Anemia is a significant complication of chronic inflammation and may be related to dysregulated activities among erythroblastic island (EBI) macrophages. GM-CSF was reported to be upregulated and attracted as a therapeutic target in many inflammatory diseases. Among EBIs, we found that the GM-CSF receptor is preferentially and highly expressed among EBI macrophages but not among erythroblasts. GM-CSF treatment significantly decreases human EBI formation in vitro by decreasing the adhesion molecule expression of CD163. RNA-sequence analysis suggests that GM-CSF treatment impairs the supporting function of human EBI macrophages during erythropoiesis. GM-CSF treatment also polarizes human EBI macrophages from M2-like type to M1-like type. In addition, GM-CSF decreases mouse bone marrow (BM) erythroblasts as well as EBI macrophages, leading to a reduction in EBI numbers. In defining the molecular mechanism at work, we found that GM-CSF treatment significantly decreases the adhesion molecule expression of CD163 and Vcam1 in vivo. Importantly, GM-CSF treatment also decreases the phagocytosis rate of EBI macrophages in mouse BM as well as decreases the expression of the engulfment-related molecules Mertk, Axl, and Timd4. In addition, GM-CSF treatment polarizes mouse BM EBI macrophages from M2-like type to M1-like type. Thus, we document that GM-CSF impairs EBI formation in mice and humans. Our findings support that targeting GM-CSF or reprogramming EBI macrophages might be a novel strategy to treat anemia resulting from inflammatory diseases.The online version contains supplementary material available at 10.1186/s12967-021-03214-5. GM-CSF impairs human EBI formation by decreasing CD163 adhesion molecule expression.GM-CSF decreases mouse BM erythroblasts and EBI macrophages as well as impairs EBI formation via decreasing surface expression of CD163 and Vcam1.Granulocyte–macrophage colony-stimulating factor (GM-CSF), a significant myelopoietic growth factor and pro-inflammatory cytokine, has been shown to be upregulated and has attracted increasing interest as a therapeutic target for many inflammatory diseases, including coronavirus disease 2019 . In genMarcel Bessis in 1958 ) in 1958 , was fun in 1958 , who fou in 1958 ). Recent in 1958 , 17, 18. in 1958 , 21. TheJoshua Tay et al. found that G-CSF reduces EBI frequency in the BM by more than 100 times [In previous studies, both G-CSF and GM-CSF were used to mobilize HSCs from the BM into the blood in order to harvest large quantities of HSCs for subsequent transplantation in humans , 23. Bot00 times . In contIn the present study, we demonstrate that GM-CSF significantly decreases human EBI formation in vitro. Bioinformatics analysis of RNA sequencing (RNA-seq) on GM-CSF-treated and control EBI macrophages further confirmed the impaired EBI formation, as evidenced by decreased adhesion molecule expression of CD163. In particular, GM-CSF injection into mice also significantly decreases BM erythroblasts as well as EBI numbers by decreasing both the number of EBI macrophages and adhesion molecule expression of CD163 and of Vcam1. Our study presents novel data that GM-CSF impairs erythropoiesis by disturbing EBI formation and proposes targeting EBI macrophages as a potential treatment option for AI.All antibodies related to this study are listed in Additional file About 30–50 μl of peripheral blood was collected through the orbital vein using an Eppendorf tube containing 1 μl of 0.5 M ethylenediaminetetraacetate  after anesthesia. Blood was diluted 1:10 in phosphate balanced saline (PBS) and analyzed via an advia120 hematology analyzer. The RBC numbers, hemoglobin, hematocrit (HCT), and reticulocytes of mice before and after GM-CSF injection were analyzed using GraphPad Prism 9.0 software.Under terminal anesthesia using isoflurane, mice were killed via cervical dislocation. The BM and spleen (SP) were collected and processed for single-cell preparation for flowcytometry. The single cell suspensions were prepared as previously described . In brie6 cells were blocked with 50 μL PBS + 0.5%BSA containing rat anti-mouse CD16/CD32 (dilution 1:100) for 15 min at 4℃, then stained with BV-421-Lin (1μL/5 × 106cells), BV-421-CD41(1μL/5 × 106cells), PE-Cy7-CD34(1μL/5 × 106cells), PE-Cy7-CD16/32(1μL/5 × 106 cells), APC-Cy7-Scal1(1μL/5 × 106 cells), APC-CD117(1μL/5 × 106 cells), and Percp-CD71(1μL/5 × 106 cells) for 30 min on ice in the dark. For murine erythroblast staining, 3 × 106cells were blocked with 25 μL PBS + 0.5%BSA containing rat anti-mouse CD16/CD32 (dilution 1:100) for 15 min at 4℃, then stained with anti-CD11b-APC-Cy7 (0.1 µg/106cells), anti-Gr1-APC-Cy7 (0.1 µg/106cells), anti-CD45-APC-Cy7 (0.1 µg/106cells), anti-Ter119-PE (0.5 µg/106cells) or FITC-Ter119(0.5 µg/106cells), and anti-CD44-APC (0.2 µg/106cells) for 30 min on ice in the dark. For macrophage staining, 5 × 106 cells were blocked with 50 μL PBS + 0.5%BSA containing rat anti-mouse CD16/CD32 (dilution 1:100) for 15 min at 4℃, then stained with anti-CD11b-APC-Cy7 (0.1 µg/106cells), anti-Gr1-APC-Cy7 (0.1 µg/106cells), anti-AF647-F4/80 (5 µg/106 cells), anti-Percpcy5.5-CD106 (0.3 µg/106 cells), anti-FITC-CD169 (4 µg/106 cells), anti-Percp-CD163 (0.2 µg/106 cells), anti-FITC-Timd4 (0.25 µg/106 cells), anti-PE-Cy7-Mertk (0.5 µg/106 cells), anti-PE-Cy7Axl (0.125 µg/106 cells), anti-PE-Cy7-MHC-II (0.5μL/5 × 106 cells), anti-FITC-CD206 (1μL/5 × 106 cells), anti-Percpcy5.5-CD14 (1μL/5 × 106 cells), anti-FITC-CD86 (1μL/5 × 106 cells), and anti-PE-Cy7-CD80 (1μL/5 × 106 cells) for 30 min on ice in the dark. After staining, the cells were washed once with PBS plus 0.5%BSA and 2 mM EDTA. DAPI were used to gate out dead cells. Then, cells were resuspended with PBS plus 0.5%BSA and 2 mM EDTA, and run on a BD Air III (BD bioscience). Flow Jo software (BD) was used to analyze the data.For burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E) staining, 5 × 10For P-STAT5 staining, the BD Transcription Factor Phospho Buffer Set (BD Cat#565575) was used. In short, the BM cells were starved for 4 h, and were stimulated with GM-CSF for 15 min. Then, the cells were fixed with TF Fix/Perm Buffer(1X) for 50 min at 4℃ and washed using 1 × TFP Perm/Wash Buffer. After fixation, cells were incubated with BD Phosflow™ Perm Buffer III, and the cells were washed once to remove Perm Buffer III using 1 × TFP Perm/Wash Buffer. Finally, cells were stained with CD11b, Gr-1, F4/80, and P-stat5  for 50 min on ice in the dark, washed using 1 × TFP Perm/Wash Buffer, and run on a BD Air III. Flow Jo software (BD) was used to analyze the data.Jessica A. Hamerman’s group [6cells), anti-Gr1-APC-Cy7 (0.1 µg/106cells), anti-AF647-F4/80 (5 µg/106 cells), and anti-FITC-Ter119 (0.5 µg/106cells) for 30 min on ice in the dark to detect cells that had phagocytosed RBCs. BD Air III was used to collect events and Flow Jo software (BD) was used to analyze the data.We followed the same method as ’s group . BM singEBIs in mouse BM were enriched and the numbers were quantified. Our EBI enrichment method matches that of previous studies , 21, 29.SP cells were obtained from both GM-CSF treated and control mice and immediately embedded into an optimal cutting temperature (OCT) compound. The tissues were kept in a − 80 ℃ freezer. Then, hematoxylin and eosin (HE) staining was performed as previously described .