In silico calculation of Ca++ coordination bond length and solvent accessibility surface area revealed that the “humanized” R197A substitution alters topology and solvent accessibility of the Ca++ coordination residues of the CRD domain. Binding assays in mouse AMs that were exposed to either endogenous SP-A or hSP-A1 (6A2) and hSP-A2 (1A0) isoforms in vivo revealed that mouse SP-A is a functional hybrid of hSP-A1 and hSP-A2 in regulating SP-A receptor occupancy and binding affinity. Binding assays using neonatal and adult human AMs indicates that the interaction of SP-A1 and SP-A2 with AMs is developmentally regulated. Furthermore, our data indicate that the auxiliary ion coordination loop encompassing the conserved E171 residue may comprise a conserved site of interaction with macrophages, and SP-R210 specifically, that merits further investigation to discern conserved and divergent SP-A functions between species. In summary, our findings support the notion that complex structural adaptation of SP-A regulate conserved and species specific AM functions in vertebrates.Surfactant protein A (SP-A) provides surfactant stability, first line host defense, and lung homeostasis by binding surfactant phospholipids, pathogens, alveolar macrophages (AMs), and epithelial cells. Non-primates express one SP-A protein whereas humans express two: SP-A1 and SP-A2 with core intra- and inter-species differences in the collagen-like domain. Here, we used macrophages and solid phase binding assays to discern structural correlates of rat (r) and human (h) SP-A function. Binding assays using recombinant rSP-A expressed in insect cells showed that lack of proline hydroxylation, truncations of amino-terminal oligomerization domains, and site-directed serine (S) or alanine (A) mutagenesis of cysteine 6 (C6S), glutamate 195 (E195A), and glutamate 171 (E171A) in the carbohydrate recognition domain (CRD) all impaired SP-A binding. Replacement of arginine 197 with alanine found in hSP-A (R197A), however, restored the binding of hydroxyproline-deficient rSP-A to the SP-A receptor SP-R210 similar to native rat and human SP-A.  SP-A belongs to the collectin family of proteins consisting of four structural domains that include an amino-terminal tail, a collagen-like domain (CDM) with Gly-X-Y repeats, an α-helical coiled-coil neck domain with heptad repeats, and a Cain (CRD) –3. The n ligands , 4–7. Th ligands –10. Dele ligands . SP-A bi ligands –21. In h ligands , mycobac ligands , 24, and ligands , 26. The ligands , 26–29.  ligands , 30–32,  ligands , 27, 33 SFTPA1 and SFTPA2  C57BL/6J mice were purchased from JAX labs and bred locally. Transgenic SP-A−/− and humanized mSftpahSFTPA1(6A2)−/−, and mSftpahSFTPA2(1A0)−/−, mice were generated as described previously  and SP-A2 (variant 1A0) were expressed in CHO-K1 cells via the pEE14 expression vectors  consisting of the neck and CRD domain with a mutation at the glycosylation site consensus asparagine 187 (N187S) and lacking the amino-terminal and collagen-like domains (ΔN1-80) using Swiss-Pdb Viewer (++ coordination residues of the CRD carbohydrate-lipid-LPS (CLL) binding pocket, E195A, R197A, N214A, and D215A, and E171A in the auxiliary metal ion coordination loop on Ca++- coordination bond length and solvent accessibility surface area. All residues tested are conserved between rodent and human SP-A, except for R197 which is naturally substituted by an alanine in both human SP-A1 and SPA2.The starting coordinates for molecular dynamics (MD) simulations were obtained from the recombinant rat SP-A crystal structure 1R13  (http://b Viewer . Moleculb Viewer . We test++- coordination residues of rat SP-A and human SP-A2 (variant 1A0) are shown on P08427) (0 (Uniprot Accession No: Q8IWL1). For the CDM alignments numbering is from the start of respective signal sequences  , 73. In  P08427) , here weequences  to avoid125I-labeled SP-A as described in detail previously , 1 mM CaCl2, 0.2 mM MgCl2, 1% bovine serum albumin) and incubated at 37°C in a humidified chamber. Plates were washed in binding buffer, incubated with HRP-conjugated anti-rat affinity purified polyclonal rabbit anti-SP-A antibody for 1 hr. at room temperature, and then washed. Bound antibody was visualized using 3, 3′, 5, 5′-tetramethylbenzidine (TMB) HRP substrate at 450 nM.Macrophage binding assays were performed using mouse or human AMs, or the RAW264.7 macrophage cell line and eviously , 32. Theeviously  was usedmax) and binding affinities (Kd). The hill equation embedded in Prizm software was used to calculate the Hill coefficient of cooperativity for human SP-A binding to murine and human alveolar macrophages.Graphical analyses of binding data were performed with GraphPad Prism software . Binding data were analyzed by non-linear regression of saturation curves fitted to a single site equilibrium binding equation using Prism software to calculate the number of SP-A binding sites/cell (Bhyp) and also exhibits an altered pattern of glycosylation  impaired binding to both macrophages and SP-R210 compared to rSP-Ahyp. Point mutation of cysteine 6 to serine in the amino terminal peptide  diminished Bmax with similar binding affinity to both macrophages and SP-R210 compared to rSP-Ahyp. The partial CDM deletion rSP-Ahyp,ΔG8−G40 and rSP-Ahyp,ΔG40−G80 mutants had increased high affinity binding at reduced Bmax in macrophages compared to rSP-Ahyp. The rSP-Ahyp,ΔG40−G80 mutant, however, exhibited reduced affinity to SP-R210, whereas rSP-Ahyp,ΔN1−G80 bound SP-R210 with increased affinity compared to rSP-Ahyp. The Hughes-Klotz double reciprocal plots  of the CRD α-carbon atoms compared to the NCRD  exhibited increased binding at high concentrations to macrophages compared to rSP-Ahyp  and D215 , two invariant residues that coordinate Ca++, demonstrated critical roles in Ca++-dependent binding and aggregation of DPPC, and binding to alveolar type II epithelial cells  214, D215, and E202 side chains relative to the Ca++ ion  mutagenesis of residues that influence geometry and CaD domain , 5 usingD domain . CompareCa++ ion . The inviary ion , 5. The rSP-Ahyp . The dourSP-Ahyp  of rSP-ArSP-Ahyp , suggesthic data . The aveed to WT , 5. In pal cells . E195 cog curves . The dourophages , whereasrophages . In the Ca++ ion . These cresidues , 5, suggrSP-Ahyp , 2. The  Alanine . The D21ccupancy –5. The a the CDM . The comrom Ca++ . The hydzed” CRD , 5.−/− mice. Binding assays were carried out in AMs from WT (SP-A+/+), SP-A-deficient (SP-A−/−), or SP-A−/− mice carrying either expression of human SP-A genes SFTPA1 (6A2) or SFTPA2 (1A0) via the alveolar epithelial Sftpc promoter  and one for SP-A2 (1A0). The amino-terminal half of the CDM of rat SP-A   . This sud humans . The pred humans , 84. UndSP-A plays versatile roles in host defense through concomitant interactions with pathogens, surfactant lipids, and immune cells . SP-A coex vivo studies but also indicate genetic origin of SP-A preparations as an important variable in understanding versatility of SP-A function in humans. In this regard, the presence of SP-A1 may attenuate receptor occupancy, which likely regulates functional interactions with AMs at physiologically high levels of SP-A at homeostasis. Furthermore, although the present studies are limited by the small numbers of samples, the findings in newborn and adult AMs suggest increased receptor complexity that is developmentally regulated in humans.Human SP-A isolated from alveolar proteinosis fluid has been utilized extensively to discern function utilizing rodent alveolar macrophages without knowledge of the donor SP-A genotype. These studies generated invaluable information but also produced controversial as well as disparate results on SP-A immune functions and identity of SP-A receptors. The present findings in mouse AMs exposed to human SP-A isoforms not only support cross-species conservation and validity of in silico analysis of the R197A mutant in combination with our binding results showing that this species adapted residue modulates binding to the receptor opens exciting questions on future structure-function studies to understand the basis of molecular adaptation of the human SP-A molecules.Finally, our simulation and binding data indicate that the auxiliary ion coordination loop encompassing the conserved E171 residue may comprise a conserved site of interaction with macrophages, and SP-R210 specifically, that merits further investigation to discern conserved and divergent SP-A functions between species. In this context, we recognize that there are limitations inherent to MD simulations that impact the behavior of ions. MD simulations, however, have the potential to unveil how structural differences alter dynamic properties that are crucial for understanding the interplay of structure and function as well as providing guidelines for experiments. The purpose of the simulations, however, was not to reproduce the exact theoretical or experimental ion interactions but instead, to carry out a comparative study of wild type and mutated structures and specifically investigate the distances and their stability. This comparative The raw datasets supporting the conclusions of this manuscript will be made available by the authors upon request.The animal study was reviewed and approved by Institutional Animal Care and Use Committee, Pennsylvania State University College of Medicine. All procedures with isolation of human alveolar macrophages were approved by the Penn State College of Medicine Institutional Review Board.AN acquired and generated figures and tables for all the molecular dynamics simulation data. TU performed all binding assays with hSP-A1 and hSP-A2, isolated macrophages from mouse and lung BAL, and purified human SP-A proteins. C-HY performed all binding assays with recombinant rat SP-A, purified recombinant SP-R210, and cultured cells. PS and NT coordinated the procurement and logistics of donated human lung and contributed to the isolation of human alveolar macrophages. JF provided humanized SP-A1 and SP-A2 mice for the isolation of alveolar macrophages, and edited the manuscript critically. FM produced all recombinant rat SP-A proteins. ZC conceptualized, designed, led the study, and wrote 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 RTECs (NRK-52E cells) were divided into six groups as follows: NG group, HG group, NG + H/R group, HG + H/R group, NG + Pio + H/R group, and HG + Pio + H/R group, among which cells in H/R groups were subjected to 4 h of hypoxia followed by 12 h of reoxygenation. After that, the cells were evaluated using the Cell Counting Kit-8 assay for the determination of their viability and flow cytometry assay for the detection of apoptosis. The levels of superoxide dismutase (SOD), glutathione reductase (GSH), catalase (CAT), and malondialdehyde (MDA) were determined via colorimetric chemical assays. In addition, the expression of ERS-associated proteins, i.e. ATF4, ATF6, GRP78, and CHOP, was determined via western blotting. A HG environment could reduce the viability and increase the apoptotic rate of NRK-52E cells with increased MDA levels and decreased SOD, CAT, and GSH levels, and upregulate the expression of ERS-associated proteins, i.e. ATF4, ATF6, and GRP78. H/R injury could further aggravate changes in the above indicators, but pioglitazone could significantly reverse such changes and alleviate cell injury. Thus, Pioglitazone exhibits a cytoprotective effect on RTECs against H/R injury under NG or HG culture conditions by inhibiting oxidative stress and ERS.Renal ischemia-reperfusion injury is a major cause of acute kidney injury. In the present study, we investigated the effects of pioglitazone on hypoxia/reoxygenation (H/R) injury in rat renal tubular epithelial cells (RTECs) under normal- (NG) or high-glucose (HG) culture conditions  It has NRK-52E cells were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Pioglitazone solution (purity 99%) was purchased from Jiangsu Hengrui Medicine Co., Ltd. (China). Rabbit anti-mouse ATF4, ATF6, GRP78, and CHOP antibodies, as well as β-actin antibody were purchased from Abcam plc. (USA). Superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSH), and malondialdehyde (MDA) assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute (China).2  incubator.NRK-52E cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 1 g/L of D-glucose and 10% newborn calf serum (NBCS) or HG DMEM (containing 4.5 g/L of D-glucose) in 50-mL culture flasks at 37°C in a CORTECs (NRK-52E cells) (four to six passages passages) grown to the logarithmic phase were randomly divided into the following six groups: 1. NG group: Cells cultured in NG medium (containing 1 g/L of D-glucose); 2. HG group: Cells cultured in HG medium (containing 4.5 g/L of D-glucose); 3. NG + H/R group: Cells cultured in NG medium and subjected to H/R treatment ; 4. HG + H/R group: Cells cultured in HG medium for 48 h followed by H/R treatment; 5. NG + Pio + H/R group: Cells cultured in NG medium followed by the addition of pioglitazone solution   1 h pri2 to remove the O2 was equilibrated in a closed container for more than 1 h into a hypoxic solution. After removing the oxygen-containing medium via aspiration, the hypoxic solution was added into the culture flask, which was then hypoxically incubated at 37°C for 4 h in an anaerobic chamber equilibrated with 5% CO2 and 95% N2. A metal catalyzer  was used to maintain a constant low oxygen concentration (< 0.1%) in the chamber. Subsequently, the hypoxic solution was replaced with normal or HG medium and the culture flasks were returned to the incubator for 12 h of re-oxygenation.D-Hanks solution was employed for preparing the hypoxic solution. Briefly, the D-Hanks solution bubbled with N450).The Cell Counting Kit-8 (CCK-8) assay was carried out in accordance with the manufacturer's instructions. Briefly, the cells were inoculated into a 96-well plate and 10 μL of CCK-8 reagent was added to each well, followed by incubation at 37°C for 4 h prior to the measurement of absorbance at a wavelength of 450 nm  each time and centrifuged at 1,500 rpm for 3 min. The resulting cell pellet was re-suspended in 300 µL of pre-chilled 1× binding buffer, and each tube of the cell suspension was mixed gently with 3 µL of Annexin V-FITC and 5 µL of propidium iodide-phycoerythrin (PI-PE), followed by incubation in the dark at room temperature for 10 min. Each tube was subsequently mixed evenly with 200 µL of pre-chilled 1× binding buffer prior to being loaded onto the flow cytometer.via the Coomassie Brilliant Blue assay (Bradford assay). After that, the intracellular levels of SOD, GSH, CAT, and MDA were determined via colorimetric chemical assays according to the manufacturer's instructions.The harvested cells were lysed in a lysis buffer and centrifuged to obtain the protein samples, which were then quantified via the Coomassie Brilliant Blue assay (Bradford assay). The protein samples were loaded at 200 μg per well and separated via electrophoresis in a 10–15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Subsequently, the proteins were transferred onto a nitrocellulose membrane, which was then blocked in Tris Buffered Saline with Tween 20 (TBST) buffer containing 5% skimmed milk powder and incubated overnight at 4°C with the primary antibody. Finally, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody  and imaged using the LAS-3000 Chemiluminescence Image Analyzer for western blotting . The protein bands were quantitatively analyzed using Fujifilm Multi-Gauge software with β-actin serving as the internal control.The harvested cells were lysed in a lysis buffer and centrifuged to obtain the protein samples, which were then quantified P < 0.05 indicates the presence of statistically significant differences.Statistical analyses in this study were performed using GraphPad Prism 5.0 statistical software. All statistical data were expressed as means ± SE. The comparison between groups was carried out using one-way analysis of variance (ANOVA) plus the Tukey post-hoc multiple-comparisons test. The results of the CCK-8 assay showed that the HG group had lower cell viability than the NG group, and there was a statistically significant difference between the two groups. After being subjected to H/R treatment, the viability of cells in both NG and HG groups showed statistically significant declines. However, the viability of H/R-treated cells was significantly improved by pioglitazone treatment  . PreviouIn conclusion, The HG culture conditions aggravated the H/R injury in RTECs, and its underlying mechanism may be associated with oxidative stress and ERS. Pioglitazone can alleviate H/R injury in RTECs under NG and HG conditions by inhibiting oxidative stress and ERS.The datasets generated for this study are available on request to the corresponding author.The authors contributed in the following way: CZ, ZZ, YT, WW, TC and HH: acquisition, analysis, and interpretation of data, and drafting the paper. CZ and HH: study conception and design, analysis and interpretation of data, and drafting the paper.This study was funded by grants from the National Natural Science Foundation of China (No. 81560311). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cutaneous squamous cell carcinoma (SCC) is a common cancer that significantly decreases the quality of life. It is known that external stimulus such as ultraviolet (UV) radiation induces cutaneous SCC via provoking oxidative stress. NAD(P)H dehydrogenase 1 (NQO1) is a ubiquitous flavoenzyme that functions as a guardian against oxidative stress. However, the effect of NQO1 on cutaneous SCC is not clearly elucidated. In this study, we investigated the effect of NQO1 on cutaneous SCC cells using the recombinant adenoviruses that can upregulate and/or downregulate NQO1 expression. Overexpression of NQO1 resulted in significant decrease of cell proliferation and colony forming activity of SCC lines (SCC12 and SCC13 cells). By contrast, knockdown of NQO1 increased the cell proliferation and colony forming activity. Accordingly, the levels of proliferation-related regulators, such as Cyclin D1, Cyclin E, PCNA, SOX2, and p63, were decreased by the overexpression of NQO1, while those were increased by knockdown of NQO1. In addition, NQO1 affected the invasion and migration of SCC cells in a very similar way, with the regulation of epithelial-mesenchymal transition- (EMT-) related molecules, including E-cadherin, N-cadherin, Vimentin, Snail, and Slug. Finally, the overexpression of NQO1 decreased the level of phosphorylated AKT, JNK, and p38 MAPK, while the knockdown of NQO1 increased the level of phosphorylated signaling molecules. Based on these data, NQO1 has tumor suppressive function in cutaneous SCC cells. It is the second most frequent type among the nonmelanoma skin cancers, influencing the quality of life considerably , 2. Manyeous SCC –9.NAD(P)H dehydrogenase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes the two-electron reduction of quinones to hydroquinones using NAD(P)H as an electron donor , 11. NQOAs the development and progression of cutaneous SCC are closely related to the UV-induced oxidative stress, we speculate that NQO1 may have roles in SCC. In this study, we demonstrate the tumor suppressive function of NQO1 in cutaneous SCC cells.2O2 for 10 min at room temperature, and blocked in 0.1% Tween-20 (v/v) and 1% bovine serum albumin (BSA) in PBS (w/v) for 30 min, and this was followed by reaction with anti-NQO1 antibody  for 1 h. Sections were incubated sequentially with peroxidase-conjugated secondary antibody (Dako) and visualized with a ChemMate EnVision detection kit (cat# K5007) (Dako).Skin tissues were fixed in 10% (v/v) formaldehyde and embedded in paraffin. The paraffin-embedded sections of skin specimens were dewaxed, rehydrated, and washed three times with phosphate-buffered saline (PBS). Sections were then incubated with proteinase K  for 5 min at 37°C, treated with HSCC12 and SCC13 cells are the human squamous cell carcinoma line, established from SCCs of the facial epidermis . Both thThe NQO1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR). Briefly, total RNA was isolated using the Easy-blue RNA extraction kit . Two micrograms of total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase . An aliquot of the RT mixture was subjected to PCR with the primer set for NQO1 (5′-ATGGTCGGCAGAAGAGCACTGA and 5′-CAGATCAAAGCTAGAAAATGA). The amplified full-length NQO1 cDNA was subcloned into the pENT/CMV vector, and replication-incompetent adenoviruses were created .For knockdown experiments, we prepared recombinant adenoviruses expressing a microRNA targeting NQO1. The target sequences for NQO1 were designed using BLOCK-iT™ RNAi Designer . The double-stranded DNA oligonucleotides were synthesized and cloned into the parental vector pcDNA6.2-GW/EmGFP-miR (Thermo Scientific). The expression cassette for microRNA was inserted into the pENT/CMV vector, and then the adenovirus was prepared using the method described above. The microRNA sequences are as follows: top strand 5′- TGCTGTTCAGTTTACCTGTGATGTCCGTTTTGGCCACTGACTGACGGACATCAGGTAAACTGAA, bottom strand 5′-CCTGTTCAGTTTACCTGATGTCCGTCAGTCAGTGGCCAAAACGGACATCACAGGTAAACTGAAC.β-Actin (cat# SC-47778) ; Cyclin D1 (cat# 2978S), Cyclin E1 (cat# 4129S), SOX2 (cat# 2748S), E-cadherin (cat# 3195S), N-cadherin (cat# 4061S), Vimentin (cat# 5741S), Snail (cat# 3895S), p-JNK (cat# 4668S), p-AKT (cat# 4060), and p-p38 (cat# 9216S) ; and p63 (cat# ab53039) and Slug (cat# ab27568) .Cells were lysed in PRO-PREP solution (Intron). Total protein concentrations were measured using a BCA protein assay kit . Samples were run onto SDS-polyacrylamide gels and transferred onto nitrocellulose membranes . After blocking with 5% skim milk (w/v), the membranes were incubated with primary antibodies. Blots were then incubated with peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence (Intron). The following primary antibodies were used: PCNA (cat# SC-7907), NQO1 (cat# SC-32793), p-ERK (cat# SC-7383), and 4 cells were reseeded on each 100 mm culture dishes. At the indicated time points, cells were trypsinized and cell numbers were counted using hemocytometer.For cell growth assay, SCC12 and SCC13 cells were transduced with 10 multiplicity of infection (MOI) of adenovirus for overnight. Cells were replenished with fresh medium and incubated for a further 2 d. Cells were then trypsinized and 1 × 103 cells were reseeded on each 100 mm culture dishes. Cells were grown for 2–3 weeks and stained with crystal violet .For colony forming assay, 1 × 10Invasion assay was performed using the Chemicon Cell Invasion Assay Kit (cat# ECM550) . Briefly, after transduction with adenovirus, cell suspension was prepared in serum-free medium and added to upper chamber. Lower chamber received 5% FBS containing medium. After incubation for 48–72 h, noninvading cells and ECMatrix were removed using cotton-tipped swabs. Invading cells were visualized by staining solution.