCMV infection fail to generate Tfh cells illustrating that IProx plays a critical positive role in Tfh differentiation , CD28, the ICOS-driven PI3k activation, is critical for Tfh cell as suggested by impaired Tfh generation in mice selectively defective in ICOS downstream signaling of the PI3k activation . Moreoventiation . Importantiation . Recentlntiation . KLF2 nentiation . Althougntiation .The adaptor molecule SAP is very critical for the generation of antigen-specific long-lived plasma and memory B cells  by stabiPlasmodium infection wherein it is constantly expressed  and its ligand OX40L (CD252) is critical for T cell survival, proliferation, and differentiation –83. Clasxpressed , 85. Enh Blimp-1 , 87. Wheneration . Howeverneration . Thus, ineration . Aberran pathway . Thus, r−/− mice. Moreover, administration of GITR-Fc protein greatly reduced CIA and joint damage by inhibiting Tfh generation and thereby autoantibody production (During chronic LCMV infection and in collagen-induced arthritis (CIA) mice, the expression of glucocorticoid-induced TNF receptor family-related protein (GITR) was elevated on Tfh cells than non-Tfh cells , 91. Impoduction . MechaniProgramed death 1, a surface molecule expressed on exhausted T cells, is also expressed by Tfh cells. PD-1/PD-L1 and PDL-2 signaling is associated with the dephosphorylation of molecules downstream of TCR signaling. Mechanistically, PD-1 engagement inhibits ICOS signaling thereby regulating Tfh generation . Howevermilieu dependent or it may have new Tfh regulatory signaling pathways which are yet to be identified.The cytotoxic T lymphocyte antigen 4 (CTLA-4), a regulatory T cell marker involved in regulation of effector T cells, was recently shown to be expressed by Tfh  and Tfr Pdcd1 gene promoter thereby regulating PD-1  plays a critical role as a positive regulator in Tfh differentiation . Mice wiing PD-1 . Thus, i+ T cells , a basic helix-loop-helix (bHLH) transcription factor, is specifically upregulated in Tfh cells. It positively regulates Tfh generation as blockade of Ascl2 signaling with Id3 leads to impaired Tfh generation . Ascl2 iIrf4−/− CD4+ T cells failed to express BCL-6, thus regulating the Tfh generation (Interferon regulatory factor 4 (IRF4) controls expression of BCL-6, as neration .2+ regulates NFAT1, NFAT2, NFAT3, and NFAT4. The Ca2+ signaling-regulated NFAT1, NFAT2, and NFAT4 is critical for T cell activation, differentiation, and maintenance of tolerance , NFAT2 (NFATc or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx or NFATc3), and NFAT5. Signaling through Caolerance –112. Recolerance . In contolerance .in vivo inhibition of STAT5 signaling leads to enhanced Tfh cell and GC formation. In the absence of Blimp-1, STAT 5 failed to inhibit Tfh cell formation, whereas over-expression of Blimp-1 in STAT5-deficient T cells resulted in Tfh differentiation. Thus, IL-2/STAT5 axis negatively regulates Tfh differentiation by regulating Blimp-1 , play a critical role in Tfh differentiation. The generation of Tfh cells was increased upon depletion of Id2  protein . Ectopic protein .+ T cell differentiation into effector function by antagonizing FOXO1 and also through inhibition of MEK and ERK signaling  binds to BCL-6 and negatively regulates Tfh differentiation by ubiquitination of histone proteins. Knocking down E3 ligase Cul3 from CD4 cells resulted in over-expression of basic leucin zipper transcription factor, ATF-like (Baft), a target of BCL-6 leading to exaggerated or enhanced Tfh generation during antigen challenge .san mice, substitution of M199R within ROQ domain of Roquin 1 leads to enhanced expression of ICOS, thereby promoting aberrant Tfh generation causing lupus like autoimmunity  and Roquin 2 (Rc3h2) are paralogous proteins made up of really interesting new gene (RING) finger, ROQ domain, and zinc finger domain and regulates Tfh differentiation at posttranscriptional level. In Roquinimmunity . Roquin immunity , 135. Unmolecule . Roquin molecule , 135. Inmolecule .microRNA regulates gene expression at posttranscriptional level and thereby is crucially involved in regulation proliferation, differentiation, and programed cell death. Studies depict critical positive and negative roles of microRNA in the Tfh differentiation. In 2009, Yu et al. showed that the microRNAs, including miR-17–92 involved in CXCR5 repression, are repressed by BCL-6-driving cells toward Tfh differentiation , while dFollicular helper T cells are known for their critical help in GC formation, and B cell maturation for infection clearance is also implicated in the autoimmune diseases and virus replication. In order to clear infection and to prevent immunopathology, precise manipulation of Tfh regulatory molecular network is needed to generate adequate Tfh cell number. Adequate Tfh cell number may be generated by manipulating Tfh regulatory molecules such as cytokines, transcription factors, surface receptors, ubiquitin ligase, and miRNAs. Further research is needed to ascertain regulatory molecules either positive or negative for generation of Tfh from effector T cells. The generation of adequate Tfh number and its critical help for optimum GC formation and B cell maturation will be helpful for effective vaccination strategy.GJ and SD discussed and revived the literature; GJ drafted the manuscript. GJ, SD, and SM prepared illustrations, discussed, and revived the manuscript. All the authors critically revised the manuscript for intellectual content and approved it for publication.The authors declare that the research conducted has no commercial or financial involvement that could be considered as potential conflict of interest.