+ cells. The detailed cultured method was same as our previously described [+ cells were purified by CD34 MicroBeads  from human healthy donors at Zhengzhou University. The cell culture procedure was comprised of 3 phases. In the present, two phases of cell culture were used. Composition of the base culture medium was Iscove's Modified Dulbecco's Medium , 2% human peripheral blood plasma (Stem Cell Technologies), 3% human AB serum , 200 μg/mL Holo-human transferrin (Sigma Aldrich), 3 IU/mL heparin , and 10 μg/mL insulin . In the first phase (day 0 to day 6), CD34+ cells at a concentration of 105/mL were cultured in the presence of 10 ng/mL stem cell factor , 1 ng/mL IL-3(Stem Cell Technologies), and 3 IU/mL erythropoietin . In the second phase (day 7 to day 11), IL-3 was omitted from the culture medium. Then, the D11 erythroblasts were harvested for the next step of co-culture experiment. Human “EBI-like” macrophages were also derived from CD34+ cells. In the Day 0 to Day 7, CD34+ cells were cultured in IMDM containing 2% human peripheral blood plasma, 3% human AB serum, 3 IU/mL heparin, 10 µg/mL insulin, 10 ng/mL SCF, 1 ng/mL IL-3, 100 ng/mL M-CSF and 50 ng/mL FLT3, 1 × penicillin–streptomycin. At day 7, the suspensions were removed and IMDM containing 2% human peripheral blood plasma, 3% human AB serum, 3 IU/mL heparin, 10 µg/mL insulin, 100 ng/mL M-CSF and 50 ng/mL FLT3 were added. Then, the adherent cells were cultured for another 4 days. At D11, all the adherent cells will differentiate into macrophages as our previously described [2+, and 5 mM Ca2+. 1 × 105 cells were collected for cytospin analysis.Erythroblasts and “EBI-like” macrophages were derived from cord blood CD34escribed , 17, 31.https://www.biosino.org/node) under accession number OEP002596.RNA-seq was prepared and analyzed as previously described , 31, 32.Absolute cell count was measured via flowcytometry using 123count eBeads™ Counting Beads (Cat#: 01–1234-42) according to the manufacturer’s protocol.+ macrophages were enriched via F4/80 microbeads . BM cells were incubated with F4/80 microbeads for 15 min on ice in the dark. The cells were washed with PBS plus 2% FBS and 2 mM EDTA, centrifugated at 300 g for 10 min, and resuspended with 2 mL PBS plus 2% FBS and 2 mM EDTA. Finally, the BM F4/80+ macrophages were enriched by Quadro MACS following the manufacturer's instructions. More than 90% purification was achieved, as previously described [Control and GM-CSF-treated mouse BM F4/80escribed . These cCD163, CD169, EMP and αV-integrin used in the present study are the same in our previous work [IL1-β, TNF-α, iNOS, TGF-β, IL-10 and Arg1 are listed in Additional file QRT-PCR, cytospins and Giemsa-Wright staining were performed as in our previous papers , 31, 32.ous work . The priP < 0.05 was considered to indicate a statistically significant difference.GraphPad Prism 9.0 software  was used to perform the statistical analysis. All the experiments were replicated at least three times. All data were reported as mean ± SEM. Comparisons between different groups were performed by Student’s t test. + cells as previously described [To begin to study the potential roles of GM-CSF on human EBI formation, we performed human EBI formation assay using “EBI-like” macrophages and late-stage erythroblasts derived from human cord blood CD34escribed . “EBI-liescribed , 14, 17.escribed , 36–38. To define transcriptional changes following GM-CSF treatment, we performed RNA-seq analysis. As expected, GM-CSF-treated human “EBI-like” macrophages clustered distinctly from control human “EBI-like” macrophages in principal component analysis  expressed by EBI macrophages. ChIP-X Enrichment Analysis Version 3 (ChEA3) indicates that several key molecules significant for the function of EBI macrophages such as CD163, VCAM1, HMOX1,MERTK,AXL, and IGF1 may be regulated by NR1H3 or MAF , II (CD117+CD71low/medi), and III  and M2  surface markers on mouse BM EBI macrophages using flowcytometry. Notably, GM-CSF significantly increased the expression of MHC-II Fig. A, F but GM-CSF is barely detectable in the peripheral blood of healthy people but significantly increases in the presence of inflammatory conditions, such as, COVID-19, SCD, and so on , 3, 9. IIn the present study, we tested the roles of GM-CSF in human and mouse EBI formation, finding that GM-CSF significantly decreases EBI formation both in vitro and in vivo. G-CSF also impairs EBI formation in vivo , 21. ConMarcel Bessis in 1958 [GM-CSFR (CSF2R) is not expressed by late-stage erythroblasts but is highly expressed in human EBI macrophages, suggesting that GM-CSF may be unable to directly signal erythroblasts. Alternatively, the low expression of GM-CSFR in early-stage erythroblasts (FPKM < 10) may be insufficient, raising questions about the indirect effects of GM-CSF on erythropoiesis in vitro and in vivo. EBI, first described by  in 1958  is compo in 1958 , 45. Usi in 1958 , we foun in 1958 , 37, 39.Because GM-CSF can stimulate numerous inflammatory processes and increase expression of pro-inflammatory chemokines, an anti-GM-CSF strategy might have broader effects than other immunomodulatory approaches in immunosuppressive therapy . Indeed,Primitive and definitive erythropoiesis in mouse embryos requires the signal transduction of GM-CSF . HoweverMertk−/− mice showed significantly decreased engulfment of pyrenocytes by EBI macrophages [Axl−/− mice showed decreased a 50% decrease in phagocytotic apoptotic cells [Phagocytosis of senescent RBCs represents another important role of EBI macrophages during erythropoiesis. In general, RBC clearance is thought to occur mainly in the spleen, where senescent RBCs are phagocytosed by splenic macrophages . Our