μg/ml) for 2 h. After changing the medium supplemented with 5% FBS, thin wound was introduced by scratching with a pipette tip. Cell migration was evaluated using ImageJ program. Wound closure was determined by calculating the proportion of wound size at 12 h to initial wound.After adenoviral transduction, cells were cultured for 2 d in growth medium. Cells were then replenished with serum-free medium, incubated for 6–8 h, and treated with mitomycin C  and incubated with ROS Deep Red dye (cat# ab186029) (Abcam) for 1 h. Cells were then observed under the fluorescent microscopy.t-test using SPSS software v 22.0 . Statistical significance was set at p < 0.05.Data were evaluated statistically by one-way ANOVA or Student's We examined the expression level of NQO1 by immunohistochemistry in the normal and SCC lesional area obtained from the same patient. NQO1 immunoreactivity was observed in the epidermis (red arrows) and vessels (red asterisks) of normal region of SCC patient. By contrast, NQO1 was barely detected (blue arrows) or partially detected (red arrows) in the lesional area of SCC. NQO1 immunoreactivity was also observed in immune cells surrounding SCC lesion (red arrowheads) . In cultTo investigate the potential role of NQO1 in SCC cells, we created the recombinant adenovirus expressing NQO1 (Ad/NQO1). We also created the recombinant adenovirus expressing a microRNA targeting NQO1 (Ad/miR-NQO1) to downregulate its expression. After transduction with the Ad/NQO1, NQO1 was expressed at high level in SCC cells compared with the control adenovirus (Ad/LacZ)-treated group. By contrast, the level of the NQO1 protein was markedly decreased after the transduction of Ad/miR-NQO1 compared to cells transduced with the control adenovirus (Ad/miR-Scr) . We thenWe assessed whether NQO1 affected the cell proliferation-related regulators. The overexpression of NQO1 significantly decreased the level of several regulators, such as Cyclin D1, Cyclin E, PCNA, SOX2, and p63. By contrast, miR-mediated downregulation of NQO1 increased the level of cell proliferation-related regulators .As the invasive growth and migration are the important manifestations of tumor progression, we next investigated whether NQO1 affected those characteristics of SCC cells. The overexpression of NQO1 significantly reduced the invasion of SCC cells, while the knockdown of NQO1 increased the invasion of SCC cells . SimilarSeveral signaling pathways are implicated in EMT. For example, AKT and mitogen-activated protein kinase (MAPK) pathways are involved in EMT of esophageal SCC . Thus, wTo investigate the putative mechanism underlying NQO1-induced tumor suppressive effect, we determined the cellular ROS level after the overexpression or knockdown of NQO1. When NQO1 was overexpressed, cellular ROS level was markedly decreased. By contrast, the knockdown of NQO1 resulted in significant increase of ROS level . These rμM. Consistent with these data, dicoumarol increased the colony forming activity  knockout mice is mediated through the activation of AKT pathway [β1-induced fascin1 facilitates the migration and invasion of kidney carcinoma cells through ERK and JNK signaling pathways [The potential role of AKT in SCC has been demonstrated several times in other systems. For example, codeletion of p53 and n of AKT . In othen of AKT . Finally pathway . Similar pathway . Other epathways . In thisIn summary, we demonstrate that NQO1 has tumor suppressive function in cutaneous SCC cells. Our results provide new insights on which we base further investigations of the molecular events underlying SCC development.


Massive occurrences of interstitial loss of heterozygosity (LOH) likely resulting from gene conversions were found by us in different cancers as a type of single-nucleotide variations (SNVs), comparable in abundance to the commonly investigated gain of heterozygosity (GOH) type of SNVs, raising the question of the relationships between these two opposing types of cancer mutations.In the present study, SNVs in 12 tetra sample and 17 trio sample sets from four cancer types along with copy number variations (CNVs) were analyzed by AluScan sequencing, comparing tumor with white blood cells as well as tissues vicinal to the tumor. Four published “nontumor”-tumor metastasis trios and 246 pan-cancer pairs analyzed by whole-genome sequencing (WGS) and 67 trios by whole-exome sequencing (WES) were also examined.Widespread GOHs enriched with CG-to-TG changes and associated with nearby CNVs and LOHs enriched with TG-to-CG changes were observed. Occurrences of GOH were 1.9-fold higher than LOH in “nontumor” tissues more than 2 cm away from the tumors, and a majority of these GOHs and LOHs were reversed in “paratumor” tissues within 2 cm of the tumors, forming forward-reverse mutation cycles where the revertant LOHs displayed strong lineage effects that pointed to a sequential instead of parallel development from “nontumor” to “paratumor” and onto tumor cells, which was also supported by the relative frequencies of 26 distinct classes of CNVs between these three types of cell populations.These findings suggest that developing cancer cells undergo sequential changes that enable the “nontumor” cells to acquire a wide range of forward mutations including ones that are essential for oncogenicity, followed by revertant mutations in the “paratumor” cells to avoid growth retardation by excessive mutation load. Such utilization of forward-reverse mutation cycles as an adaptive mechanism was also observed in cultured HeLa cells upon successive replatings. An understanding of forward-reverse mutation cycles in cancer development could provide a genomic basis for improved early diagnosis, staging, and treatment of cancers.The online version of this article (10.1186/s40246-018-0170-6) contains supplementary material, which is available to authorized users. The progressive development of cancer has been investigated extensively at the cytochemical and genetic levels, leading to the recognition of early premalignant stages characterized by precancerous changes in DNA sequence, gene expression, protein structure, and microscopic rearrangement –14. GenoWhile forward mutations converting wildtype sequences into mutant forms and reverse mutations restoring the wildtype sequences from the mutant forms have been compared in microbial studies regarding their differential sensitivities to various mutagens –20, suchAccordingly, in the present study, “nontumor” tissue isolated at > 2 cm from the tumor, “paratumor” tissue isolated at ≤ 2 cm from the tumor, and tumor from different types of cancers were compared with same-patient white blood cell controls based on massively parallel sequencing. Somatic mutations in both directions, i.e., GOH and LOH types of SNVs and CNV gains and losses, were examined residue-by-residue and window-by-window in order to detect the presence of mutation reversals during the development of cancer cells and to assess their biological significance. The results obtained from both clinical cancer samples and cultured HeLa cells indicated that forward-reverse (FR) mutations together with directional selection constitute important determinants of the mutation profiles of stage-specific cell populations in cancer development.Tumor purity in all B-N-P-T tetra and B-N-T trio samples was estimated using VarScan software  and “absFor histological and immunohistochemical staining  propane sulfonic acid (MOPS) from Sigma-Aldrich, USA. Cultures were incubated at 37 °C in a humidified environment containing 5% CO2. To start the HeLa cell culture, frozen cells were thawed and plated at a density of approximately 4 × 105 cells per Petri dish and allowed to reach confluence whereupon they were harvested by treatment with trypsin-EDTA , and 105 cells were replated every 2 to 3 days on fresh Petri dishes at intervals of 2 to 3 days. Genomic DNA was isolated from the cells harvested on days 1, 3, 5, 8, 10, 12, 14, 16, 21, 25, 27, and 29 by extraction with TES , and centrifuged at 12,000 rpm for 10 min at room temperature. The supernatant was transferred to a new vial and precipitated with an equal volume of ethanol. These successive HeLa cell DNA samplings were subjected to AluScan sequencing.HeLa cell line was obtained from American Type Culture Collection . Cells were cultivated in DMEM media supplemented with 10% heat-inactivated fetal bovine serum , and medium pH was adjusted to 7.4 by 3-(www.bioline.com), 3 mM MgCl2, 0.15 mM dNTP mix, 1 unit Taq polymerase, 0.1 μg DNA sample, and 0.075 μM each of the four following Alu consensus sequence-based PCR primers:AluY278T18 (5′-GAGCGAGACTCCGTCTCA-3′);AluY66H21 (5′-TGGTCTCGATCTCCTGACCTC-3′);R12A/267 (5′-AGCGAGACTCCG-3′);L12A/8 (5′-TGAGCCACCGCG-3′).AluScans of genomic regions flanked by Alu repetitive sequences were obtained by means of inter-Alu PCR as described , 24, empPCR was carried out at 95 °C, 5 min for DNA denaturation, followed by 30 cycles each of 30 s at 95 °C, 30 s at 50 °C, and 5 min at 72 °C, plus finally another 7 min at 72 °C. Amplicons were purified with ethanol precipitation, sequenced on the Illumina HiSeq platform at Beijing Genomics Institute  and mapped to the hg19 reference human genome for downstream bioinformatic analysis.https://www.synapse.org/#!Synapse:syn2887117). These included the Pilot-63 set and 86 hepatocellular carcinoma (LIHC), 75 non-small-cell lung cancer (NSCLC), and 22 intrahepatic cholangiocarcinoma (ICC) cases with information accessible through the ICGC Data Portal (https://dcc.icgc.org). In addition, raw WGS data generated by Ouyang et al. [Whole-genome sequencing (WGS) data generated from tumor-blood paired samples with the Illumina system by the International Cancer Genome Consortium (ICGC) and TCGA were downloaded in bam format with permission  , SAMtoolFor cancer cases with more than two samples from each patient, i.e., the B-N-P-T tetra sample set of 12 cases, the B-N-T trio sample set of 17 cases, and the N-T-M trio sample set of 23 cases, the abovementioned calling of SNVs was first performed for each sample of each case in the multiple sample sets. For each of the multiple-sample cases, only nucleotide positions conformed to all the above SNV calling criteria in every sample of the same patient were included in further analysis. Sites not covered in the further analysis were arising from either lack of sequencing reads or failure to meet the filtering criteria in any one of the samples of the same patient.https://github.com/hutaobo/ProfilePlots).Mutational profiles of SNVs were analyzed following the procedure developed by Alexandrov et al. . For eacFrom AluScan data, the AluScanCNV software  was emplhttp://grch37-cancer.sanger.ac.uk/cosmic/download). The human genome was divided into tandem 2000 bp windows, and the average densities of CNVT breakpoints and base pairs in CpGe or MeMRE in each window were calculated. Thereupon, the windows with zero CpGe or MeMRE density were removed to avoid error caused by missing data, and the remaining windows were separated into ten groups based on the percentile of CpGe or MeMRE density. Finally, the average CNVT breakpoint densities in the groups were plotted against the percentile CpGe or MeMRE density.CpGe and MeMRE entries were downloaded from UCSC Genome Browser as described , and somgrch37.ensembl.org” host. The resultant gene list was uploaded to DAVID Bioinformatics Resources 6.7 [p value, Benjamini-corrected p value, and FDR q value all less than 0.05 were considered statistically significant.The results of variant analysis of AluScan data of the 12 tetra sample cases were uploaded to BioMart of the Ensembl database to generate a list of their gene contents under R environment using the “biomaRt” R package . For thehttp://www.r-project.org). The significance probability (p) values were calculated by the two-tailed t test or chi-square test functions in R, and the Pearson correlation coefficients (r) were calculated by the cor function in R. Figures were drawn using the ggplot2, lattice, or ellipse package under R environment, except for Fig. Statistical analyses were performed using