M. tuberculosis are not fully understood. In this study, we examined host macrophage immune responses of 3 race/ethnic groups to 3 genetically and geographically diverse M. tuberculosis lineages.The roles of host and pathogen factors in determining innate immune responses to M. tuberculosis whole cell lysates of clinical strains Beijing HN878 (lineage 2), Manila T31 (lineage 1), CDC1551 (lineage 4), the reference strain H37Rv (lineage 4), as well as with Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA) and TLR4 agonist lipopolysaccharide (TLR4/LPS). Following overnight incubation, multiplex assays for nine cytokines: IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFNγ, TNFα, and GM-CSF, were batch applied to supernatants.Monocyte-derived macrophages from healthy Filipinos, Chinese and non-Hispanic White study participants  were challenged with Filipino macrophages produced less IL-1, IL-6, and more IL-8, compared to macrophages from Chinese and Whites. Race/ethnicity had only subtle effects or no impact on the levels of IL-10, IL-12p70, TNFα and GM-CSF. In response to the Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA), Filipino macrophages again had lower IL-1 and IL-6 responses and a higher IL-8 response, compared to Chinese and Whites. The TLR2/LTA-stimulated Filipino macrophages also produced lower amounts of IL-10, TNFα and GM-CSF. Race/ethnicity had no impact on IL-12p70 levels released in response to TLR2/LTA. The responses to TLR4 agonist lipopolysaccharide (TLR4/LPS) were similar to the TLR2/LTA responses, for IL-1, IL-6, IL-8, and IL-10. However, TLR4/LPS triggered the release of less IL-12p70 from Filipino macrophages, and less TNFα from White macrophages.Both host race/ethnicity and pathogen strain influence the innate immune response. Such variation may have implications for the development of new tools across TB therapeutics, immunodiagnostics and vaccines. Mycobacterium tuberculosis (M. tuberculosis) has shown that lineages of the organism have predilections for certain geographic distributions and dominate certain geographic areas .–20.M. tuM. tuberculosis strains, comprising 3 predominant lineages identified in San Francisco , and one laboratory reference strain (H37Rv), or with agonists of TLR2 (lipoteichoic acid LTA) or TLR4 (lipopolysaccharide LPS). A total of 9 cytokines were measured. This study adds to the growing body of evidence that differential innate immune responses contribute to ethnicity-associated differences in TB susceptibility.Our study explores the interaction of host and pathogen factors on the innate immune response. Based on the molecular epidemiology of TB in San Francisco, which includes a sizable population of Asian patients , 21, we The “Innate Immunity to TB” (IITB) study protocol was reviewed and approved by the committee on human research at the University of California, San Francisco (CHR# H45279-29459). Written informed consent was obtained from all study participants.Healthy volunteers, aged 18 to 55, self-identifying as Chinese, Filipino, or non-Hispanic White were invited to participate in the study. Recruitment was through bulletin board postings at the Schools of Dentistry, Medicine, Nursing, Pharmacy, Physical Therapy of the University of California, San Francisco, and at the undergraduate and graduate schools of San Francisco State University. Between 2009 and 2011, all consenting volunteers meeting inclusion and having no exclusion criteria were consecutively enrolled into IITB. Recruitment continued until 50 participants per race/ethnic group were enrolled. All volunteers completed a standardized questionnaire reviewing demographics, health history as well as information on potential risk factors for TB. All volunteers reported the race/ethnicity of their biological parents and grandparents, and only those identifying both generations as Chinese, Filipino or non-Hispanic White were eligible for participation in the study. Those reporting a history of active TB, evidence of prior TB on chest radiograph, vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine in the past 15 years, co-morbid conditions including diabetes, renal disease, cancer, hepatitis, HIV/AIDS, or other moderate or high risk characteristics associated with TB, such as homelessness or history of incarceration, significant alcohol use (greater than 2 drinks per day), tuberculin skin testing within past 6 months, or treatment with any immunomodulating agents such as corticosteroids, were not eligible to participate. All participants had IFN-γ release assay testing, specifically Quantiferon-Gold® (QFT), for latent TB infection at the time of study enrollment.http://www.beiresources.org/; catalog numbers: NR-14820, NR-14821, NR-14824, NR-36496).Whole cell lysates of Beijing HN878, Manila T31, CDC1551, and H37Rv, were obtained from gamma-irradiated cells as described previously . The tot5/ml in 10% vol/vol human serum (HuS), and then plated at 500,000/well in ultralow adherence plates, without stimulation, and incubated at 37°C with CO2 for 72 hours to obtain enrichment of monocyte-derived macrophages (MDMs) through adherence. Upon harvesting of MDMs, viability was assessed by absence of uptake of trypan blue, and purity (ranging 96–98% MDMs) was determined by fluorescence cell sorter (FACS). We further assessed viability on the basis of HLA-DR and CD11c, and T cell contamination on the basis of CD3. MDMs from each study participant, were incubated at 1x105 for 18 hours with 10 ug / ml whole cell lysate of M. tuberculosis strains, and to agonists of TLR2 (10 ug / ml Lipoteichoic acid (LTA) from S. aureus, Sigma, L 2515) and TLR4 (50 ng/ml lipopolysaccharide (LPS) from E. coli Sigma, L-2880); and culture medium (negative control). Following overnight incubation, supernatants were collected and frozen at -80°C and batch tested using MSD Human ProInflammatory 9-Plex Tissue Culture Kit K15007B-1  run on a Meso Scale Diagnostics MESO QuickPlex SQ 120 instrument.For each study participant, phlebotomy was performed to fill four BD Vacutainer® CPT™ Cell Preparation Tube with Sodium Citrate . Whole blood was processed according to manufacturer guidance for the separation of mononuclear cells. Monocytes were prepared from freshly drawn blood samples through negative selection using the Miltenyi Biotec Monocyte Isolation Kit II, which provides an indirect magnetic labeling approach for the isolation of untouched monocytes from human peripheral blood mononuclear cells (PBMCs). Monocytes were counted and resuspended at 5x10M. tuberculosis strains: H37Rv “reference strain,” CDC1551 “EuroAmerican,” HN878 “East Asian,” and T31 “Indo-Oceanic;” and 2 stimulants on TLR pathways: LTA and LPS) and by race . For analyses where stimulant was held constant and responses were compared by race, linear regression was used, adjusting for independent factors age and gender. For analyses where race was held constant and participant responses to each stimulant were compared, a general estimating equation (GEE) was added to the linear regression to adjust for responses clustered by participant, in addition to adjusting for age and gender [p < 0.20 level [Categorical demographic factors were analyzed using chi squared tests; age was compared by race using the Student’s t-test. Geometric mean cytokine responses were compared in response to 6 stimulants (4 d gender . Multiva20 level .Cytokines were measured in picograms (pg), logged to approximate a normal distribution for analysis, and presented as geometric means with 95% confidence intervals. The upper limit of detection (ULOD) was 10,000 pg for all cytokines; values above the ULOD were set to 10,000 pg. The lower limits of detection (LLOD) were unique for each cytokine; values below the LLOD were left unchanged. All statistical analyses were performed using SAS version 9.4 .The study population included 137 healthy donors with mean age 30±8 (range 19–55) years of age. Most were female , and 39% were foreign-born (n = 54). A positive QFT test was uncommon (8%) and more likely to occur among the foreign-born . Self-identified race was divided approximately evenly: non-Hispanic White (n = 46), Chinese (n = 47), or Filipino (n = 44). Filipinos were on average older, and Whites were less likely to be foreign-born .M. tuberculosis lysates. The concentration of cytokines released in response to the bacterial lysates were measured using 2 different platforms ; Meso Scale Discovery, Gaithersburg, Maryland) according to manufacturer instructions. Since the cytokine levels and trends were identical for these 2 platforms, we describe our data generated with the MSD kit. We measured a total of 9 cytokines: IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFNγ, TNFα, and GM-CSF. As the study was designed to assess macrophage stimulation, the T cell cytokines IFN-γ and IL-2 were included as controls. IFNγ and IL-2, at the lower limit of detection was noted, possibly due T cell contamination.We prepared monocyte-derived macrophages (MDMs) from healthy study participants (n = 137), and incubated the cells with M. tuberculosis strains for IL-1b, IL-6 and IL-8 , Manila T31 (lineage 1) and CDC1551 (lineage 4), and reference strain H37Rv  . This SNM. tuberculosis strains produce different disease phenotypes and immune responses [M. tuberculosis strains is likely attributable to differing gene expression [Many epidemiological and experimental studies have shown that esponses , 49–51. pression , metabolpression , cell wapression  and growpression . We showpression . As IL-8pression , 59, andpression . These dM. tuberculosis pathogen lineages are largely unknown. However, comparative genomics, and ongoing efforts to understand the unusual biology of these bacteria, including unbiased systems biology approaches [The molecular bases for these innate immune response differences across host ethnicity and proaches , 61, areM. tuberculosis. We recognize that the use of cellular lysates represents a proxy of infection useful for comparisons rather than definitive determination of immunogenicity of strains. Also, whereas macrophages for all assays were enumerated at 50,000 cells/well, we did not enumerate circulating monocytes in volunteers nor evaluate for cell surface expression, which may have affected induced cytokine differences. Additionally, due to the multiplexed nature of assays and resource limitations, we were unable to conduct diluted assays, resulting in a censored upper limit of detection. Finally, ethnicity was determined based on self-report rather than genetic testing. Whereas, it has been shown that self-definitions of ethnicity are highly accurate reflection of true ethnic identity in Chinese and Whites, in particular, there is the potential for misclassification of ethnicity [M. tuberculosis were evaluated using cells from healthy individuals and focused on innate immunity, thus reducing the influence of adaptive immunity, history of TB exposure, as well as environmental and socioeconomic factors. Second, our controlled experimental design allowed us to examine the interaction of host and pathogen genetics. Third, our study population of 137 healthy macrophage donors lends considerable robustness to our results.Our study has limitations, principally that our experimental system did not include a model in which macrophages were infected with live thnicity . Our stuIn sum, our data show that that both host race/ethnicity and pathogen factors influence the innate immune response and such variation has implications for the development of new tools across TB therapeutics, immunodiagnostics and vaccines , 45, 63.S1 FigM. tuberculosis lysates from four strains. Comparisons are adjusted for age and gender. Brackets indicate FDR-adjusted p < 0.20.Cytokines released by macrophages derived from healthy individuals of three ethnicities in response to (DOCX)Click here for additional data file.S1 TableP values adjusted for multiple testing using the false discovery rate (FDR) method.(DOCX)Click here for additional data file.S2 TableP values adjusted for multiple testing using the false discovery rate (FDR) method.(DOCX)Click here for additional data file.S3 TableGeneral estimating equation is included to adjust for cytokine values clustered by participant. P values adjusted for multiple testing using the false discovery rate (FDR) method.(DOCX)Click here for additional data file.