prerophages . Macrophic cells . Phosphaic cells . As suchMany proinflammatory cytokines  are known to inhibit steady-state BM erythropoiesis . Heat-kiIn summary, we have identified previously unknown roles of proinflammatory cytokine GM-CSF in erythropoiesis. Based on our findings, we propose that GM-CSF significantly impairs erythropoiesis by affecting the functioning of EBI macrophages. This conclusion is supported by several lines of evidence. (1) GM-CSF impairs human and mouse EBI formation by decreasing the expression of adhesion molecule CD163. (2) GM-CSF reduces mouse BM erythroblast numbers by decreasing the numbers of EBI macrophages and EBI formation. (3) GM-CSF impairs phagocytosis of senescent RBCs by decreasing the engulfment-related expression of Mertk, Axl and Timd4. Although we cannot exclude the effect of GM-CSF on erythropoiesis through influencing on other myeloid cells, our findings nonetheless provide new insights into GM-CSF’s role in impairing terminal erythroid differentiation at least in part by affecting EBI macrophages, and our findings can help better understanding of elevated GM-CSF levels of inflammatory diseases. Targeting GM-CSF or GM-CSF inhibitors might be a novel option for treating AI. This conclusion must be confirmed in mice in which the GM-CSFR gene is specifically depleted by EBI macrophages.Additional file 1: Figure S1. Targeting gene expression regulated by transcription factors analyzed using ChIP-X Enrichment Analysis Version 3 (ChEA3).Additional file 2: Figure S2. The gating strategy of BFU-E and CFU-E: the gating strategy of lin-CD16-CD32CD41-CD34-Scal- cell populations.Additional file 3: Figure S3. The gating strategy of terminal erythroid cells: the gating strategy of CD11b-Gr1-CD45-Ter119+ cells.Additional file 4: Figure 4. Peripheral Blood Routine of control and GM-CSF-treated mice: (A) RBCs assessments of control and GM-CSF-treated mice. (B) Hemoglobin assessments of control and GM-CSF-treated mice. (C) Hematocrit assessments of control and GM-CSF-treated mice. (D) Reticulocyte assessments of control and GM-CSF-treated mice. N=6.Additional file 5: Figure 5. GM-CSF does not induce stress erythropoiesis in SP. (A) The SP weight index (mg/g body weight) of control and GM-CSF-treated mice. (B) Representative flowcytometry image of terminal erythroid cells in SP in control and GM-CSF-treated mice. (C) Representative HE staining image of SP in control and GM-CSF-treated mice(20X). (D) Representative HE staining image of SP in control and GM-CSF-treated mice(100X).Additional file 6: Figure 6. The gating strategy of macrophages: the gating strategy of the CD11b-Gr1-SSClow cell population.Additional file 7: Figure 7. (A) Representative flowcytometry image of terminal erythroid cells in liposome-control and clodronate-loaded liposomes treated mouse BM. (B) Quantitative analysis of erythroblast numbers in in liposome-control and clodronate-loaded liposomes treated mouse BM (N = 6).Additional file 8: Figure 8. GM-CSF increased the mRNA expression of IL-1β, TNF-α, and iNOS and decreased the mRNA expression of IL-10, TGF-β, and Arg1. (A) The relative mRNA expression of IL-1β, TNF-α, and iNOS among GM-CSF treatment enriched BM EBI macrophages. (B) The relative mRNA expression of IL-10, TGF-β, and Arg1 among GM-CSF treatment enriched BM EBI macrophages (N = 3).Additional file 9: Table S1. The list of antibodies used in this study.Additional file 10: Table S2. Preparation of EBI enrichment buffer with different concentrations.Additional file 11: Table S3. The list of primers used in this study.Additional file 12: Table S4. The differentially expressed genes between control and GM-CSF treated human macrophages.


Both R- and S-enantiomers are connected into infinite helical chains by weak C—H⋯O hydrogen bonds between the phenyl ring of the benzoyl group and the carbamate carbonyl group.The title compound, C 21H25NO3, crystallized as a racemic twin in the Sohnke space group P21. In the molecular structure of the title compound, both enantiomers show a highly similar conformation with the urethane function and the benzoyl group showing an almost perpendicular arrangement [the dihedral angle is 72.46 (8)° in the S-enantiomer and 76.21 (8)° in the R-enantiomer]. In the crystal structure, molecules of both enantiomers show infinite helical arrangements parallel to the b axis formed by weak C—H⋯O hydrogen bonds between the phenyl ring of the benzoyl group and the carbamate carbonyl group. In case of the R-enantiomer, this helix is additionally stabilized by a bifurcated hydrogen bond between the carbonyl function of the benzoyl group towards both phenyl groups of the molecule.The title compound, C The same is true for the benzoyl groups . In case of the S-enantiomer, the carbamate and the benzoyl planes subtend a dihedral angle of 77.46 (8)° whereas for the R-enantiomer an angle of 76.21 (8)° is observed  Å], C3A—O2A [1.360 (7) Å], C3B—N1B [1.350 (7) Å] and C3B—O2B [1.363 (6) Å] being slightly shorter than a typical C—O or C—N single bond due to the partial double-bond character of the respective bonds in a carbamate.The carbamate functional moieties —Ph group attached to the carbamate oxygen atom —Ph group instead of the CH(Ph)—C(O)—Ph unit in the title compound . These two interactions lead to a double-strand arrangement of molecules parallel to the a axis.In the Cambridge Structural Database  dissolved in a small amount of DMSO were added in one portion. The consumption of the 2-bromo-1,2-diphenylethan-1-one was monitored by TLC, and after 30 min the reaction mixture was poured onto ice to quench the reaction. After extraction with dichloromethane (3 × 40 ml), the combined organic phases were washed with brine, separated and dried over Na2SO4. The solvent was removed in vacuo and the crude product was recrystallized from n-hexane/ethylacetate  to afford the title compound  as a colourless crystalline solid. M.p. 485 K; 1H NMR  [ppm]: δ = 7.96 , 7.50–7.47 , 7.39–7.32 , 6.88 , 4.05 , 3.86 , 1.28 ; 13C NMR  [ppm]: δ = 195.4 (C=O), 154.8 (NC=O), 135.2, 134.5, 133.3, 129.0, 129.0, 128.9, 128.7, 128.7 (CPh), 77.7 (C benzylic), 46.8, 45.9 [(H3C)2CH–], 21.6, 21.4 [(H3C)2CH–].Diisopropylamine  and one equivalent of caesium carbonate  were placed in a Schlenk tube and dissolved in anhydrous DMSO (150 ml). The tube was sealed with a septum, and two balloons filled with COUiso(H) = 1.2(C) for methine and hydrogen atoms of the phenyl group or 1.5×Ueq(C) for methyl groups. The crystal studied was refined as a two-component twin with fractions of 29% vs 71%.