R software (White blood cells (B), tumor tissue (T), paratumor tissue (P) immediately adjacent to the tumor, and more remote nontumor tissue (N) were collected in 12 same-patient tetra sample cases consisted of four breast carcinomas (BRCA), five stomach adenocarcinomas (STAD), and three hepatocellular carcinomas (LIHC) . Reversals of N-stage SNVs and CNVs (viz. sum of types IVa and IVb patterns) were common, amounting to ~ 70% of N-stage SNVs or ~ 40% of N-stage CNVs, and far more of such reversals took place in P-stage (type IVa) than in T-stage (type IVb), see Additional files Figure When another 17 B-N-T trio sample sets consisted of 1 BRCA, 2 LIHC, and 14 non-small cell lung cancers (NSCLC) were analyzed with respect to the GOH and LOH changes in the N- and T-stage cells relative to B-stage cells  among the ∆TN changes, the LOH/GOH-M ratios of 2.5 for the WGS-based samples Fig. , and 2.1LOH > RGOH-m > RGOH-M, with LOH rate being the highest. The massive interstitial LOH rates observed earlier based on AluScan data were thus confirmed by the ICGC Pilot-63 WGS dataset.A total of 246 tumor-control pairs from the International Cancer Genome Consortium (ICGC) collection of WGS data  were anaFor the ∆TB SNVs of Pilot-63 WGS dataset, Fig. Moreover, for the B-N-P-T tetra samples, 95.5% of the forward LOHs in the B-to-N transition —comprising six different orders, where a U status occurred in two of the N-, P-, and T-stages.II.Class II (U ≤ 1)—comprising eight different orders, where the U status occurred in no more than one of the N-, P-, and T-stages.III.Class III (disadvantaged)—comprising 12 different orders, where 10 of them (viz. outside of LUG and GUL) included an abrupt double-dose change directly from G to L, or L to G in the order.In the B-N-P-T tetra sample cases, the status of any CNV in the N-, P-, and T-stages could be CN-unaltered (U), CN-gain (G), or CN-loss (L) relative to its status in B-stage. Arranging in serial order, the CN-status found in the N-P-T stages  of GUG/GUL and LUL/LUG greatly exceeded unity (marked by dashed red line), yielding significant lineage effects of p < 10−16 for all sizes of sequence windows ranging from 50 to 500 kb.The double-dose disadvantage explained the low frequencies of GLU, LGU, UGL, ULG, LGL, GLG, LGG, GGL, GLL, and LLG, but not the low frequencies of LUG and GUL which fell into class III even though they did not incur any double-dose copy number changes, in contrast to GUG and LUL which belonged to the more abundant class II. The contrast indicates that lineage effects were important not only to LOH partitions Figs. , and 5b Residue-by-residue tracing of these mutations through the B-, N-, P-, and T-stages pointed to much higher LOH rates and GOH-m rates than GOH-M rates Fig. , in accoThe diminishing LOH-M/LOH-m ratios from L13/L14 = 5.7 to L15/L16 = 2.9 and on to L21/L22 = 1.7, again also observable with both aggregate Fig.  and indiWhile the steeply unequal CNV frequencies between GUG and GUL and between LUL and LUG Fig.  were agaPremalignant and precancer cells have been observed in a variety of cancers –14. The Accordingly, these convergent lines of evidence derived from the B-N-P-T tetra samples, and also supported by those from the B-N-T trio samples Fig. , the N-TσN) comprised different proportions of GOHs (red) and LOHs (orange) and underwent substantial reversions in P-stage cells (light green), followed by a much smaller number of additional reversions occurring in the T-stage cells (dark green). Altogether, that more N-stage SNVs were reversed in P-stage than in T-stage in all 12 samples amounted to a highly non-random observation (p = 3.1 × 10−12), in confirmation of the stage-specific difference in mutational activities between P- and T-stage cells. The LOH/GOH ratios in tumor tissues relative to same-patient white blood cell samples as controls were different for different cancer types: 0.32 for BRCA, 1.16 for STAD, and 4.29 for LIHC  or 36-fold (p < 0.0001) observed in normal kidney cortices of the subjects that were smokers or exposed to the environmental carcinogen aristolochic acid, respectively [Earlier, comparison of different cancer-control pairs indicated that the LOH rates were greater than GOH-m rates, which were in turn greater than GOH-M rates in different types of cancers, pointing to widespread interhomolog chromosomal gene conversions arising from defective DNA double-strand break repair in cancers to cause massive LOHs and tag-along GOHs . In addiectively .The necessity of mutation load reduction in cancer development was also consistent with the slower multiplication of cultured transformed cells than untransformed cells at low population density, e.g., upon transformation of a C3H/10T1/2CL8 fibroblast cell line derived from C3H mouse embryos by 3-methylcholanthrene, the transformed cells exhibited a saturation density 2–3 times that of untransformed cells, but generation times of 22 and 27 h, viz. 40–70% longer than the 15.5 h for the untransformed cells . LikewisDrosophila melanogaster population induced by extreme starvation was allowed to readapt to the ancestral culture environment, reversions of SNPs back to ancestral allele genotypes over 50 generations of evolution amounted to about 50% [The pronounced reversions of N-stage SNVs in P-stage, P-stage SNVs in T-stage, and T-stage SNVs in M-stage Figs. , and 5b bout 50% , comparaFRMD4A gene [CAGE1 for cancer antigen 1 [The D4A gene  was mutantigen 1  was mutaIn conclusion, the occurrences of a wave of forward mutations followed by their reversals are observed in both cancer development samples and serial samples of cultured HeLa cells. Because cancers are driven by mutations, the nature of the mutations in the evolving cancer cells furnishes an appropriate basis for delineating the major stages of carcinogenesis. In the present study, the mutational profiles of the cell populations in the N-, P-, and T-stage samples showed that N-stage cells surprisingly harbored large numbers of SNV mutations, more GOHs than LOHs, which were enriched with NCG>NTG type of GOHs with associated CNVs. The P-stage cells displayed, relative to N-stage cells, more LOHs than GOHs. A major fraction of their LOHs represented reversals of the forward GOH mutations found in N-stage and was enriched with NTG>NCG type of LOHs with associated CNVs. In the T-stage cells, the ratio between LOHs and GOHs was even higher than P-stage cells Additional file 2:Table S2. Tumor purities of B-N-P-T tetra and B-N-T trio samples estimated by absCN-seq. (XLSX 10 kb)Additional file 3:Table S3. Summary of SNV mutations in B-N-P-T tetra samples. (XLSX 14 kb)Additional file 4:Table S4. The exact residue-by-residue SNV mutations in each sample of the B-N-P-T tetra sample cases. (XLSX 652 kb)Additional file 5:Figure S1. Total numbers of different dinucleotide sites in the human genome. Numbers of CG as well as other 15 types of dinucleotides in human reference genome hg19 are plotted out. (PDF 100 kb)Additional file 6:Table S5. Summary of CNV mutations in B-N-P-T tetra samples. (XLSX 11 kb)Additional file 7:Table S6. Summary of SNV mutations in B-N-T trio samples. (XLSX 14 kb)Additional file 8:Table S7. The exact residue-by-residue SNV mutations in each sample of the B-N-T trio sample cases. (XLSX 3018 kb)Additional file 9:Table S8. Summary of SNV mutations in N-T-M trio samples. (A) AluScan, (B) WES-Non-Lung, (C) WES-NSCLC-L, (D) WES-NSCLC-H, and (E) WGS-Liver-M. (XLSX 48 kb)Additional file 10:Table S9. The exact residue-by-residue SNV mutations in each sample of the AluScan N-T-M trio sample cases. (XLSX 214 kb)Additional file 11:Figure S2. SNV mutations in the AluScan-capturable regions of WGS samples in the WGS-Liver-M group. In this figure, AluScan-capturable sequences, corresponding to all the AluScan-captured sequences analyzed in Fig. a Genotypic changes in T-stage and M-stage cells. The numbers of genotypic changes in T- or M-stage sequences relative to N-stage sequences are represented by ΔTN and ΔMN, respectively. b Patch diagrams tracing SNVs between the N-, T-, and M-samples. c Mutational profiles for the ∆TN and ∆MT SNV changes as numerically indicated in the patch diagrams in part b. In each vertical bar in the ∆TN tier, the solid segment represents the SNVs that were reversed in the ∆MT tier, whereas the open segment indicates the unreversed SNVs. (PDF 1230 kb)Additional file 12:Table S10. Numbers of GOHs and LOHs in 22 ICC, 86 LIHC, and 75 NSCLC samples. (XLSX 14 kb)Additional file 13:Figure S3. Mutation-rate diagrams of Pilot-63 samples from ICGC analyzed by WGS. a Mutation-rate diagrams for GOHs. Each of the ten diagrams of triplet duplexes corresponds to a context group, labeled 1–10 as in Fig. b Mutation-rate diagrams for LOHs. The arrows employed are similar to those in part a. All arrows in parts a and b are shown as dashed lines for transitions (TSs) or solid lines for transversions (TVs). In the ten diagrams in part a or part b, the boxed TS/TV ratio given for each diagram represents the ratio pertaining to all the TS and TV mutations in the diagram, e.g., in diagram 1 of part a, TS equals the sum of the four TS rates in the diagram, and TV the sum of the eight TV rates, yielding TS/TV = 1430/2804 = 0.51. The different rates in the diagrams in parts a and b are color-coded as in Fig. Additional file 14:Figure S4. Most of the LOHs observed in the course of cancer development occurred in copy-neutral regions of the genome. a Upper panel: reverse LOHs occurring in the N-to-P transition (viz. ∆PN). Pie chart indicates that 1850 out of 1875 (98.7%) of the reverse LOHs via L1 and L8 steps analyzed in Fig. b Upper panel: reverse LOHs occurring in the P-to-T changes (viz. ∆TP). Pie chart indicates that 1095 out of 1150 (95.2%) of the reverse LOHs via L3 and L10 steps occurred in copy-neutral regions. c Upper panel: forward LOHs occurring in the B-to-N transition (viz. ∆NB). Pie chart indicates that 986 out of 1028 (95.9%) of the forward LOHs via L13 and L14 steps occurred in copy-neutral regions. In parts a–c, the lower panels show for reference the proportions of CN-neutral, CN-gain, and CN-loss in the course of the B-to-N, N-to-P, and P-to-T transitions, respectively. (PDF 853 kb)Additional file 15:Table S11. The exact window-by-window CNV mutations in each sample of the B-N-P-T tetra sample cases. (XLSX 783 kb)Additional file 16:Table S12. List of genes harboring SNV mutations in B-N-P-T tetra sample cases. (XLSX 317 kb)Additional file 17:Table S13. SNV mutations enriched pathways and genes in B-N-P-T tetra sample cases. (XLSX 109 kb)