Olea europaea L. cv. Picual). Here, in the plasma-membranes of live protoplasts, we used fluorescence to examine various specific lipophilic stains in sphingolipid-enriched regions and investigated the composition of the sphingolipid long-chain bases (LCBs) as well as the expression patterns of sphingolipid-related genes, OeSPT, OeSPHK, OeACER, and OeGlcCerase, during olive-fruit development and ripening. The results demonstrate increased sphingolipid content and vesicle trafficking in olive-fruit protoplasts at the onset of ripening. Moreover, the concentration of LCB  increases during fruit development to reach a maximum at the onset of ripening, although these molecular species decreased during fruit ripening. On the other hand, OeSPT, OeSPHK, and OeGlcCerase were expressed differentially during fruit development and ripening, whereas OeACER gene expression was detected only at the fully ripe stage. The results provide novel data about sphingolipid distribution, content, and biosynthesis/turnover gene transcripts during fleshy-fruit ripening, indicating that all are highly regulated in a developmental manner.Plant sphingolipids are involved in the building of the matrix of cell membranes and in signaling pathways of physiological processes and environmental responses. However, information regarding their role in fruit development and ripening, a plant-specific process, is unknown. The present study seeks to determine whether and, if so, how sphingolipids are involved in fleshy-fruit development and ripening in an oil-crop species such as olive ( Olea europaea L.) is one of the most economically important fruit trees worldwide for the oil of its fruit. During development and ripening, the olive fruit undergoes considerable physical and biochemical changes, with five distinguishable phases: (1) fruit set following fertilization, (2) seed development, (3) endocarp lignification, (4) mesocarp development, and (5) ripening ], while LOH2 is selective for dihydroxy-LCBs and C16 acyl chains (a long chain-fatty acid or LCFA). The combination of 9 different LCBs and 32 different FAs yields 288 potential ceramide backbones , which can be either dephosphorylated by LCB-P phosphatases or cleaved by LCB-P lyases to yield a long-chain aldehyde and ethanolamine phosphate.Pseudomonas syringae and in salt tolerance. AtACER shows no substrate preference based on the length of fatty acid moieties but according to LCB moieties, it acts preferentially on hydroxyceramides , sphingosine kinase (OeSPHK), alkaline dihydroceramidase (OeACER), and glucosylceramidase (OeGlcCerase), during fruit development and ripening. The results demonstrate that endogenous sphingolipid levels are intricately controlled during fruit development, mainly involving the sphingolipids containing the LCBs t18:1(8Z), t18:1 (8E), t18:0, d18:2 (4E/8Z), d18:2 (4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E). This detailed temporal profile of the LCB composition and the sphingolipid metabolic gene expression in the olive fruit offers new insights for understanding the physiological role(s) of plant sphingolipids. This is the first report available describing the regulation of sphingolipid distribution, content, and metabolic gene expression during fleshy-fruit development and ripening.The purpose of this study was to investigate the developmental regulation of the sphingolipid biosynthesis and turnover pathways during fruit development and ripening in olive. Although the results of lipid analyses during olive-fruit development from several cultivars have been available for many years , a charaO. europaea L. cv. Picual) grown under drip irrigation and fertirrigation during the 2013–2014 growing seasons in an orchard near Badajoz (Spain) were studied. To investigate fruit development, five different stages were differentiated attending to macroscopic differences. Harvested whole fruits were sampled at 70 (stage 1), 125 (stage 2), 168 (stage 3), 182 (stage 4), and 210 (stage 5) days post-anthesis (DPA) . A total of 250 fruits from 10 olive trees were used for each developmental stage. In order to minimize the effects related to asynchronous fruits ripening within the same tree, fruits with similar pigmentation were picked from all around the external parts of the tree canopy. Fruit firmness was measured at four equatorial regions of the fruit using a penetrometer  fitted with an 5 mm plunger. For protoplast isolation, an initial group of pericarp (fruit mesocarp and epicarp) samples was used from different developmental stages, while a second group of pericarp (fruit mesocarp and epicarp) samples was frozen immediately using liquid nitrogen and then stored at -80°C for LCB analysis after sphingolipid hydrolysis, total lipids, and RNA extraction.Twenty-year-old olive trees  in a digestion buffer using a shaker at 30 rpm. The digestion buffer was composed of pectolyase (0.1% w/v), cellulase (0.8% w/v), bovine serum albumin (0.5% w/v), and polyvinylpyrolidone (0.5% w/v), all from Sigma–Aldrich, Spain. Lastly, calcium chloride (1 mM) was added. Also, the pH was held to 5.6 (MES-Tris), and, using sorbitol, the osmolarity was adjusted to 280 milliosmol/kg. Following incubation, a 10-min centrifugation was needed (80–100 rpm at 4°C) for the separation of protoplasts from pericarp fragments.Protoplast images were taken by a FluoView 1000 spectral confocal microscope , which had 405, 488, 543, and 633 nm laser lines. The selection of fluorescence acquisition was made to maximize the fluorochrome emission in each case by the use of spectral detectors. Each sample comprised about 20 protoplasts, the data being compiled by FV10 4.2 software (Olympus).For the detection of neutral (apolar) lipids (Thermofisher), bodipy , a strongly lipophilic neutral fluorophore, was used. Bodipy is used for labeling a broad range of lipids, including phospholipids, fatty acids, ceramides, cholesterol, and cholesteryl esters . For 30 Nile Red (9-(Diethylamino)-5H benzo [α] phenoxazin-5one) staining was used to detect neutral and polar lipids. This compound is a fluorescent lipophilic dye with an emission shift from red to yellow according to the degree of hydrophobicity of lipids. Nile Red is an ideal probe for detecting lipids as it exhibits high affinity, specificity, and sensitivity to the degree of hydrophobicity of lipids . When thN- sphingosyl phosphocholine) is composed of a sphingosine moiety linked by an amide bond to a fatty acid. The sphingomyelin itself is covalently linked to a Bodipy fluorophor (Thermofisher). Although sphingomyelin is not present as such in plants, the structure has the features of a complex sphingolipid. Plants have an enormous variety of these lipids and they show a physicochemical behavior expected for a complex sphingolipid, therefore, BD-SM molecules incorporate into the plasma-membrane and mixed with native sphingolipids -4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridiumdibromide) is based on a polyethylene structure. FM4-64 is employed extensively for studying vesicle organelle organization, trafficking, and endocytosis in eukaryotic cells .As a cell-viability indicator, Trypan blue stain was used at 0.4% . The dye was solved in 0.6 M mannitol to 1 mg/ml. Cells that are non-viable take up the dye, appearing blue, whereas intact cells and membranes resist staining. After the dye was applied directly to the protoplast buffer, the suspension was thoroughly mixed. For sharp black-and-white contrast needed for imaging, Trypan blue was used at concentrations reaching 20% (v/v). Incubation for 10 min at room temperature sufficiently stained