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021010367/wm5618sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021010367/wm5618Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021010367/wm5618Isup3.cmlSupporting information file. DOI: 2114278CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Discolaimium dubium are studied, including their morphological and morphometric characterization, molecular analysis (LSU-rDNA) and the description of the male for the first time. For comparative purposes, this species is distinguished by its 1.10 to 1.40 mm long body, lip region offset by constriction and 8 to 10 µm wide, odontostyle 7.5 to 10.5 µm long with aperture occupying 59 to 76% of total length, neck 300 to 362 µm, pharyngeal expansion 127 to 181 µm long or 44 to 46% of the total neck length, uterus simple and 38 to 53 µm or 1.2 to 1.5 times the body diameter long, V = 52 to 58, tail conical  with rounded tip and a hyaline portion occupying 14 to 15% of tail length, spicules 30 to 32 µm long, and two or three widely space ventromedian supplements with hiatus. Both morphological and molecular data support its belonging to the genus Aporcella, whose monophyly is confirmed and to which the species is formally transferred as A. dubia (Three Iranian populations of A. dubia  comb. n. DiscolaimoidesDiscolaimium dubium based on the holotype plus 25 females collected from several European enclaves in Italy, the Netherlands and Switzerland. Later, it was recorded from the Netherlands again . Morphometrics included Demanian indices and the usual measurements and ratios. Line illustrations were prepared using CorelDRAWFor the molecular phylogenetic studies, DNA samples were extracted from one or two females selected from each population, studied individually on temporary slides, placed on a clean slide containing a drop of distilled water or worm lysis buffer (WLB), and crushed by a sterilized scalpel. Then, the suspension was transferred to an Eppendorf tube containing 25.65 μl ddH2O, 2.85 μl 10 × PCR buffer and 1.5 μl proteinase K (600 μg/ml) . The tubes were incubated at −80°C (1 hr), 65°C (1 hr) and 95°C (15 min). Each sample was regarded as an independent DNA sample and stored at −20°C until used as polymerase chain reaction (PCR) template. Primers for 28S rDNA D2-D3 amplification/sequencing were forward primer D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and reverse primer D3B (5´-TCGGAAGGAACCAGCTACTA-3´) . The 25 Paravulvus hartingii  homology search program was used to compare the newly generated sequences with other available sequences in GenBank database. The recently generated sequences were aligned with the other segments of 28S rDNA gene sequences retrieved from the database using MEGA6 software . Paravulingii de   nematodes of medium size, 1.10 to 1.40 mm long. Body cylindrical, tapering toward both ends, but more so toward the posterior extremity as the tail is conical. Upon fixation, habitus regularly curved ventrad, to an open C shape in females and nearly J shape in males. Cuticle two-layered, 1 µm thick at anterior region, 1.5 to 2.5 µm in mid-body and 2.5 to 3.5 µm on tail, consisting of thin outer layer bearing fine but conspicuous transverse striation, and thicker inner layer, more appreciable at caudal region. Lateral chord 7 to 10 µm broad, occupying 21 to 28% of mid-body diameter, lacking any differentiation. Lateral pores obscure. Lip region somewhat truncate anteriorly, slightly expanded (but not discoid), that is, slightly wider than its adjoining body, 2.6 to 3.3 times as wide as high and ca one-third (26-37%) of body diameter at neck base; lips mostly amalgamated, with weakly protruding papillae. Amphidial fovea cup-like, its aperture occupying 4.5 to 6 µm or more than one-half (50-63%) of lip region diameter. Cheilostom comparatively short and broad, with thick walls. Odontostyle relatively short and robust, 4.1 to 4.6 times as long as wide, nearly equal (0.9-1.0 times) to the lip region diameter, and 0.66 to 0.76% of the total body length; aperture large, 5.5 to 6.5 µm long, occupying more than one-half (59-76%) of the odontostyle length. Guiding ring simple, but conspicuous, situated at 4.5 to 5.5 µm or 0.5 to 0.6 times the lip region diameter from the anterior end. Odontophore rod-like, 1.7 to 2.0 times the odontostyle, lacking any differentiation. Pharynx entirely muscular, consisting of a slender section gradually enlarging into the basal expansion that is 7.7 to 9.5 times as long as wide, 4.5 to 5.5 times the body diameter at neck base and occupies 44 to 46% of the total neck length; gland nuclei located as follows (n = 2): DO = 58, 60; DN = 63; S1N1 = 72, 75; S1N2 = ?82, S2N = 88, 90. Pharyngo-intestinal junction with a rounded conoid cardia 5.5−9.5 × 8.5–12.5 µm, surrounded by intestinal tissue. Tail conical, with rounded tip, ventrally nearly straight, dorsally regularly convex; inner core not reaching the tail tip, therefore a hyaline portion is present occupying 5 to 5.5 µm or 14 to 15% of tail length; caudal pores two pairs, at the middle of tail, one nearly dorsal, other subdorsal.Slender to very slender  of body diameter: pars proximalis 7–10 × 8–11 µm, with slightly convergent walls surrounded by weak circular musculature and pars distalis 3.5 to 4.5 µm long. Vulva a somewhat posterior, transverse slit. Prerectum 3.0 to 6.6, rectum 1.2 to 1.5 times the anal body diameter long.Genital system didelphic-amphidelphic, with both branches equally and moderately developed, the anterior 111 to 203 µm long or 9 to 16% of body length, the posterior 108 to 202 µm or 8 to 17% of body length. Ovaries reflexed, relatively small, often not reaching the oviduct-uterus junction, the anterior 52 to 74 µm, the posterior 54 to 90 µm long. Oviduct 44 to 74 µm or 1.3 to 2.1 times the body diameter long, consisting of a slender distal portion made of prismatic cells and a moderately developed Genital system diorchic, with opposed testes. In addition to the adcloacal pair, located at 8 to 9 µm from the cloacal aperture, there are two or three, widely spaced 13.5 to 16 µm apart, ventromedian supplements, the most posterior of them situated far from the ad-cloacal pair, at 59 to 79 µm. Spicules dorylaimid, regularly curved ventrad and relatively robust, 3.9 to 4.9 times as long as wide; head small, 2.5 to 3 μm long, occupying 8 to 10% of total spicule length, with its dorsal side slightly longer than the ventral one; ventral side bearing visible hump and hollow, median piece occupying 