High efficient electrocatalytic activity and strong stability to both oxygen reduction reaction (ORR) and oxygen evolution (OER) are very critical to rechargeable Zn-air battery and other renewable energy technologies. As a class of promising catalysts, the nanocoposites of transition metal nanoparticles that are encapsulated with nitrogen-doped carbon nanoshells are considered as promising substitutes to expensive precious metal based catalysts. In this work, we demonstrate the successful preparation of high-density cobalt nanoparticles encapsulated in very thin N-doped carbon nanoshells by the pyrolysis of solid state cyclen-Co-dicyandiamide complex. The morphologies and properties of products can be conveniently tuned by adjusting the pyrolysis temperature. Owing to the synergetic effect of hybrid nanostructure, the optimized Co@N-C-800 sample possesses outstanding bifunctional activity for both ORR and OER in alkaline electrolyte. Meanwhile, the corresponding rechargeable zinc-air battery that is based on Co@N-C-800 air cathode also has excellent current density, low charge-discharge voltage gap, high power density, and strong cycle stability, making it a suitable alternative to take the place of precious metal catalysts for practical utilization. The growing environmental degradation and the increasing requirements for renewable energy have stimulated great interest in exploring sustainable energy storage and conversion devices from hydrogen production by water splitting to metal-air battery . Among v2 in form of the parallel diatomic adsorption to improve electrocatalytic activity [Currently, the noble metals with the high catalyst activity, good conductivity, and favorable mechanical properties are used as the state-of-the-art benchmark catalysts for ORR and OER. Unfortunately, a good catalyst for ORR usually has poor activity toward OER and vice versa. For example, Pt-based catalysts exhibit excellent ORR activity, while Ir (or Ru)-based catalysts show remarkable OER activity ,10. Furtactivity ,27; and,activity ,28. Althactivity , the laractivity ,30,31. C2 catalyst. Moreover, the rechargeable zinc-air battery employing the optimal Co@N-C-800 catalyst as the air electrode exhibited high performance and cycling stability when compared to other counterparts.Recent studies prove that various solid phase precursor can be used to synthesize M@N-C materials by the facile pyrolysis process. This synthesis strategy has many advantages, such as simple equipment and process, high yield, and convenience of N doping in carbon matrix, making it a prospective method to the large scale commercialization. Herein, we demonstrate that cobalt nanoparticles encapsulated in N-doped carbon nanoshells (named as Co@N-C) as efficient and durable bifunctional electrocatalysts have been prepared by a convenient pyrolysis route of the solid-phase cyclen-Co-dicyandiamide complex precursor. The structure and morphology of prepared samples exhibited strong dependence on pyrolysis temperature. With an increased temperature from 700 to 900 °C, the morphology of carbon changed from carbon nanoshells to carbon nanotubes. Due to the synergistic effect of nitrogen-doping and Co encapsulating, the optimized Co@N-C-800 catalyst displayed outstanding bifunctional activities for ORR and OER in alkaline solution, which is comparable to commercial Pt/C and IrO3)2∙6H2O), 1,4,7,10-tetraazacyclododecane , dicyandiamide (C2H4N4), perchloric acid (HClO4), potassium hydroxide, isopropanol, and ethanol are analytical grade without further purification. Distilled water was used throughout.Cobaltous nitrate hexahydrate (Co(NO3)2∙6H2O (145 mg) was completely dissolved in 10 mL distilled water by ultrasonic and then slowly dropped into above 1,4,7,10-tetraazacyclododecane solution with constant stirring for 12 h. After that, dicyandiamide (3 g) was first dispersed in hot distilled water (60 °C) and also dropped into the above mixture solution with constantly stirring for another 6 h. After being thoroughly dried, the obtained powder was calcinated at 800 °C for 1 h . To remove unstable cobalt species, the powder was then immersed in 80 °C HClO4 solution  for 8 h. Finally, the black powder was thoroughly cleaned with distilled water and ethanol, dried at 60 °C in vacuum, and marked as Co@N-C-800, in which “800” represents the pyrolysis temperature. To study the influence of different pyrolysis temperature on the morphology and electrochemical performance of Co@N-C hybrid catalysts, the Co@N-C-700 and Co@N-C-900 samples were also synthesized by the same process.Typically, 1,4,7,10-tetraazacyclododecane (172 mg) was diffused in 10 mL distilled water. Subsequently, Co(NO−1).The products were characterized by a Rigaku/Max-3A diffractometer with Co Ka radiation , a scanning electron microscope  equipped with an energy dispersive X-ray spectrometer , a transmission electron microscope , and an X-ray photoelectron spectrometer . The thermo gravimetric (TG) was operated on a Thermo Gravimetric Analyzer  from 25 °C to 800 °C in air , and silver-silver chloride electrode (Ag/AgCl) were served as the counter electrode, working electrode, and reference electrode, respectively. The potentials were normalized to the reversible hydrogen electrode (RHE) by using the following equation [All electrochemical measurements were recorded by an electrochemical workstation  connected with a rotating disk electrode (RDE) in a three-electrode system with Oequation :ERHE = E−2. The commercial Pt/C  and IrO2  catalysts with the same loading were used as the benchmark for ORR and OER, respectively.The fabrication of working electrode: 2.4 mg of obtained catalyst was added in a mixture solution, including distilled water (735 μL), isopropanol (205 μL), and 5 wt % Nafion  by ultrasound to form a homogeneous ink. After that, 3 μL of the above catalyst ink was dropped onto the GC electrode and naturally dried at room temperature. The loading was 100 μg cm−1. The linear sweep voltammograms (LSV) was executed from 1.0 V to 0.1 V (vs. RHE) at 10 mV s−1 with a series of rotation speed from 600 to 2000 rpm. Koutecky-Levich (K-L) plots were estimated at diverse potentials (0.1–0.4 V vs. RHE). The transfer electron number (n) was calculated by the following K-L formula:j, jL, jK, and ω are the measured current density, diffusion-limiting current density, kinetic-limiting current density, and angular velocity, respectively. n, F, Co, Do, ν, and k are the transferred electron number of the ORR, the Faraday constant, the bulk concentration of O2, the diffusion coefficient of O2, the kinematic viscosity of the electrolyte, and the electron-transfer rate constant, respectively.For the ORR test, the static cyclic voltammogram (CV) was operated between 0.1 and 1.0 V (vs. RHE) at the scan rate of 50 mV s2-saturated electrolyte . All polarization curves were corrected with 95% iR-compensation.For the OER test, the LSVs were tested from 1.0 V to 1.86 V (vs. RHE) in O−2. 9 mg of catalyst was dispersed in the solution of isopropanol (900 μL) and 5 wt % nafion solution (100 μL) by ultrasonic to form a homogeneous ink. Subsequently, the catalyst ink (100 μL) was dropped on the above air diffusion layer and slowly dried in vacuum at an ambient temperature. The commercial Pt/C based air electrode was also prepared under the same method. The loading of the catalyst was all about 1 mg cm−2.The Vulcan XC-72R (CABOT) and Polyvinylidene Fluoride (PVDF) with a mass ratio of 8:1 was mixed and coated onto the carbon paper  to obtain the air diffusion layer with the loading of ~2 mg cmThe performances of zinc-air battery were investigated on a home-made cell. Zinc plate, prepared air electrode, and 6 M KOH with 0.2 M zinc acetate solution were used as the anode, cathode, and electrolyte, respectively. Polarization and galvanostatic charge/discharge measurements were performed on the CHI 660E electrochemical station and LAND testing system, respectively. All of the tests were provided continuous oxygen from ambient air.2 atmosphere. For comparison, Co@N-C-700 and Co@N-C-900 show similar XRD patterns but different morphologies, indicating that the temperature is the key factor to tune the morphology of catalysts  in N 1s spectrum [The chemical states and surface composition of the Co@N-C-800 were investigated by XPS. As provided in spectrum D can be spectrum .2+ ion under constantly stirring, the unique cavity of the cyclen molecule can bind and anchor the Co2+ ion by the four-NH groups occupying the partial coordination sites of Co2+ ion to form Co-cyclen complex. After the dicyandiamide solution was introduced to the above reaction system, the –CN group derived from dicyandiamide can coordinate with Co-cyclen complex by occupying the residual sites of Co2+ ion to form the cyclen-Co-dicyandiamide complex subsequently. Furthermore, the dicyandiamide molecules can link with each other by hydrogen bonds between –NH2 (or =NH) groups and –NH– groups to form a network on which the Co-cyclen complex molecules were uniformly distributed and anchored. Finally, after pyrolyzed at high temperature in N2, the cyclen molecule was transformed to nitrogen-doped thin carbon nanoshells to encapsulate Co nanoparticles, resulting in the formation of Co@N-C hybrid catalyst. More importantly, the dicyandiamide molecule units also transformed into N-doped graphene-like carbon layers during the pyrolysis process [As a common macrocyclic ligand, 1,4,7,10-tetraazacyclododecane  was chosen to be the complexing agent in our experiment due to its strong and selective binding ability to metal cations and high nitrogen content in molecule. The synthetic procedure and possible formation mechanism of Co@N-C catalysts could be described by  process ,41 and s2-saturated and N2-saturated electrolyte, respectively. As given in −2, which was almost three times than that of commercial Pt/C (1.37 mA cm−2) , which indicates how well the system performs under a specific current, is even 29 mV more positive than Pt/C catalyst (0.842 V vs. 0.813 V), and the limiting current density of Co-N/C-800 (6.49 mA cm−2) is also higher than the Pt/C catalyst (6.14 mA cm−2) (j−1) and the reciprocal of square root of angular velocity (ω−1/2) at various potentials. The electron transferred number (n) is calculated to be 3.85  between ORR process at the current density of 3 mA cm−2 and OER process at the current density of 10 mA cm−2. Refs. [E value will exhibit good bifunctionality for ORR and OER. The low ΔE value of 0.80 V for Co@N-C-800  . The ORRmA cm−2) B. In ord be 3.85 D, implyicatalyst E. The bi2. Refs. ,36 Usual@N-C-800 F is smal@N-C-800 , indicat−2 at 1.0 V, while the Pt/C based battery only reach a current density of 120 mA cm−2 at the same voltage. During the charge process, Co@N-C-800 based Zn-air battery also demonstrates a better performance than Pt/C based battery. Moreover, the maximum peak power density of the Zn-air battery based on Co@N-C-800 cathode achieves 255 mW cm−2, which is far greater than Pt/C catalyst (156 mW cm−2). The cycle stability of the rechargeable Zn-air battery based on Co@N-C-800 catalyst was investigated and characterized by the fluctuation of the charge potential (ECP) and discharge potential (EDP) [−2 and is shown in ECP increased only 0.9% and EDP decayed 0.8% in total 300 cycles. After that, long-term charge-discharge cycling durability (a cycle includes 30 min discharge followed by 30 min charge) was further investigated at a higher current density of 10 mA cm−2 (ECP and limited voltage drop for EDP (7.4% decay) in total 100 h cycle period. All of the above results demonstrate the outstanding performances of Co@N-C-800 catalyst in rechargeable Zn-air battery are comparable or even superior to the state-of-the-art Pt/C catalyst and many previously reported air cathodes  . A fast  mA cm−2 D. Obvioucathodes . The supIn summary, a novel nanocomposite of high-density cobalt nanoparticles encapsulated in very thin N-doped carbon nanoshells was successfully synthesized by simple thermal decomposition of cyclen-Co-dicyandiamide solid-phase precursor. The morphologies and properties of products can be easily tuned by changing the pyrolysis temperature. Due to the synergetic effect of nanocomposite, the optimized Co@N-C-800 catalyst showed outstanding bifunctional catalytic activities for both ORR and OER in alkaline electrolyte. Furthermore, the rechargeable Zn-air battery that is based on Co@N-C-800 air cathode presented excellent electrochemical performance and strong cycle stability. The work demonstrated here provides a new strategy and approach to design and prepare advanced nanocomposites of transition metal nanoparticles and N-doped carbon materials for highly efficient bifunctional electrocatalysts in the high-performance rechargeable Zn-air battery and the other catalytic field.


Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling.To test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems—as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture.Together these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation.The online version of this article (10.1186/s12915-019-0625-9) contains supplementary material, which is available to authorized users. The actin cytoskeleton plays a major role in the regulation of cell shape and tissue organization in animals , and as While few signaling systems other than cell-cell and cell-ECM contacts have been shown to be force sensitive, recent work has implicated mechanical force in the activation of Notch receptor –12. In cDrosophila pupal notum, the activation of Notch is induced via contact with a neighboring cell expressing Delta. This represses the pro-neural fate so that epithelial cells in which Notch is activated tend to remain epithelial in character sfGFP  or in both signal sending and receiving cells (using pannier-GAL4) [In order to reduce myosin II activity, we expressed UAS-spaghetti squasher-GAL4) , 30. StrDrosophila S2R+ cells expressing either a synthetic Notch ligand or receptor. Once these form cell-cell contacts, myosin II is inhibited by pharmacological inhibitors or dsRNA-mediated knockdown of zipper or spaghetti squash expression  of basal protrusions can lead to defects in long-range Notch signaling and, therefore, to decreased SOP cell spacing , 34. We Additional file 3: Movie S1. Basal protrusion dynamics in a control SOP cell. SOP cell expressing UAS-GMCA under the neur-GAL4 driver. Movie represents a maximum projection over 2.1 μm. Time in seconds. Scale bar, 10 μm. (AVI 4726 kb)Additional file 4: Movie S2. Basal protrusion dynamics in a SOP cell with decreased myosin activity. SOP cell expressing UAS-GMCA and UAS-spaghetti squashAA under the neur-GAL4 driver. Movie represents a maximum projection over 2.1 μm. Time in seconds. Scale bar, 10 μm. (AVI 8361 kb)How might the actomyosin cortex influence Notch signaling? Endocytosis requires deformation of the cell cortex, whose stiffness is regulated by actomyosin contractility , 39. At zipperDN expressing cells without these being a visible difference in the numbers of these puncta , we observed severe patterning defects. These appeared to be a combination of the two phenotypes observed in the single mutants, i.e., there was an increase in the variability of bristle spacing together with an enhanced number of GFP-positive cell clusters relative to the expression of either RNAi alone  , led to zipper or spaghetti squash expression in signal sending or receiving cells in the presence or absence of Dynasore  and GFP11 in signal sending cells , so that the reconstituted GFP fluorescence would be visible only when the two cell types came into contact with one another  . Membranence Fig. c′. In cope Fig. c″. Howevope Fig. c. These pannier-GAL4 to express UAS-LifeActRuby in both signal sending and receiving cells in the notum, we observed a complex network of protrusions that criss-crossed the basal surface of the epithelium . Scale bar, 10 μm. (AVI 1802 kb)spaghetti squashAA construct under the pnr-GAL4 driver to reduce myosin II activity throughout the notum, we observed a twofold decrease in the mean initial recoil velocity, from 6.6 ± 2.5 μm/s . Taken together, these data support a model in which myosin II-dependent tension in basal contacts that connect epithelial cells in the notum facilitate robust activation of Notch response during pattern formation in the notum.Finally, to test whether these basal protrusion contacts are subject to mechanical tension tension, we performed laser ablation to disrupt them. Using a focused UV-laser, we made ~ 10-μm cuts within the basal surface of the notum, disrupting the protrusion network without affecting more apical planes Fig. e. Cuts iDrosophila, the pattern of bristles on the dorsal thorax of the fly is set up by Notch-Delta signaling through a process of lateral inhibition [In hibition . As we shibition . While ahibition , 51, 52 How might Notch receptor be activated on protrusions? Our data suggests that one way this could occur is through the contact, engagement, and retraction of basal protrusions in Notch and Delta expressing cells. Importantly, since the contact area between protrusions can be extensive, Notch/Delta signaling molecules localized along the protrusions could potentially contribute to signal activation. In addition, when contact has been made, the ligand and receptor could diffuse along the length of the protrusion and become trapped at the site of contact—as suggested previously . Once NoWhile our data do not rule out the possibility that endocytosis contributes directly to protrusion-mediated Notch activation. However, the structure of filopodial-like protrusions, with bundled actin closely associated with the plasma membrane, may be refractory to endocytosis. Indeed there is evidence for the existence of endosomal ‘hot spots’ at the base of cellular protrusions rather than along them . A closeHow much does protrusion-mediated Notch-Delta signaling contribute to the cell fate decision making process of epithelial cells? Previously, it was shown that stochastic noise can be a feature of signaling mechanisms and contribute to the plasticity of bristle patterning . More geRecent evidence shows that the patterning of bristle rows begins developing in the notum far earlier than 12 h APF, where we begin our analysis . AlthougOur results show that actomyosin contractility plays a role in Notch signaling both in a cell culture model of signaling and in bristle patterning in the developing fly. By tuning the activity of myosin II to affect contractility without affecting protrusion dynamics and morphology, we show that protrusion interactions are subject to contractile forces and that myosin II activity is required for robust signaling between cells whose only contact is via basal protrusions. Our data also show that, actomyosin contractility in cells that contact one another through extensive lateral contacts promotes the Notch. In these cells endocytosis and myosin-dependent pulling may both contribute to force-dependent Notch activation. These results add to our understanding of protrusion-mediated signaling in the patterning of self-organized tissues. Importantly, they also suggest a new mechanism of mechanotransduction via which forces generated within the actomyosin cytoskeleton impact signaling to help pattern a tissue.GAL4 Drivers: tub-GAL80ts; neu-GAL4, UGM. Neu-GMCA; pnr-GAL4. NsfGFP; neu-GAL4. NsfGFP; pnr-GAL4. shotgunGFP; pnr-GAL4. shotgunGFP, neu-GMCA; neu-GAL4. UAS-lifeActRuby; pnr-GAL4. UAS Responders: UAS-zipperdn, gfp. UAS-squashAA. UAS-LifeActRuby. UAS-liquid facets RNAi. UAS-ROK RNAi. UAS-white RNAi UAS-zipper RNAi. Other: zipperVenus, Flag. GRASP: ase-LexA-p65/CyO(Kr:GFP); LexAOp-GAL80, UAS-CD4spGFP(1–10)/TM6BTb. LexAOp-CD4spGFP(11)/CyO; pnr-GAL4/TM6BTb.sfGFP, ex vivo experiments, and filopodia imaging were performed on either a Leica SPE confocal, × 40 oil immersion objective (1.15 NA) at room temperature or a Nikon EclipseTi, × 20 (0.75 NA) or × 60 (1.4 NA). Localization of myosin in live pupae was performed on a Zeiss LSM880 with AiryScan, × 63 (1.4 NA) oil immersion objective. Fixed nota were imaged on a Leica SPE confocal, × 63 (1.3 NA) oil immersion objective. Fixed SIM images were obtained on a Zeiss Elyra PS.1, × 63 (1.4NA) oil immersion.White pre-pupae were picked and aged to ~ 12–24 h AP at 18 °C. Live pupae were removed from pupal case and mounted on a slide as previously described . Final pLive images were acquired on a Zeiss Axio Imager.M2 m, × 40 (1.2NA) water objective, using Micromanager  acquisition software. An Nd:YAG UV laser (Continuum) was interfaced with the confocal microscope to allow steered laser incisions . We madePrimaries: chicken anti-GFP ; rabbit anti-pS19-MRLC ; mouse anti-Delta extracellular domain ; mouse anti-Notch extracellular domain ; guinea pig anti-Delta ECD . Secondaries: Rhodamine Red-X anti-guinea pig  and Cy5 anti-mouse ; AlexaFluor 488 anti-chicken  and AlexaFluor 568 anti-rabbit ; Texas Red-X Phalloidin  were used to visualize F-actin. Nota were mounted in 50% glycerol or Vectashield  for labeling nuclei.2 glass bottom dishes (Matek) using a thrombin/fibrinogen (Sigma) clot, then cultured in 250-μL modified Clone8 medium (Schneider’s insect medium (Sigma), 2.5% fly extract, 2% fetal bovine serum (ThermoFisher Scientific) as previously described [2O) or 0.1 mM Y27632  was added to the dish.Live nota were dissected and attached to 35-mmescribed . After ihttps://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl). Gene-specific amplicons of sqh, zipper, and white genes were amplified from fly genome by PCR, inserted into pDONR221 vector, and subcloned into pWALIUM10 RNAi vector. All constructs verified by sequencing. The primers used for the synthesis of dsRNA were as follows , sequence 5′-3′:sqh F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCGGCTCCATTTAGCTCCATTAsqh R: GGGGACCACTTTGTACAAGAAAGCTGGGTGGCAGGACGCCCATATTCTCzipper F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCACAGGGTACAGCCGATAAA;zipper R: GGGGACCACTTTGTACAAGAAAGCTGGGTGCTTGTGCTTGCAGCTTCTTC;w F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTGCCCAGTGTCCTACCA;w R: GGGGACCACTTTGTACAAGAAAGCTGGGTGTACGAGGAGTGGTTCCTTGA.Long hair-pin dsRNA was designed using SnapDragon  supplemented with 10% fetal bovine serum and 0.5% Pen/Strep (Gibco). Cells were transfected using Effectene transfection reagent (Qiagen) , with DNqPCR: S2R+ cells plated in six-well plate were transfected with indicated dsRNAi plasmid (0.6 μg) together with pAct-GAl4 (0.2 μg) using Effectene. dsRNA against white used as control. The transfection procedure was repeated two more times every 4 days to maximize transfection efficiency. Total RNA was extracted from S2R+ cells after the 3rd transfection using TRIzol Reagent (ThermoFisher Scientific). Raw RNA was treated with DNase I, purified by QIAGEN RNeasy kit, and converted to cDNA template using iScript cDNA Synthesis Kit (Bio-Rad). Y27632 treatment: Cells expressing either synNotch or GFP-ligand were treated with 20 μM Y-27632 for 1 h and then mixed and cultured in the presence of 20 μM Y-27632 for 1 day before analysis.All statistical analysis was performed in Prism 6 (GraphPad). Specific tests used are indicated in the text and figure legends, see Additional file sfGFP signal was measured as previously described [sfGFP