the cells . The samples were eluted (at 0.4 ml/min) with 20% RA solvent , 80% solvent RB (100% methanol) for 7 min, rising to 90% solvent RB at 15 min, after which isocratic flow was applied for 10 min prior to increasing to 100% solvent RB at 30 min and a 3-min 100% solvent RB wash preceding a return to 80% solvent RB and 2 min of re-equilibration. At 340 nm, fluorescence was excited and then detected at 455 nm. The results were both analyzed as well as integrated by Openlab.As previously described, total sphingolipids were analyzed using their released LCB . The samFrom olive pericarp (fruit mesocarp and epicarp) tissues, total RNA was extracted following OeSPT/LCB1, Unigene ID: OL002160), sphingosine kinase , alkaline dihydroceramidase  and glucosylceramidase . With random hexamers and Superscript III (Invitrogen), total RNA (2 mg) was reverse transcribed, following the manufacturer’s indications. For qRT–PCR assays, purified cDNA (2 ng) served as a template, using gene-specific primers. Primer sequences were 5′-GGTGTTGGTTCCTGTGGTCCTCGTGGA-3′ (forward) and 5′-AGACATAACCAGAGCTACTGAGGCGTT-3′ (reverse) for OeSPT/LCB1; 5′-GGCCCTTTTGTTTCAATCTGGCT T-3′ (forward) and 5′-ACCTCTGGCCAGAACCTCACCATC-3′ (reverse) for OeSPHK; 5′-ATGGAAGATGGATTATCAAGC-3′ (forward) and 5′-AGGAAGAATGACATAATTTGG-3′ (reverse) for OeACER, and 5′-GGTCAATGTAATGGACAGGAT-3′ (forward) and 5′-CGGAATTGTCAAAGCACTTTGC-3′ (reverse) for OeGlcCerase. A SYBRGreen-PCR Master kit (Applied Biosystems) containing an AmpliTaq Gold polymerase on an iCycler (BioRad Munich) was used for cDNA amplification following the supplier’s protocol. The samples were submitted to thermal-cycling conditions to activate DNA polymerase . A final 7-min elongation step was performed at 72°C. The melting curve was programmed to rise 0.5°C every 10 s, starting at 62°C. The amplicon, submitted to electrophoretic analysis, was sequenced for identity confirmation. An estimation of qRT-PCR efficiency was made by a calibration dilution curve and slope calculation. The expression levels were established as the number of cycles that the amplification needed before reaching a threshold set for the exponential phase of the PCR (CT). The data were normalized based on the amount of the O. europaea ubiquitin (OeUB) gene (Previously published RNA-Seq data  were minUB) gene . DuplicaFigure 1 shows representative images of the olive fruit harvested at 70 , 125 , 168 , 182 , and 210  DPA used for this work. In olive fruit, firmness decreased following the onset of ripening and reached the oversoft stage at 210 DPA . The content of total lipids (% DW) was determined throughout these stages in the fruit pericarp (mesocarp and epicarp). We found that the amount of the total lipid content progressively increased during fruit development . Small amounts of lipids accumulated during the first phase of development (70 DPA), but the lipid level maintained a continued increase throughout ripening until reaching 7.5-fold the departing lipid level in the fruit .Olive  fruits completed their development and ripening in 210 days under the experimental conditions used. Five major developmental stages were established to better scrutinize olive-fruit development. Figure 2, during olive-fruit development, the Bodipy fluorescence intensity gradually increased , reaching a maximal at the onset of ripening (stage 3)  and then, significantly decreased during the late stage of fruit development  . For white-light images, see Figures J. Intensity of the signal is shown in Figure 2K, wherein the biphasic pattern is appreciated.Next, we investigated the level of lipids during fruit growth and ripening through the use of several fluorescent probes that allow differentiating the type of lipid and its location. This was performed in live protoplasts, which improved plasma-membrane accessibility to the individual lipid analogs supplemented from outside the cell, and also helped to diminish non-specific fluorescence signals, ascribed mainly to the non-specific staining of cell-wall components. Neutral and polar lipid contents were located in live protoplasts from olive fruit during development by Bodipy and Nile Red staining, respectively. As shown in Figures 3E,P). Polar lipids, which are present mostly in membranes, are stained in red  whereas neutral lipids (esterified cholesterol and triglycerides), which are present in lipid droplets, are stained in green . Therefore, the maximum concentrations of polar lipids were significantly correlated with the onset of fruit ripening in olive . Figures 3K–O depict white-light images from protoplasts.Likewise, the use of fluorescent Nile Red for lipid measurement in olive-fruit protoplast revealed that the onset of ripening (stage 3) induced a high increase in polar-lipid concentration, while an enrichment of non-polar lipids was slightly detected in the fully ripe fruit (stage 5) . The signal was detectable inside the protoplast (intracellular membranes) and in plasma-membrane. At the veraison (stage 4), the fluorescence intensity gradually decreased . These results suggest that sphingolipid domains formation is required during olive-pericarp cell expansion and at the onset of ripening, most probably to mediate translocation of cargo proteins to plasma-membrane involved in endo- or exocytosis.To determine the distribution pattern of sphingolipids during fruit development and ripening, live olive protoplasts were stained with Bodipy Sphingomyelin FL C12 (BD-SM). As shown in Figure 5, the FM4-64 dye was internalized and a substantial number of punctuated fluorescent vesicles were detected in the cytosol of olive protoplasts during fruit development. Conversely, few fluorescent vesicles were observed in fruit protoplasts at stage 1, indicating that endocytosis was stimulated in protoplast cell derived from olive fruit during cell expansion and ripening . Quantification of FM4-64 showed a significant increase of the probe in fruit protoplasts at the fully ripe stage compared with fruit protoplasts at the veraison stage , indicating that endocytosis was stimulated during olive-fruit ripening.Because sphingolipids have been reported to be involved in endomembrane trafficking , we analFigure 6). The total LCB content did not change significantly during the cell-expansion stage (stage 2), whereas it increased up at the onset of ripening (stage 3) and then, declined during the last phases of olive-fruit ripening (stage 4 and 5) . The total LCB increase was due essentially to the increase of trihydroxy LCBs . In fact, the fraction of trihydroxy LCBs was the main contributor to the total LCB levels at the five development stages. Therefore, the behavior of trihydroxy LCB content was similar to the trend observed for the total LCB, and the highest peak of accumulation was observed at the onset of fruit ripening (stage 3). However, dihydroxy LCB levels were not altered during olive-fruit development, except in the last phase of fruit ripening , when the lowest levels were measured . Thus, at the onset of olive fruit ripening, the main LCB species showing an augment were trihydroxylated LCBs in their saturated and unsaturated forms (8Z and 8E) . Altogether, these results indicate, first, that the trihydroxy LCBs potentially play a role at the onset of ripening, and second, that levels of sphingolipids must drop to reach fruit ripening.To investigate the possible relationship between sphingolipid composition and fruit development, we analyzed the LCB profiles in the olive fruit during development and ripening. The amount of LCB hydrolyzed from total sphingolipids changed during olive-fruit