85 to 87% of spicule length. Lateral guiding pieces 8.5 to 9 µm long. Prerectum 2.4 to 4.5, cloaca 1.4 to 1.5 times body diameter at level of cloacal aperture.ca 800 bp long. Their analysis has allowed the elucidation of evolutionary relationships of the species. The phylogenetic results are presented in Three D2-D3 28S rDNA sequences were obtained, Prunus cerasus L.); (ii) Osku, Teymourlu (GPS coordinates: N 37°48′ 35′′E 45° 53′ 37′′), collected from rhizosphere of Almond trees (Prunus dulcis L.); and (iii) Azarshahr, Qazi Jahan village (GPS coordinates: N 37°46′ 27′′E 45° 55′ 51′′), from the rhizosphere of walnut trees (Juglans regia L.).This species has been collected in three locations of East-Azarbaijan province, northwestern Iran: (i) Sufiyan, Roodghat area, Zeinabad village , in soil around roots of black cherry trees  subclade with members of the genus Aporcella . Consequently, it is transferred to Aporcella as A. dubia , tail conical  with rounded tip and a hyaline portion occupying 14 to 15% of tail length, spicules 30 to 32 µm long, and two or three widely spaced, ventromedian supplements with hiatus.For comparative purposes and taken into consideration the results provided in the present study, a-ratio ≥ 39), short odontostyle (< 17 µm long) and comparatively short pharyngeal expansion , A. dubia resembles A. debruinaeA. minorA. tropica , narrower lip region , shorter odontostyle  and much shorter spicules . From A. minor, a similar species recently described from Iran too, in its more slender body , narrower lip region (8-10 vs 11-13 µm), shorter odontostyle (7.5-10.5 vs 12.5-14 µm), and more anterior vulva (V = 52-58 vsV = 58-61).By having slender body  and discolaims  of this clade becomes controversial and problematic. The results obtained in the present contribution suggest that some species currently classified under Discolaimium (and perhaps other species under Discolaimoides too) might belong to Aporcella and that, as consequence, a re-evaluation and/or re-definition of these genera are recommendable and necessary.The transference of


Salmosalar). Thus, we examined how: (1) moderate hypoxia(∼70% air saturation) at 12°C for 3 weeks; (2)an incremental temperature increase from 12°C to 20°C (at1°C week−1) followed by 4 weeks at20°C; and (3) treatment “2” combined with moderatehypoxia affected transcript expression in the liver of post-smolts as comparedto control conditions . Specifically, we assessed theexpression of 45 genes related to the heat shock response, oxidative stress,apoptosis, metabolism and immunity using a high-throughput qPCR approach(Fluidigm Biomark™ HD). The expression profiles of 27“stress”-related genes indicated that: (i) moderate hypoxiaaffected the expression of several stress genes at 12°C; (ii) theirexpression was impacted by 16°C under normoxic conditions, and thiseffect increased until 20°C; (iii) the effects of moderate hypoxia werenot additive to those at temperatures above 16°C; and (iv) long-term(4 weeks) exposure to 20°C, with or without hypoxia, resulted ina limited acclimatory response. In contrast, the expression of 15 immune-relatedgenes was not greatly affected until temperatures reached 20°C, and thiseffect was particularly evident in fish exposed to the added challenge ofhypoxia. These results provide valuable information on how these two importantenvironmental factors affect the “stress” physiology andimmunology of Atlantic salmon, and we identify genes that may be useful ashypoxia and/or temperature biomarkers in salmonids and other fishes.The marine environment is predicted to become warmer, and more hypoxic, and theseconditions may negatively impact the health and survival of coastal fishspecies, including wild and farmed Atlantic salmon ( Increasing water temperatures, and de-oxygenation of the oceans (hypoxia), as aresult of global warming may pose critical challenges to the performance, health andsurvival of marine organisms , high-fish density/crowding,low-rates of water exchange, inter-tidal changes and/or algal blooms  or acclimated to constant temperatures .Given that multi-stressor experiments are essential to more accurately predicting theeffects of foreseen environmental scenarios  related to theheat shock response, oxidative stress, apoptosis, metabolism and immunity frompost-smolt Atlantic salmon of the same experimental groups used in #16-90-KG) and followed guidelines set by the Canadian Council on AnimalCare. Furthermore, all sections of this study adhered to the ARRIVE Guidelines forreporting animal research  and mortality are publishedin All experimental procedures described herein were approved by the InstitutionalAnimal Care Committee of Memorial University of Newfoundland  at 15 L minute−1. The fishwere acclimated for four weeks under optimal conditions and werefed a ration of 1% body weight day−1 with commercialsalmon feed during this period. All fish in this experiment were implanted with PassiveIntegrated Transponder (PIT) tags  approximately 2 months before the experiment for identificationpurposes.The experiment was performed from March to August 2017 at the Laboratory forAtlantic Salmon and Climate Change Research (LASCCR), Memorial University, St.John’s, Newfoundland, Canada. Post-smolt Atlantic salmon of Saint JohnRiver  origin obtained from Northern Harvest Sea Farms were randomlydistributed into six 2.2 m−1) as illustrated in −1) at ∼100% air sat., and thenmaintained at 20°C for 27 days (∼4 weeks); and(3) Warm & Hypoxic (WH): decrease in water oxygen content to∼70% air sat. over 1 week, followed by 2 weeks of acclimation tothis oxygen level, and then the same temperature regimen(12°C–20°C at a rate of 1°Cweek−1) at 70% air sat. Throughout thisexperiment, the salmon were carefully fed by hand to satiation twice daily withthe commercial salmon feed as indicated above.Three hundred and sixty Atlantic salmon wererandomly assigned to the following three treatment groups . Based on a priori multiple regression powercalculations, and experiments with similar designs, 24 samples at TP 1–5had sufficient statistical robustness and power (80%) to detectsignificant differences atP < 0.05 with anestimated medium–large effect size (f2 = 0.43) (−1) of the anesthetic MS-222 followedby cranial concussion. For the current qPCR study, a 200 mg piece ofliver was quickly collected from each fish, flash-frozen in liquid nitrogen, andstored at −80°C until further processing.In this experiment, fish were sampled at 12°C, 3 days afterreaching 16°C, 18°C and 20°C, and finally