transgene includes a nuclear localization signal. GFP fluorescence localized to nuclei either adjacent to (1 N) or one cell removed (2 N) from an SOP (which lack GFP expression) was measured by drawing an ROI in the nucleus and recording mean intensity for the ROI for each time point until nuclear envelope breakdown (NEB). Since epithelial cells are staggered in their cell fate decision making process, and we previously established that exit from G2 phase of the cell cycle is Notch signaling dependent, we use NEB as a fixed time point for comparison. For the purposes of these graphs, t = 0 at NEB. The data was normalized to the fluorescence at the first time point of the movie .Figure escribed . Brieflyy-axis to result in a 1D histogram along the bristle row (x-axis). Distance between SOPs is defined as the centroid to centroid distance between signal peaks along the histogram; apical diameter is defined as the edge-to-edge measurement of a signal peak . To verify that we were only measuring the distance between true SOP cells, we captured 12 h time-lapse movies (starting at 12 h APF). We therefore measured the distance between GFP+ cells which become SOPs and not GFP+ cells that switch back to epithelial fates. However, in measuring grouping, we measured any GFP+ cells adjacent to each other.Figures Figure Figure Supplement: Filopodia dynamics were measured by manually tracing protrusions in z-projected time-lapse images. Myosin localization: basal images (up to 1 μm maximum projection). ROIs were drawn to include only basal filopodia areas. All other measurements were performed as described in main text figures.Additional file 1:Figure S1. Contribution of Myosin II activity to Notch response. (A) RNAi against an activator of Myosin II activity, Rho kinase (ROK) in signal sending cells alone does not disrupt protrusion morphology. (B-C) Decreased ROK activity in signal sending cells alone (via neur-GAL4) leads to decreased Notch response in both (B) adjacent and (C) distant wildtype neighboring cells . (D) Decreased ROK activity in all cells (via pnr-GAL4) does not affect the rate of signaling between adjacent cells, but does decrease the total signal . (E) The rate of Notch response in distant neighbors is significantly affected by ROK RNAi expression . (F) S2R+ cells expressing synNotch in the absence of ligand expressing cells and cultured in the presence of Y27632 and/or Dynasore do not exhibit changes in their expression of synNotch in response to drug treatment. (G) Acute inhibition of ROK does not significantly alter the basal synNotch activity measured in S2R+ cells expressing synNotch in the absence of ligand expressing cells. (H) Basal synNotch activity is affected by transfection of zip and spaghetti squash siRNA, but not by acute treatment with Dynasore. However, because fold changes of synNotch activity in the presence of GFP-ligand is calculated based on each treatment respectively, the relative fold changes should still primarily reflect the efficiency of synNotch cleavage under different conditions. (PDF 1657 kb)Additional file 2:Figure S2. Localization of Notch and Delta with decreased Myosin II activity. In tissues expressing control (LifeActRuby) or sqhAA constructs in SOP cells  we observe no differences in (A) Notch localization or (B) Delta localization at a single apical section and basal projection (over 2 μm). Scale bars, 10 μm and 5 μm (sqhAA anti-Delta panels). (PDF 4856 kb)Additional file 5:Figure S3. Role of ligand endocytosis in Notch signaling. (A) Delta ligand endocytosis assay in SOP cells expressing LifeActRuby (control) or zipperDN under tubGAL80ts; neur-GAL4, UAS-GMCA. Endocytosed Delta is specifically visualized through uptake of the anti-Delta (ECD) monoclonal antibody, red puncta (arrows). GFP is stained to visualize the SOP cell body. We find no significant difference in numbers of Delta positive puncta between the control or myosin II perturbed genotypes (graph). (B-C) RNAi against key regulator of ligand endocytosis pathway (liquid facets) lead to a pairing phenotype (indicated by yellow brackets). Pairs of neuralized expressing cells are seen with less frequency in control pupae. (B-C) Scale bars, 25 μm. (D-E) Quantifications of patterns for genotypes in (B-C). (D) Apical ‘cell’ diameter, measuring grouping/pairing. ***p < 0.001 by unpaired t-test. (E) Measurements of the distance between neuralized expressing cells. ***p < 0.001 by unpaired t-test. (n), number of distances measured, N ≥ 3 nota measured for each genotype at ~ 14 h AP. (F) Grouping is observed in cells co-expressing RNAi targeting zipper and liquid facets in SOP cells, compared to either RNAi expressed in SOP cells alone. We note that the grouping phenotype is slightly different in neur-GAL4, UAS-GMCA (D) and tubGAL80ts; neur-GAL4, UAS-GMCA (F) background, for unclear reasons. (PDF 5104 kb)Additional file 6:Figure S4. Data supporting main Fig. Additional file 8:Raw data file. Data analyzed for the main and supplemental figures. (XLSX 245 kb)


IL23R may have crucial role in pathogenesis of Crohn’s disease (CD). Thus, we sought to assess the IL23R SNPs contribution to susceptibility and phenotype of CD. We recruited 117 CD subjects and 117 controls from Poland and 30 CD subjects and 30 controls from Bosnia and Herzegovina (B&H). Two common IL23R SNPs: rs1004819, rs7517847 were genotyped using TaqMan SNP assays. In the Polish population it was found that allele rs1004819: A increases the risk of CD, while allele rs7517847: A is protective against disease development. In Poles the co-carriage of two IL23R risk genotypes was associated with increased risk of CD. A significantly increased risk of CD early onset was observed in Poles carrying at least one rs7517847: G allele. It was also found that IL23R SNPs may be associated with structuring/penetrating CD behavior, as alleles rs1004819: A and rs7517847: G were significantly less frequent in patients without complications, from Poland and B&H, respectively. Allele rs1004819: A was also significantly more frequent in Poles with penetrating CD. These results confirm IL23R SNPs contribution to CD susceptibility in the Polish population and suggest their impact on early age of onset and more severe disease course.It is suggested that IL-23/IL-17 axis and single nucleotide polymorphisms (SNPs) of  Crohn’s disease (CD) is an incurable chronic autoimmune disorder determined by environmental, immunological and genetic factors, characterized by remitting and relapsing inflammation that may occur anywhere along the length of the gastrointestinal tract (GIT) ,2.Recently, the crucial role of the interleukin 23 (IL-23) signaling pathway (IL-23/IL-17 axis) in CD pathogenesis has been suggested. According to the leading hypothesis the chronic intestinal inflammation is stimulated by pro-inflammatory cytokines including IL-17A, IL-17F, IL-21, and IL-22 produced by activated Th17 CD4+) lymphocytes + lymphoc,6. It waIL23R gene  is located on chromosome 1p31 and contains at least 11 exons [IL23R have been linked with CD susceptibility in European-descent populations [IL23R gene  and its influence on the function of IL23R remains uncertain [IL23R mRNA, resulting in the generation of a different IL-23R protein isoforms [The 11 exons . Apart f11 exons . To dateulations . Two of ulations ,17,18,19ulations ,21. The ncertain . Howeverisoforms ,25.One of the most promising therapeutic approach in CD is based on the monoclonal antibodies targeting IL-23 (p40 and p19) subunits . HoweverIL23R biological and potential therapeutic significance in CD, the investigation of the IL23R SNPs gene may be beneficial for the optimization of the future therapeutic strategies. Here we aimed to assess the contribution of the IL23R SNPs in determining susceptibility and phenotype of CD in population from Poland and Bosnia and Herzegovina (B&H).Therefore, given the IL-23 and This study was approved by local Ethics Committees . All study subjects provided written informed consent before the enrolment.We recruited 117 CD patients and 117 controls from Poland and 30 CD patients and 30 controls from B&H. Crohn’s disease diagnosis was determined according to the clinical, radiological, endoscopic, and histological criteria . SubjectIL23R rs1004819 and rs7517847 SNPs genotyping was performed using LightCycler 96 Real-Time PCR System , TaqPath ProAmp Master Mix  and commercial pre-designed TaqMan SNP assays , according to the manufacturer’s instruction. Samples were first heated for 10 min at 95 °C, before amplification as follows: 40 cycles of two-step PCR program at 95 °C for 15 s and 60 °C for 1 min.In total, 294 whole blood samples were collected from CD subjects and controls from both populations. Genomic DNA was isolated from peripheral blood leukocytes by standard procedures using the QIAamp DNA Mini Kit . The IL23R genotype and allele frequencies were determined by direct counting. Deviations from Hardy-Weinberg equilibrium (HWE) were estimated using a chi-square (χ2) test from R package “HardyWeinberg” (v. 1.5.9). Overall differences in genotype distributions between CD patients and controls from both studied populations were evaluated using χ2 test. For the statistical analysis of the IL23R SNPs genotypes and alleles association with CD susceptibility and phenotype Fisher’s exact test was applied. Above mentioned statistical analyses were carried out using GraphPad Prism v. 5.03 software . All p values were two-sided and considered statistically significant at p < 0.05.The IL23R rs1004819 and rs7517847 genotypes between the Polish CD patients and controls . There were no significant differences in the overall distribution of both IL23R SNPs genotypes between the Bosnian CD patients and controls (p > 0.05). The association of IL23R genotypes with CD was analyzed using co-dominant, dominant and recessive genetic models of inheritance. A significant association between rs1004819 SNP and CD risk was found in the Polish population, under dominant  = 2.15 (1.27–3.62)) and both co-dominant  = 3.19 (1.27–8.02) and p = 0.0188; OR (95% CI) = 1.95 (1.13–3.39)) genetic models. In Poles, a significant associations between the rs7517847 SNP and protection against CD was found under dominant  = 0.54 (0.32–0.92)), recessive  = 0.36 (0.16–0.82)) and a single co-dominant  = 0.28 (0.12–0.68)) genetic models. Allelic analysis revealed that rs1004819: A allele was significantly over-represented in the Polish CD group compared to controls  = 1.85 (1.25–2.75)), while allele rs7517847: G was