development (E) > t18:1(8Z) > t18:0 > d18:2 (4E/8Z) > d18:2 (4E/8E) > d18:1(4E) > 1,4-anhydro-t18:1(8E) levels that coincided with a fall in t18:1(8Z)-Glc, t18:1(8E)-Glc, d18:2(4E/8Z)-Glc, and d18:2(4E/8E)-Glc levels . Conversely, in the olive fruit at the fully ripe stage (stage 5), a rise was registered in the t18:1(8Z)-Glc, t18:1(8E)-Glc, d18:2(4E/8Z)-Glc, and d18:2(4E/8E)-Glc levels, while t18:1(8Z), t18:1(8E), t18:0, d18:2(4E/8Z), d18:2(4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E) levels fell in comparison with the fruit at the onset of ripening . Likewise, our data show an increase of d18:0 and d18:1(8E) content in olive fruit at the veraison stage .In particular, at onset of ripening or stage 3, olive fruit showed the highest peak of LCB accumulation with a rise in t18:1. The OeSPT, OeSPHK, and OeGlcCerase genes were differentially expressed throughout the course of olive-fruit development , whereas OeACER gene expression was detected only at the fully ripe stage , when the content of total LCBs (dihydroxy and trihydroxy forms) diminished . The highest OeSPT and OeGlcCerase expression was found in olive fruit at the onset of ripening (stage 3), while the highest OeSPHK and OeACER expression was found in olive fruit at the end of fruit ripening (stage 5) .To determine whether mRNA levels of sphingolipid-metabolism-related genes were differentially regulated during olive-fruit development and ripening, we assessed the expression profiles of OeSPT, OeSPHK, and OeGlcCerase expression increased in olive fruit , suggesting that OeSPT, OeSPHK, and OeGlcCerase genes were up-regulated during pericarp cell expansion and at the onset of ripening. However, from 168 (stage 3) to 182 (stage 4) DPA, OeSPT, and OeGlcCerase expression decreased in olive fruit  while OeSPHK gene expression did not significantly change . Finally, in ripe fruit at 200 DPA (stage 5), OeSPT, OeSPHK, and OeGlcCerase transcript levels rose in comparison with those observed at 182 DPA (H) . Thus, our data indicate that the genes OeSPT, OeSPHK, and OeGlcCerase were positively associated with the pericarp cell expansion and the onset of ripening, while OeACER gene was up-regulated in olive fruit at the late stage of ripening. In particular, OeSPT and OeGlcCerase showed significantly higher expression in association with the onset of ripening and in parallel to the increase of LCB content.From 70 (stage 1) to 168 (stage 3) DPA, Recent research has elucidated many specific aspects concerning the functions of sphingolipids in plants , althougFigure 8).No reports are available on the sphingolipid content and gene expression analysis during fleshy-fruit development. Here, the neutral:polar lipid ratio and sphingolipid content are studied employing dye, fluorescence labeling, and confocal fluorescence microscopy image analysis, as well as the sphingolipid LCB composition and sphingolipid-related gene expression during fruit development and ripening in olive. It is shown that the measured proportion both of polar lipids and sphingolipids in live protoplasts from olive fruits as well as sphingolipid LCB composition and the expression of some genes related to sphingolipid metabolism display significant fluctuations during key points of the stages of olive-fruit development and ripening  of Sphingoid Base Hydroxylase (SBH) genes, exhibiting a complete loss of trihydroxy LCBs, presented reduced growth caused by defects in cell elongation and cell division  and accumulate in the fleshly pericarp. A connection of sphingolipid and glycerolipid metabolism through the fatty acid flux has previously been suggested . This isZ) were found specifically in olive fruit at the stage 2 (cell expansion) and those with d18:1(8Z)-Glc appeared specifically in olive fruit at the stage 5 (fully ripe fruit), suggesting that these sphingolipids may play a specific role during olive-fruit development and ripening. This difference is clear when the relative distribution of the LCBs is compared during olive-fruit ripening. At the onset of ripening, an increase in the t18:1(8E), t18:1(8Z), t18:0, d18:2 (4E/8Z), d18:2 (4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E) levels, and increased OeSPT, OeSPHK, and OeGlcCerase expression parallels a decline of the t18:1(8Z)-Glc, t18:1(8E)-Glc, d18:2(4E/8Z)-Glc, and d18:2(4E/8E)-Glc levels. Conversely, the olive-fruit ripening shows, on the one hand, an increase in the t18:1(8Z)-Glc, t18:1(8E)-Glc, d18:2(4E/8Z)-Glc, and d18:2(4E/8E)-Glc levels and, on the other hand, a significant decrease in the t18:1(8Z), t18:1(8E), t18:0, d18:2(4E/8Z), d18:2(4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E) levels. The data indicate that these LCB species maintain an inverse correlation during olive-fruit ripening, suggesting an antagonistic action of these LCB species during olive-fruit ripening.Because changes in sphingolipid metabolism during fruit development and ripening may be part of a mechanism controlling the transitions from pericarp-cell expansion to ripening, sphingolipid levels should be maintained within strict limits. The onset of ripening, as mentioned before, is associated with strong increase in total LCB content in olive fruit. However, the profiling of sphingolipid LCB measurement revealed a specific association between the individual LCBs and the stages of fruit development and ripening. The sphingolipids containing d18:1(8OeACER expression has been found exclusively in fully ripe olive fruit and showed a specific association with the d18:1(8Z)-Glc accumulation. On the basis of mutant phenotypes, in Arabidopsis, the enzyme AtACER was found to influence turgor pressure within pollen tubes as well as guard cells of siliques  of plant sphingolipids.CI and MP-L performed the experiments. MP and JL sampled the material and contributed to sphingolipid analysis. MG contributed to data analysis. MS-G and MG-R contributed to analyze and discuss the results and prepare the manuscript. MG-J conceived and supervised the study, wrote and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ATP-binding cassette (ABC) reporter family functions to regulate the homeostasis of phospholipids and cholesterol in the central nervous system, as well as peripheral tissues. ABCA7 belongs to the A subfamily of ABC transporters, which shares 54% sequence identity with ABCA1. While ABCA7 is expressed in a variety of tissues/organs, including the brain, recent genome-wide association studies (GWAS) have identified ABCA7 gene variants as susceptibility loci for late-onset Alzheimer’s disease (AD). More important, subsequent genome sequencing analyses have revealed that premature termination codon mutations in ABCA7 are associated with the increased risk for AD. Alzheimer’s disease is a progressive neurodegenerative disease and the most common cause of dementia, where the accumulation and deposition of amyloid-β (Aβ) peptides cleaved from amyloid precursor protein (APP) in the brain trigger the pathogenic cascade of the disease. In consistence with human genetic studies, increasing evidence has demonstrated that ABCA7 deficiency exacerbates Aβ pathology using in vitro and in vivo models. While ABCA7 has been shown to mediate phagocytic activity in macrophages, ABCA7 is also involved in the microglial Aβ clearance pathway. Furthermore, ABCA7 deficiency results in accelerated Aβ production, likely by facilitating endocytosis and/or processing of APP. Taken together, current evidence suggests that ABCA7 loss-of-function contributes to AD-related phenotypes through multiple pathways. A better understanding of the function of ABCA7 beyond lipid