after4 weeks at 20°C. For each of the five temperature exposure timepoints (TP 1–5), we randomly collected four fish per tank, resulting ineight fish per treatment group  following the manufacturer’s protocol.For RNA extraction, 100 mg of flash-frozen liver tissue per sample washomogenized in 800 µl of QIAzol-Lysis Reagent  for 2 minutes at 20 Hz using aTissueLyzerII system with 5 mm stainless steel beads  according to the manufacturer’s instructions.To remove potential protein, lipid and genomic DNA contamination, we performedRNA precipitation followed by DNase I treatment and column purification asexplained in (® 44 Kmicroarray study (ampk), apoptosis regulator BAX (bax),glucocorticoid receptor 1 (gr1) and superoxide dismutase 1(sod1). All of these 45 GOIs were related to one ormore of the following functional categories: heat shock response, stressresponse, oxidative stress response, apoptosis, cellular metabolism, theimmune response or transcriptional regulation (DNA methylation) (http://bioinfo.ut.ee/primer3/) and were quality tested asdescribed in (rprl32 and BT043656), eukaryotic translationinitiation factor 3 subunit D (eif3d and GE777139) andpolyadenylate-binding protein 1 (pabpc1 and EG908498)](We quantified the mRNA expression of 41 genes of interest (GOI) that werepre-selected based on our previous Agilentylation) .ParalogThe relative transcript expression values of the 45 GOIs and the threereference genes were assessed for the 120 liver samples using the real-timeqPCR Fluidigm Biomark™ HD system  that is based on a 96.96 Dynamic Array™ IFC (GE-arrays), andaccording to the protocol described in (TC), standard deviation (SD), and thecoefficient of variation (CV) were calculated. As a quality control forpipetting errors, TC values (duplicates) with aCV ratio higher than 4%  andeif3d (geNormM = 0.313) were identified as themost stable combination (geNormV = 0.115). The RQ of each GOI wasdetermined through normalization to the geometric mean of the two endogenousreference genes (rpl32 and eif3d),including the amplification efficiencies (Supplementary Table S1), and bysetting the sample with the lowest expression level as the calibrator sample . For each lmermodel, the residual distribution and the model fit were examined. Modelsshowing significant results were followed by an estimated marginal means(emm) posthoc test with the FalseDiscovery Rate (FDR) set atP < 0.05 andusing the emmeans function in R , within andbetween each of the five sampling points . Then, thepercentage of the total variance contribution (%) for each gene wasevaluated and illustrated in factor maps by applying thefactoextra package in R  cluster algorithm.All statistical tests and figures were computed in the R environment (v.3.5.1) .To explage in R . Thescoion in R . For eacage in R . Finallylmermodels and emmeans posthoc tests with the FDR set atP < 0.05 asdescribed in the previous paragraph. Each lmer model wasgraphically examined (histogram and qqplots), and the residuals were testedfor normality  andequal variance (Levene test). The RQ-values weretransformed with either log2 or box-cox transformation whennecessary to fulfill the assumptions of normality and equal variance.The differences in transcript expression between the treatment groups weredetermined for each gene individually by performing https://doi.org/10.1594/PANGAEA.913696). Supplemental files are available atfigshare (https://figshare.com/s/893b1284a406be4aeb38). Supplementary Table S1contains details about qPCR primers. Supplementary Table S2 contains the completeresults of the multivariate statistical approach. Supplementary Table S3 containsthe complete results of the univariate statistical approach. Supplementary Table S4contains the fold-change (FC) ratios of the 45 target genes. Supplementary Figure S1is a clustered and hierarchical ordered heatmap based on fold-change expressionvalues of the 45 target genes. Supplementary material is available at figshare:https://figshare.com/s/893b1284a406be4aeb38.The obtained threshold cycle (CT) values for the samples of the current study areaccessible on-line at the PANGAEA data sharing server (−1)that reflects summer conditions in Atlantic Canada at 18°C(P < 0.0001) and a5.51-fold increase at 20°C(P < 0.0001) ascompared to CT-fish held at 12°C (Oncorhynchusmykiss) (serpinh1 mRNA in the liver may have assisted in thestabilization of collagen molecules within the extracellular matrix (ECM), andfurther enabled the elimination of generated ROS to maintain cellularhomeostasis during this thermal challenge. This hypothesis agrees with what hasbeen observed for Atlantic salmon exposed to 19°C for 21 and56 days  which had a 1.51-foldup-regulation at 18°C(P = 0.041) and a3.08-fold increase at 20°C(P < 0.0001) inWN-fish as compared to CT-fish  exhibited ∼27- and50-fold elevations in hsp90aa mRNA in the head kidney andliver, respectively (Oncorhynchus nerka and O. gorbuscha) had∼2-fold increased hsp90aa1 expression in the gillsafter 7 days at 19°C as compared to 14°C (hsp90a (in addition to serpinh1) wereup-regulated in the Chinook salmon (O. tshawytscha) gill whenthe fish were exposed to 18°C vs. 10°C or 14°C for6 days. Furthermore, we found that hsp70 (encoding HSP70) transcript expression in the WN-group was up-regulated at both 18°C and20°C  ascompared to CT-fish at 12°C  at 18°C(P = 0.0057), and1.73-fold increase at 20°C(P = 0.0048) inWN-fish  in the liver of WN-fish with warming to 18°C(P = 0.0035), andthe 3.18-fold increase at 20°C(P = 0.0001), incomparison to CT-fish (casp8(encoding caspase 8) in WN-fish at 20°C(P = 0.0001) incomparison to CT-fish at 12°C   due to increased oxidative stress(An activation of anti-apoptotic processes was apparent by the significant2.89-fold up-regulation of the transcript  CT-fish . JunD ac at 12°C  indicate at 12°C . The incsp90aa1)  could hahcn1 transcripts inthe liver of WN-fish once temperatures reached 20°C(P < 0.0001) incomparison to CT-fish (+) and potassium (K+) ions and thecontrol of the inner mitochondrial membrane potential, and these are connectedwith the activity of the respiratory chain and ATP synthesis  transcript expression at 16°C(P = 0.0019) and20°C(P < 0.0001) inWN-fish  at temperatures from 16°C to 20°C(P < 0.0001) exposed to warming temperatures(0°C–25°C)  when exposed to an increase from 15°C to25°C (rraga mRNA (encoding ras-related GTP-bindingprotein A) was also decreased by 1.89-fold with warming to 20°C(P < 0.0001) incomparison to CT-fish (P < 0.0014) to5.26-fold (P < 0.0004)down-regulation of the redox