significantly more frequent in the Polish controls than CD subjects  = 0.57 (0.39–0.84)). There were no significant differences in the genotype and allele frequencies of both IL23R SNPs between the Bosnians CD patients and controls (p > 0.05).In studied populations, all analyzed SNPs were in HWE, both in CD subjects and controls. The genotype and allele frequencies are shown in IL23R SNPs on CD development was assessed separately for homozygous (rs1004819: A/A and rs7517847: G/G) and homo- and/or heterozygous risk genotypes (rs1004819: A/A or G/A and rs7517847: G/G) co-occurrence. The frequencies of the IL23R genotypes co-occurrence in CD patients and controls from both population are shown in p = 0.0444; χ2 = 6.23; df = 2) and homo- and/or heterozygous  risk genotypes in the Polish CD group compared to controls. The carriage of one or two of the homozygous risk genotypes (rs1004819: A/A or/and rs7517847: G/G) was significantly more frequent in the Polish CD patients than controls  = 2.23 (1.19–4.17) and p = 0.0034; OR (95% CI) = 2.76 (1.44–5.31), respectively). In addition, the carriage of two homo- and/or heterozygous IL23R risk genotypes (rs1004819: A/A or G/A and rs7517847: G/G) was found to be significantly more frequent in the Polish CD patients  = 2.71 (1.10–6.69)). In the Bosnian population, there were no significant differences in the occurrence of combined IL23R risk genotypes in CD patients compared with controls (p > 0.05).Considering that both SNPs were related with CD susceptibility in Poles, the joint effect of the combined risk genotypes of rs1004819 and rs7517847 SNPs on the risk of CD was investigated. Individuals from both populations were grouped on the basis of number of carried risk genotypes. The effect of IL23R SNPs with disease sub-phenotypes was also investigated. Patients were grouped based on their age of CD onset, disease location and clinical behavior according to the Montreal classification. IL23R rs1004819 and rs7517847 genotype and allele distributions were compared between subgroups of CD patients with and without given categories. In the statistical analysis the combined counts of the minor allele containing genotypes (rs1004819: A/A + G/A and rs7517847: G/G + G/T) were used. The genotype and allele frequencies in subgroups of the Polish and Bosnian CD patients and association between genetic variants of IL23R and CD phenotype are presented in p = 0.0309; OR (95% CI) = 9.14 (1.10–76.19)) in the Polish subjects with early CD onset (A1) compared to patients with adult disease onset (A2 or A3). It was also found that allele rs1004819: A was significantly less frequent  = 0.48 (0.27–0.86)) in the Polish subjects with non-stricturing, non-penetrating CD (B1) compared to subjects with complications (B2 or B3). At the same time, allele rs1004819: A was significantly over-represented (A vs. G: p = 0.0234 OR (95% CI) = 2.22 (1.13–4.39)) in the Polish subjects with penetrating CD (B3) compared to subjects with non-penetrating non-stricturing or structuring disease phenotype (B1 or B2) (A vs. G: p = 0.0234 OR (95% CI) = 2.22 (1.13–4.39)). For the group from the Bosnian population, allele rs7517847: G was observed significantly less frequently  = 0.2105 (0.07–0.63)) in subjects with non-stricturing non-penetrating CD (B1) than subjects with CD complications (B2 and/or B3). Results revealed no significant associations between both of the investigated IL23R SNPs and age at diagnosis of CD in the Bosnian patients (p > 0.05). In both studied populations no significant associations between the IL23R SNPs and CD location were found (p > 0.05).The association of IL23R gene polymorphisms could contribute to susceptibility and phenotype of disease [IL23R rs1004819 and rs7517847 SNPs frequencies in CD subjects from Poland are limited, and in Bosnia and Herzegovina are non-existent. Thus, in this study the association of two IL23R SNPs with CD was investigated in above mentioned populations. Additionally, the correlation between IL23R SNPs and disease clinical manifestation defined according to the Montreal classification was evaluated.The incidence of IBD and particularly CD are recently rising in developing countries ,31. Alth disease ,32,33,34IL23R SNPs were associated with CD susceptibility at the genotype and allele level in the Polish, but not in the Bosnian population. In Poles the IL23R rs1004819: A variant has been found to be associated with increased risk of CD, while the rs7517847: G allele showed protection against disease development. These results are consistent with findings of the previous studies on European-descent populations. The association of rs1004819 and rs7517847 with CD was reported in German population by Glas et al. Similar to our results the rs1004819: A allele was shown to be a CD risk factor and the rs7517847: G allele was found to be a protective variant [IL23R rs1004819: A variant association with higher CD risk was also reported in two British studies [IL23R rs1004819 SNP was found in another Polish study by Jakubowska-Burek et al. Interestingly, in their study, the heterozygous risk genotype rs1004819: G/A over-representation was observed in control group, while the rs1004819: A/A genotype frequency was higher in CD subjects [Both of the investigated  variant . The IL2 studies ,36. The  studies , Austral studies , Finnish studies  and Swed studies . On the  studies . Additiosubjects . SimilarIL23R rs7517847 significant association with CD in the dominant genetic model as well as the rs7517847: G variant protective effect for disease development were found in the Slovenian [n = 19), compared to controls (n = 20) [n = 708) and controls (n = 537), the IL23R rs7517847: G variant was found to be significant protective against CD development [IL23R rs75717847: G variant (T/T and T/G) [A strong association of the rs7517847 SNP with an increased risk of CD was reported by Newman et al. in non-Jewish cohort from the Canadian population . The alllovenian , Italianlovenian  and anotlovenian . For the(n = 20) . Comparaelopment . Similarelopment ,33, Austelopment . In the and T/G) .IL23R SNPs association with CD were reported in non-European populations. In Brazilians IL23R rs1004819: A allele was associated with CD, while no association was found for rs7517847 [IL23R SNPs were revealed in the Japanese study by Yamazaki et al. [IL23R rs1004819 and rs7517847 SNPs are associated with CD mainly in populations of Caucasian origin [It is worth mentioning that contradictory results of the s7517847 . At the IL23R risk genotypes may increase the risk of CD development, as it was significantly more frequent in the Polish CD subjects than controls. Therefore, it is possible that IL23R risk variants could have an additive effect on the risk of CD. Due to small sample size, we failed to replicate these results in the Bosnian cohort.The results of this study also show that co-occurrence of homozygous and homo- and/or heterozygous IL23R SNPs correlation with CD clinical manifestation revealed a significant over-representation of genotypes with at least one rs7517847: G allele (G/G and G/T) in those Polish individuals with early CD onset (A1) as compared with patients with adult disease onset (A2 or A3). The results also showed a significantly lower MAF of rs7517847 SNP in the Bosnian subjects with non-stricturing non-penetrating CD (B1) compared to cases with stricturing and/or penetrating disease behavior (B2 and/or B3). The association with complications occurrence was also found in the Polish population for rs1004819: A variant, as it was significantly less frequent in subjects with non-stricturing non-penetrating CD (B1) compared to those with more severe disease behavior (B2 or B3). Simultaneously, the MAF of rs1004819 SNP was significantly higher in the Polish subjects with penetrating CD (B3) compared to subjects with non-stricturing non-penetrating or stricturing disease phenotype (B1 or B2). All in all, these results suggest that IL23R SNPs could affect CD clinical manifestation. To date, only a small number of studies investigated genotype-phenotype associations of the IL23R rs1004819 and 7517847 SNPs in CD and reported results were contradictory. The IL23R rs10048419 association with the disease location was firstly described by Glas et al. They have found that ileal disease location (L1 or L3) was significantly more frequent in German patients with rs1004819: A/A (T/T) genotype than in those with rs1004819: G/G (C/C) genotype. At the same time, colonic disease location (L2) was significantly less frequent in subjects with rs1004819: A/A (T/T) genotype compared to carriers of the rs1004819: G/G (C/C) genotype. The increased incidence of structuring disease behavior (B2) was found in group of rs1004819: A/A (T/T) homozygotes compared to carriers of the rs1004819 G/A (C/T) genotype [IL23R rs7517847 association with CD location was reported by Doecke et al. in the combined Australian-New Zealand cohort. They have found that the rs7517847: G allele frequency was significantly lower in individuals with colonic (L2) than ileal (L1) CD location. It was also showed that the rs7517847: G variant is significantly less frequent in groups of patients with ileal (L1) as well as with colonic (L2) or ileo-colonic disease location (L3) compared to controls [IL23R SNPs and CD phenotype were found in the Spanish [Additionally, the analysis of genotype . For thegenotype . Additiocontrols . On the  Spanish  and Brit Spanish . No asso Spanish  and anot Spanish .IL23R SNPs observed in our study are comparable to those reported in above mentioned studies from European-descent populations [A major limitation of the presented study is the small sample size of the investigated groups from both populations, the Bosnian group in particular. Thus, we will re-evaluate this results in subsequent studies with larger sample size of individuals from Polish and Bosnian populations. Nevertheless, it is worth mentioning that genotype and allele frequencies of both investigated ulations ,43,44,45IL23R SNPs: rs1004819 and rs7517847 contribution to CD susceptibility in the Polish population and suggest their impact on early age of onset and more severe disease course. This is also the first study on rs1004819 and rs7517847 in the Bosnian CD population. A major limitation of the presented study is the small sample size of the investigated groups. Therefore, these results will be re-evaluated in subsequent studies with larger sample size of individuals from both populations.Our results confirm the