metabolism in both physiological and pathological conditions becomes increasingly important to explore AD pathogenesis. APOE is the strongest genetic risk factor for late-onset AD [ABCA7 on chromosome 19p13.3 have also been recognized as novel risk factors for the disease [ABCA7 codes ATP-binding cassette (ABC) transporter A7, which is a member of the A subfamily of (ABC) transporters. Consistently, accumulating in vitro and in vivo studies support the potential contribution of ABCA7 to AD-related phenotypes. Therefore, to explore the pathogenesis of AD, a greater understanding of the role of ABCA7 in physiological and pathological conditions might be important. In this review, we summarize current evidence for the risk of ABCA7 gene variants of AD development and discuss how ABCA7 is involved in the pathogenic pathways of AD.Alzheimer’s disease (AD) is the leading cause of dementia in the elderly, accounting for 60–80% of cases. Approximately 5.5 million individuals are living with Alzheimer’s dementia in the United States. This number is estimated to increase continuously due to the expansion of the aged population . Alzheimonset AD ,14,15,16 disease . ABCA7 cABCA7 with a minor allele frequency (MAF) of more than 5% have been implicated to associate with the risk for AD [ABCA7 intron, as one of the susceptibility loci for late-onset AD  with replication among independent Caucasian cohorts through a genome-wide association study (GWAS) [ABCA7 SNP rs3752246, a missense variant (p.Gly1527Ala), is associated with the risk for late-onset AD  [ABCA7 intron  [ABCA7 rs3764650 has been associated with cortical and hippocampal atrophy in cognitively normal and mild cognitive impairment (MCI) subjects [ABCA7 is possibly responsible for both the development and progression of AD.Common variants of k for AD . In 2011y (GWAS) . Naj et 09–1.21) . FurtherABCA7 rs3764647 (p.His395Arg), located near rs3752246, has been associated with AD risk , while no or a minimal significant association was detected in rs3752246 and rs3764650, respectively [ABCA7 rs115550680 is linked to the development of late-onset AD, in which the effect size  is comparable with that of APOE ε4  [ABCA7 rs142076058 (p.Arg578Alafs) is likely rare in Caucasians, it is relatively common in African Americans and has been identified as an AD risk allele; MAF 15.2% in AD vs. 9.74% in controls  [ABCA7 gene variants are involved in AD risk in both Caucasians and African Americans, there may be ethnic-dependent effects.In an African American cohort, a coding variant of ectively . Another42–3.20) . Thus, wABCA7 have significant associations with the risk for AD. In a Belgian cohort, a low frequency variant, rs78117248, in an ABCA7 intron showed a strong association with AD even after adjustment for the common SNPs, rs3764650, rs4147929, and rs3752246  [ABCA7 missense variant  was also shown to contribute to AD risk in African Americans [ABCA7 using whole genome sequencing and demonstrated that they are associated with AD risk in an Icelandic population; when analyzed by combining those rare “loss-of-function” variants, the OR is calculated to be 2.12 [ABCA7 loss-of-function variants with increased AD risk [ABCA7 loss-of-function variants receive exon skipping or alternative splicing, which likely allows the production of functional proteins and rescues the deleterious effects [ABCA7 to AD risk is mediated by the dysfunction or reduction of ABCA7. Indeed, a common ABCA7 variant, rs3764650, likely influences ABCA7 expression levels in the brain. Whereas ABCA7 mRNA expression is increased in AD brains compared to control individuals, carrying the protective rs3764650 (T) allele is associated with higher ABCA7 expression levels [In addition to the common variants, whole genome sequencing, exome sequencing, and targeted resequencing have also demonstrated that some of the low frequency variants (MAF 1–5%) and rare variants (MAF < 1%) in mericans . On the mericans . Of note AD risk ,33,34,35n levels .ATP-binding cassette (ABC) transporters constitute a superfamily of highly conserved proteins involved in the membrane transport of various substrates, such as ions, amino acids, lipids, and sterols across cell membranes . AbsolutABCA7 and ABCA1 is differently regulated. Although ABCA1 gene expression is upregulated through heterodimerization of the liver-X-receptor (LXR) and the retinoid-X-receptor (RXR) under conditions increasing cellular cholesterol accumulation [ABCA7 mRNA levels [ABCA7 expression is positively regulated by sterol through the SREBP2 pathway, which is oppositely involved in ABCA1 transcription [In spite of their structural homology, the transcription of mulation ,46, cellA levels . The simA levels . Indeed,cription .ABCA7 mRNA is the most abundant in microglia compared to other cell types [Abca7, not only in neurons and microglia, but also in various cell types, including oligodendrocytes, endothelial cells, and astrocytes in the mouse cortex [ABCA7 expression is detected in a variety of tissues/organs, which include brain, lung, adrenal gland, kidney, spleen, thymus, lymph node, testis, keratinocytes, and pancreatic islets, as well as blood cells  ,44,49,50ll types . While ill types ,52, singe cortex .ABCA7 gene is expressed as two variants, full-length cDNA (type I) and the shorter splicing variant cDNA (type II), in a tissue-specific manner. In peripheral tissues, type I ABCA7 expression is predominantly detected in bone marrow, whilst the type II ABCA7 is mainly expressed in the spleen and trachea [ABCA7 splice variants might serve different functions depending on cell types, as well as organs/tissues.Interestingly, the  trachea . In vitr trachea . Thus, aAbca7 deficiency alters the phospholipid profile in the mouse brain [As predicted from the structure, the major function of ABCA7 is likely to regulate lipid metabolism. Serum levels of total cholesterol and high density lipoprotein (HDL) were lower in female ABCA7-knockout (KO) mice compared to wild-type mice under fasting conditions, while there was no significant difference in serum fatty acid levels . Shotgunse brain . In humase brain ,44,56,57se brain . Howeverse brain . Thus, fIn addition to lipid efflux, the forced expression of ABCA7 has been shown to increase the amounts of intracellular/cell surface ceramide and intracellular phosphatidylserine (PS) in HeLa cells, resulting in cell cycle arrest . MoreoveCaenorhabditis elegans, CED-7 is one of the major adhesion molecules mediating the engulfment of apoptotic cells during embryogenesis [ced-7 in mammalians has been predicted to encode ABC transporters from sequence similarity [Abca7 deficient peritoneal macrophages possess an impaired phagocytosis ability compared to those from wild-type mice [Staphylococcus aureus was suppressed by ABCA7 knockdown in J774 cells, reproducing the results using artificial polystyrene beads [In ogenesis . As the milarity , ABCA familarity . Indeed,milarity . While tmilarity ,65. Consype mice . Additione beads . Anotherne beads . Althougne beads . Thus, tne beads , it has While the roles of ABCA7 in lipid metabolism and macrophage-mediated phagocytosis have been actively studied, its function in the central nervous system has received relatively less attention. Nonetheless, a study has shown that ABCA7 deficiency causes slight, but significant, effects on neurobehaviors in young mice . Male ABSeveral groups, including us, have demonstrated the contribution of ABCA7 to Aβ pathology by crossing ABCA7-KO mice with the amyloid AD model J20 , TgCRND8CR1, SPI1, the MS4As, TREM2, CD33, and INPP5D, as well as ABCA7, are involved in AD [As gene network analyses have demonstrated that microglial expressing genes including ed in AD , contribed in AD . Microgled in AD . As disced in AD ,74 and med in AD ,70, ABCAed in AD . Thus, ied in AD , future Aβ is proteolytically cleaved from APP by processing through β- and γ-secretases . Of noteWhile the endocytosis of APP into endosomes is likely an important step for APP processing and Aβ generation , ABCA7 dABCA7 [In addition, ABCA7 deficiency induces endoplasmic reticulum (ER) stress, represented by the activation of the PERK-eIF2α pathway . Since tABCA7 , SREBP2 ABCA7 . InteresABCA7 , which aABCA7 ,83,84.ABCA7 gene variants as susceptibility loci of AD from human genetics studies, a better understanding of the roles of ABCA7 in the central nervous system has been of high significance to explore the pathogenic pathways in AD. As PTC variants in ABCA7 are associated with an increased risk for AD, subsequent studies have proven that ABCA7 deficiency exacerbates brain Aβ accumulation and AD-related phenotype using in vitro and in vivo models. Studies to date have mainly implicated two possible mechanisms whereby ABCA7 loss-of-function contributes to AD pathology; disturbing microglial Aβ clearance and accelerating APP processing. Furthermore, ABCA7 deficiency in microglia may compromise the elimination of diverse brain debris including apoptotic cells during AD progression. It is also possible that ABCA7 deficiency makes brain cells more vulnerable to Aβ toxicity and neuroinflammation in AD (APOE4 and TREM2. These studies could provide us with novel insights to develop effective therapeutic strategies for AD. In addition, since lentivirus-mediated ABCA7 overexpression likely relieves the neurotoxicity of Aβ by promoting cell viability and reducing ER stress [Since the discovery of on in AD . Future R stress , the uprR stress , may be


Manihot esculenta) shows strong tolerance to drought stress; however, the mechanisms underlying this tolerance are poorly understood. Ethylene response factor (ERF) family genes play a crucial role in plants responding to abiotic stress. Currently, less information is known regarding the ERF family in cassava. Herein, 147 ERF genes were characterized from cassava based on the complete genome data, which was further supported by phylogenetic relationship, gene structure, and conserved motif analyses. Transcriptome analysis suggested that most of the MeERF genes have similar expression profiles between W14 and Arg7 during organ development. Comparative expression profiles revealed that the function of MeERFs in drought tolerance may be differentiated in roots and leaves of different genotypes. W14 maintained strong tolerance by activating more MeERF genes in roots compared to Arg7 and SC124, whereas Arg7 and SC124 maintained drought tolerance by inducing more MeERF genes in leaves relative to W14. Expression analyses of the selected MeERF genes showed that most of them are significantly upregulated by osmotic and salt stresses, whereas slightly induced by cold stress. Taken together, this study identified candidate MeERF genes for genetic improvement of abiotic stress tolerance and provided new insights into ERF-mediated cassava tolerance to drought stress.Cassava ( Plants are frequently challenged by unfavorable environmental factors, such as cold, drought, and high salinity in their life cycle, which severely affects their growth, development, yield, and quality15In plants, accumulating evidence has shown that the transcriptional abundance of ERF family genes are regulated by various abiotic stressors and related signaling. A lot of ERF genes exhibited transcriptional changes when they are subjected to various stimuli, including cold, freezing, drought, salt, heat, ABA, and ethylene in various plant species791113141617181921222324Arabidopsis thaliana, 139 in Oryza sativa, 169 in Populus trichocarpa, 103 in Cucumis sativus, 90 in Prunus mume, 200 in Musa acuminata, and 243 in Musa balbisiana5Manihot esculenta), an important tropical crop. Cassava is the third most important crop after rice and maize in Africa, Asia, and Latin America2729To date, numerous ERF family members have been identified in several plant species, such as 122 ERF genes in To identify the ERF family genes from cassava, both Hidden Markov Model and BLAST searches were employed to search the cassava genome with Arabidopsis and rice ERF sequences as queries. Totally, 147 putative ERF members were identified from the cassava genome, which was further confirmed by conserved domain analysis showing that all the identified ERFs have the AP2/ERF domain. The 147 predicted ERF proteins vary from 132 (MeERF71) to 716 (MeERF12) amino acid in length, the relative molecular mass range from 14.7 (MeERF71) to 79.7 (MeERF12) kDa, and the isoelectric points are in the range of 4.51–9.8 .To characterize the evolutionary relationships between ERFs from cassava and other known ERFs from Arabidopsis, an Neighbor-Joining tree was constructed. As shown in MeERFs, intron/exon structure was detected based on the evolutionary relationships  and wild subspecies (W14). Transcriptome analysis showed a transcript abundance of 116 MeERFs in different organs, while the remaining 31 MeERFs were not detected in the RNA-seq libraries  that had high expression levels  in all the tested tissues. Additionally, 15 (12.9%), 23 (19.8%) and 17 (14.7%) genes did not show transcripts in stem, leaf, and tuberous roots, respectively.For Arg7 variety, 20 (17.2%), 21 (18.1%), and 42 (36.2%) genes exhibited high transcriptional abundance  in stem, leaf, and tuberous roots, respectively. Moreover, there were 14 MeERFs  showed high transcriptional levels  in all the tested tissues. Additionally, 16 (13.8%), 19 (16.4%) and 15 (12.9%) genes did not show transcripts in stem, leaf, and tuberous roots, respectively.For W14 subspecies, 27 (23.3%), 23 (19.8%), 44 (46.3%) genes had high transcriptional abundance  in stem, leaf, and tuberous roots, respectively. Moreover, 13 MeERFs in different organs between Arg7 and W14 showed that 76 genes (65.5%) had transcripts abundance in all tissues of Arg7, while 83 genes (71.6%) in W14, suggesting the constitutive expression patterns for these genes. On the contrary, the failure to transcript detection might represent the distinct temporal or spatial expression patterns for the remaining MeERFs. Notably, 9 MeERFs  showed high expression abundance  in all examined organs of Arg7 and W14, implying key roles for these genes in organs development. Overall, most of MeERF genes have similar expression profiles between W14 and Arg7 during organ development. Besides, Some MeERF genes exhibited differential expression profiles. Such as MeERF-32, -15, -26 and -33 had higher transcript abundance  in stem of Arg7, whereas lower in stem of W14. On the contrary, MeERF-146, -72, -8, -11, -25, -3, -86, -134, -45, -42, and -106 had higher transcriptional abundance  in stem of W14, while lower in stem of W Arg7. This phenomenon was also observed in leaves and tuberous roots of Arg7 and W14.Comparative analysis of expression profiles of MeERFs in drought stress response, cassava seedlings of three genotypes were subjected to drought treatment and their leaves and roots tissues were sampled to perform transcriptome analysis , 54 (38.3%) and 29 (20.6%) MeERFs were induced by drought in leaves and roots, respectively, in Arg7 variety; 51 (36.2%) and 22 (15.6%) MeERFs showed to be upregulated after drought treatment in leaves and roots, respectively, in South China 124 (SC124) variety; and 19 (13.5%) and 40 (28.4%) MeERFs were up-regulated by drought in leaves and roots, respectively, in W14 subspecies. These results indicated that the number of MebZIPs up-regulated by drought was greater in Arg7 and SC124 than those in W14. However, the number of drought-induced MeERFs in roots of W14 was significantly more than those in roots of Arg7 and SC124, whereas contrary in the leaves. Also, we found that 7 genes  showed induction in roots of W14, whereas downregulation or no response in roots of Arg7 and SC124 after drought treatment. Six genes  were upregulated in leaves of Arg7 and SC124, whereas downregulated or no response in leaves of W14 after drought treatment. These results suggested that the expression patterns of MeERFs responding to drought were similar between Arg7 and SC124, which differs from W14, and also suggested that the function of MeERFs underlying drought tolerance might be different between cultivated varieties and wild subspecies. In addition, some ERFs that had close relationship displayed different responses to drought stress, such as, MeERF-21/-115, MebERF-39/-40, MeERF-3/-4, and MebERF-147/-128. Together, the expression profiles of MeERFs responding to drought between cultivated varieties and wild subspecies will provide new clues to further investigation underlying mechanisms in cassava tolerance to drought.To get some clues on the function of analysis . AccordiMeERFs to various environmental stresses at transcriptional levels, 8 MeERFs  induced by drought based on transcriptome data in distinct cassava genotypes were chosen for further investigation of their expression patterns after osmotic, salt, and cold treatments. Under osmotic treatment, almost all the tested MeERFs were significantly up-regulated  during the whole treated time points  during the treatment period, whereas MeERF-1, -5, -53, and -95 did not show obvious trends, and were only up-regulated at several time points  analysis. There was a good correlation  between RNA-seq data and the qRT-PCR results . These rERF family in cassava.Although cassava is an important crop, the research progress on cassava is still lagging compared to other crops. Physiologically and morphologically researches have characterized that cassava is a kind of crop that highly resistant to drought and low-fertility2930313217ERF family members were identified and characterized from cassava. Previous studies have identified 26 ERFs in Picea sitchensis, 32 in Pinus taeda, 67 in Selaginella moellendorffii, 90 in Prunus mume, 102 in Sesamum Indicum, 103 in Cucumis sativus, 122 in Arabidopsis thaliana, 139 in Oryza sativa, 169 in Populus trichocarpa, 200 in Musa acuminata, 243 in Musa balbisiana, and 248 in Brassica rape5634ERFs in cassava had expanded in comparison to that in most species, while had shrunk relative to that in Populus trichocarpa, Musa, and Brassica rape. Evolutionary analysis indicated that the cassava ERFs was grouped into 12 subfamilies, which was further supported by analyses of gene structure and conserved motifs , soybean (Glycine max), and tomato (Lycopersicon esculentum) suggested that many ERF family genes showed transcriptional changes after high or low temperature treatments38MeERFs could transcriptionally respond to drought stress in different genotypes (MeERFs significantly induced by drought was greater in roots of W14 (28.4%) than that in roots of Arg7 (20.6%) and SC124 (15.6%), whereas less in leaves of W14 (13.5%) than that in leaves of Arg7 (38.3%) and SC124 (36.2%)  . Due to MeERFs  upregulated by drought based on RNA-seq transcriptome data in different cassava genotypes were selected to examine their expression levels after osmotic, cold and salt treatments  is a drought-induced transcription factor that can regulate drought-responsive gene expressions by binding to the GCC box and/or dehydration-responsive element (DRE) in the promoter of downstream genes3640424346474849MeERFs might be positively involved in drought and salt tolerances of cassava. On the contrary, the expression of ERFs was slightly affected by cold stress, which could be explained by cassava is native to a warm habitat and is categorized as a cold-sensitive species51Further, 8 eatments . The resERF genes from cassava have been characterized based on evolutionary, conserved protein motif, and gene structure analyses, which will supply abundant information for functional characterization of MeERF genes. The expression profiles of MeERFs in distinct organs of two cassava genotypes indicated that MeERF members showed similar or differential expression patterns between Arg7 and W14, thus assisting in understanding the molecular basis for tissue development and function. Transcriptome analysis of three cassava genotypes responding to drought stress revealed that more MeERFs were activated in the roots of W14, whereas in leaves of Arg7 and SC124, which advances the understanding of functional differentiation of MeERFs in adapting to drought between drought-tolerant and drought-sensitive cassava genotypes. Expression of MeERFs under various abiotic treatments indicated the significant responses of MeERFs to osmotic and salt stresses. Together, these data will supply abundant information for functional characterization of MeERF genes and advance the understanding of MeERF-mediated cassava tolerance to abiotic stress.In conclusion, 147 ERF genes in distinct organs. To detect the transcriptional changes of cassava ERF genes in response to drought, leaves and roots were collected from Arg7, SC124 and W14, respectively, under drought conditions for 12 d. For osmotic, cold, or salt treatments, two month old seedlings of Arg7 were suffered from 200 mm mannitol for 14 days, low temperature (4 °C) for 48 h, and 300 mM NaCl for 14 days, respectively. Time point of sampling was determined while phenotypic changes reached on a moderate level of stress . The plants were grown from April to July in 2013 under an environmentally controlled growth room with a 16 h/35 °C day and 8 h/20 °C night regime, and a relative humidity of 70%. Ninety-day-old stems, 90-day-old leaves, and 150-day-old tuberous roots were sampled from Arg7 and W14 under normal conditions to study the expression levels of cassava f stress .Protein sequences of ERFs from cassava, rice, and Arabidopsis were downloaded from Phytozome, RGAP, and UniPort databases, respectively53545759The molecular weight and isoelectric points of MeERFs were predicted by proteomics server ExPASyStems, leaves and tuberous roots of Arg7 and W14 growing on normal condition, and leaves and roots of SC124, Arg7, and W14 under normal conditions and drought treatment were used to isolated total RNA for transcriptome analysis by plant RNeasy extraction kit . Then, cDNA libraries of each sample were constructed with 3 μg of total RNA following the Illumina manufacture protocol, and sequenced by Illumina GAII. Data analysis was carried out by previously described−1) of cDNA in 10 μL solution systems was used for quantitative analysis of gene expression with SYBR Premix Ex Taq (Takara) on a Stratagene Mx3000P Real-Time PCR system. Agarose gel electrophoresis, melting curve, and sequencing analyses were performed to confirm the specificities of primer pairs. The primers of target genes were listed in −△△Ct66According to the method mentioned above, the leaf samples of Arg7 treated with osmotic, cold, or salt stress under different time point were collected to isolate total RNA. First-strand cDNA was synthesized from 1 μg of total RNA using SuperScript reverse transcriptase (Takara). One microliter .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.