gene cyp1a1 (encoding cytochromeP450 1A1) upon exposure to temperatures between 18°C and 20°C,as compared to 12°C  suggests 0.0001). A down- to25°C . Theexp CT-fish , and thi to 12°C , is conssod1 (encoding superoxide dismutase 1),cat , and txn (encodingthioredoxin) in the WN-group did not differ significantly from those of theCT-group (see Pagothenia borchgrevinki) whenexposed to an increase in water temperature (+5.6°C) for3 weeks, and this resulted in the accumulation of oxidative damage inthe liver (measured as protein carbonyls and lipid peroxides)  was notsignificantly different in WN-fish as compared to CT-fish  of juvenile Atlantic salmonexposed to a long-term increase from 12°C to 20°C  do not causesignificant phenotypic changes  orinduce a physiological stress response  in salmon of Saint John River stock. However, the increasedexpression of several genes  or decreased attemperatures beginning at ∼16°C indicates that these fishmounted a robust cellular stress response to cope with these conditions.An elevation in circulating cortisol levels is a highly conserved response tostressor exposure in vertebrates, and is essential for animals to overcomeand/or adapt to particular stressors . In our lmer-PC-2-Group:P = 0.011;Temperature:P < 0.0001;Group*Temperature:P < 0.0001;explaining 22% of the variance; emmeans-18°C: CT vs. WNP = 0.044;Supplementary Table S2, emmeans-20°C: CT vs.WN P < 0.0001;emmeans-WN: 16°C vs. 20°CP < 0.0001,18°C vs. 20°CP = 0.001;Supplementary Table S2, c1ql2 (c1q-like protein 2), which had a 6.39-fold higherexpression in the liver of WN-fish once temperatures reached 20°C(P < 0.0001) ascompared to CT-fish  resulted inthe differential expression of 15 immune-relevant genes measured in the salmonliver  incomparison to CT-fish at 12°C (tnfrsf6b (encoding TNF-R superfamily member6B) at 18°C(P = 0.026), butnot at 20°C(P = 0.228), inWN-fish as compared CT-fish at 12°C  with warming to 20°C(P = 0.0228) ascompared to 12°C (epx transcripts (eosinophil peroxidase) at20°C(P = 00042) in WN-fishrelative to CT-fish at 12°C , they induced similar, and evenhastened, innate immune responses as compared to CT-fish at 12°C  generally had up-regulated constitutive levels ofemmeans-12°C: CT vs. WHP = 0.032, WN vs.WH P = 0.007;Supplementary Table S2, emmeans-12°C: CT vs. WHP = 0.819, WN vs.WH P = 0.581,Supplementary Table S2, hif1α, calm, cld3) and cellular stress response functionswere significantly down-regulated in WH-fish as compared to WN-fish  was 1.45-fold lower in WH-fish in comparisonto CT-fish, and differed significantly from the 1.19-fold up-regulation inWN-fish at 12°C(P = 0.0287) when exposed to chronichypoxia (15 days at 30% DO), whereas its expression was notaffected in the liver. Similarly, hif1α expression in the liver of Atlanticsalmon when exposed to 4–5 mg O2L−1 for 120 days at 12°C. In this study,the significant down-regulation of hif1α at12°C might have been related to the limited period of hypoxia exposure(3 weeks) and/or the moderate nature of the hypoxia. A quite similarexpression profile was observed for calm ,which was 1.45-fold down-regulated in WH-fish as compared to CT-fish at12°C, and differed significantly from the 1.29-fold up-regulation inWN-fish (P = 0.0115)(2+), and it regulates many crucial processes such asgrowth and cell proliferation  is affectedby hypoxia  (cirbp transcript expression in thehippocampus of rats after exposure to chronic hypoxia (21 days) (hif1α expression through the direct modulation ofmicroRNAs (prdx6(P < 0.0001) asthis oxygen level is similar to summer conditions in coastal sea-cages inNewfoundland , which i WN-fish . Amongst 0.0287). HIF1-A  0.0287). In seveAlevels . For ins 0.0115). Calmoduferation . The act 0.0198). This re 0.0001). Peroxir 0.0001), and mayCollectively, these findings demonstrate that chronic exposure to moderatehypoxia (∼70% air sat.) at 12°C was a mild stressor forpost-smolt Atlantic salmon, and suggest that it likely initiated molecular andphysiological stress-related pathways in the salmon’s liver cells.emmeans-16°C: CT vs. WHP = 0.028,18°C: CT vs. WHP < 0.0001;20°C: CT vs. WHP < 0.0001;emmeans-WH: 16°C vs. 18°CP = 0.029,16°C vs. 20°CP < 0.0001,18°C vs. 20°CP < 0.0001;Supplementary Table S2, emmeans-16°C: WN vs. WHP = 0.303;18°C: WN vs. WHP = 0.861;20°C: WN vs. WHP = 0.672;Supplementary Table S2, cirbp, calm, hif1α, hspd1,ndufa1 and prdx6) as temperature increasedfrom 12°C to 20°C as compared to normoxic fish (serpinh1and jak2) after the initial temperature increases to16°C (serpinh1was 2.00-fold higher expressed in hypoxic fish after warming to 16°C(P = 0.0267) ascompared to the marginal 1.21-fold increase in normoxic fish at 16°C(P = 0.4798)(jak2 only inWH-fish at 16°C as compared to CT-fish at 12°C(P = 0.0217), and thi 0.4798). Inaddi 0.0217), suggestpdk3 [encoding pyruvatedehydrogenase kinase 3, an essential regulator for glycolysis and themaintenance of glucose homeostasis (reviewed in P = 0.0104)(gck (encodingglucokinase) expression was decreased by 5.26-fold in WN-fish(P = 0.0126) andby 7.14-fold in WH-fish(P = 0.0246) at18°C as compared to 12°C revealed that the same WN- and WH-fish (at 20°C for 3 days)showed a marked down-regulation of pathways connected to the metabolism ofcarbohydrates, proteins, fatty acids and lipids in the liver, and that theexpression of 19 biomarker genes strongly correlated with seven healthparameters in this group ; which w to 12°C  at high emmeans-18°C: CT vs. WHP = 0.044;Supplementary Table S2, emmeans-20°C: CT vs. WHP < 0.0001;Supplementary Table S2, emmeans-18°C: WN vs. WHP = 0.848;20°C: WN vs. WH P = 0.182;Supplementary Table S2, i.e., tnfrsf6b,camp-a and il8) were more strongly affected in WH than WN-fishas compared to CT-fish. For example, the expression of tnfrsf6bwas 1.29-fold higher in WH-fish as compared to WN-fish at 20°C(P = 0.0103)(camp-a inWH-fish as compared to WN-fish at 20°C(P = 0.0534) ascompared to CT-fish, whereas there was no difference between WN- and CT-fish andAcinetobacter spp. disappear , and thi 0.0534) suggests 0.0534). In salm CT-fish, and thi CT-fish. Exposuri.e., hcn1,c1ql2 and jund). More specifically, the genehcn1 returned toward initial levels after prolongedexposure to 20°C with moderate hypoxia , aperiod that would naturally correspond to August and early September on the eastcoast of Canada. Interestingly, we observed a slightly lower impact ontranscript expression changes in the warmed groups after long-term exposure to20°C, and that overall, the transcript expression of 27“stress”-related , A–C and hypoxia . Given temmeans-WH: 20°C-3dvs. 20°C-4wksP = 0.003;18°C-3d vs. 20°C-4wksP = 0.049;Supplementary Table S2, Forte V II; containing both bacterial andviral components/antigens) their capacity to mount an innate immuneresponse was not impaired in comparison to fish maintained at 12°C wassignificantly reduced in hypoxic fish maintained for 4 weeks at 20°C,while it remained up-regulated in WN-fish , a simulserpinh1, hsp90aa1, hsp70 andhspd1), oxidative stress response  and hypoxic signaling  were comparable in WN- and WH-fish exposedto 20°C for 3 days vs. 4 weeks  and collagenI, was still up-regulated in Atlantic salmon exposed to 19°C for 21 and56 days.Nevertheless, the expression of 13 target genes related to the heat shockresponse  eitherremained differentially expressed, or partially returned to basal (12°C)levels. Overall, it appears that fish that experienced the combined stressors ofhigh temperature and low oxygen levels had transcript expression levels thatwere similar to those of 18°C fish. It is unclear whether this wasbecause they had partially acclimated to the conditions of high temperature(20°C) and hypoxia, or whether prolonged exposure to these conditionsled to the exhaustion of particular cellular responses/pathways.In summary, long-term (4 weeks of) exposure to 20°C did notresult in increased transcriptional responses. Rather the investigated immuneand “stress”-related response pathways (dnmt1 (encoding DNA methyltransferase 1) expression was2.13-fold reduced in salmon challenged with moderate hypoxia(∼70% air sat.) at 12°C as compared to fish undernormoxia (100% air sat.)(P < 0.0001), butwas not further impacted by warming to 20°C (dnmt1transcript expression as compared to CT-fish at 12°C(P < 0.0001) thatreached a similar magnitude of down-regulation by 20°C (2.04-fold)(3) to the 5' carbon end ofcytosine (5mC) and is a key player in the epigenetic regulation of transcriptexpression  offive of the herein investigated treatment responsive genes , and that thesealterations correlated with transcript expression changes . DNMT1 i−1) alone, or in combination with moderate hypoxia(∼70% of air sat.); conditions that simulate summer conditions insalmon aquaculture sea-cages in Canada and the North Atlantic , apoptosis , oxidative and/or generalstress responses  and that are responsive to hypoxia.In contrast, the overall expression of 15 immune-related genes was only impactedwhen temperatures reached 20°C, and the responsive genes were mainlyassociated with innate immunity and apoptosis . Interestingly, moderate hypoxiaalone impacted transcript expression in the liver of salmon  at 12°C, andthis suggests that this condition triggered minor cellular stress or acclimationresponses. However, the transcriptional responses of these genes were not additiveor synergistic when hypoxia-acclimated salmon were subsequently challenged with anincremental temperature increase to 20°C. In fact, these transcriptexpression responses were of a similar magnitude. On the other hand, the overallexpression of 15 immune-related genes was more strongly impacted in the liver ofhypoxia-acclimated fish subjected to the temperature increase to 20°C; thus reflecting a higher state of constitutiveimmunity. Finally, after long-term (4 weeks) exposure to 20°C, thefish showed a trend toward reduced stress and immune transcript expression.Collectively, our data indicate that salmon can adjust their physiology toincreasing temperatures, but have some, yet limited, capacity to acclimate totemperatures as high as 20°C. This may not be surprising as mortality ofthese salmon started to occur when temperatures reached 22°C with∼30% of mortality by 23°C .In futu


Beauveria bassiana and Metarhizium anisopliae against whiteflies in Ethiopia.This study was aimed to assess the enzymatic activity and pathogenicity potential of Beauveria bassiana AAUMB-29, AAUMFB-77, and AAUEB-59 generated the highest chitinase (EI = 3.41), lipase (EI = 4.45), and protease activities (EI = 5.44) respectively. The pathogenicity study of isolates on whitefly nymphs and adults indicated significant variation (P < 0.05) with mortality ranging from 71.67 to 98.33% and 60 to 100% against Bemisia tabaci and Trialeurodes vaporariorum nymphs respectively. The mortality of adults was between 58 and 94.27% against B. tabaci and 59.03 to 95.37% against T.vaporariorum. The result also showed that AAUMB-29, AAUMFB-77, and AAUDM-43 were the most virulent with LC50 values of 2.7 × 104, 5.3 × 104, and 5.4 × 104 conidia/ml against nymphs of B. tabaci, and with LC50 values 6.8 × 104, 8.2 × 104, and 7.2 × 104 conidia/ml against nymphs of T. vaporariorum, respectively. The B. bassiana AAUMB-29, B. bassiana AAUMFB-77, and M. anisopliae AAUDM-43 induced the highest whitefly mortality than other isolates. These isolates can be recommended for further tests under field conditions to fully realize their potential as effective biocontrol agents against whitefly pests in tomato.The data showed that The online version contains supplementary material available at 10.1186/s13104-022-06004-4. Bemisia tabaci and Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) are notorious sap-sucking pests causing serious damage to vegetable crops through direct feeding and transmission of several plant viruses [Solanum lycopersicum) is one of the most favorable hosts of B. tabaci and T. vaporariorum [Whiteflies  viruses . Tomato rariorum , and therariorum . The conrariorum , 5. Howerariorum .Beauveria bassiana and Metarhizium anisopliae are the most environmentally-friendly bio-control agents against sucking insect pests [B. bassiana and M. anisopliae provide an advantage to effectively control sap-sucking insect species over others [B. bassiana and M. anisopliae effectively controlled B. tabaci and T. vaporariorum with mortality ranging from 71 to 96.61% [Entomopathogenic fungi (EPF) ct pests . They prct pests , and toxct pests . These tr others . Differeo 96.61% –13.Aphis gossypiin [Pachnoda interrupta [Tuta absoluta [B. bassiana and M. anisopliae against whitefly species, B. tabaci and T. vaporariorum in Ethiopia.Increased interests in the use of entomopathogenic fungi in pest management options necessitate the selection of fungal isolates with high virulence that shows significant enzyme activities on target insects. In Ethiopia, locally isolated entomopathogenic fungi showed promising results for the control of agricultural pests such as ossypiin , Pachnodterrupta , and Tutabsoluta . HoweverB. bassiana and M. anisopliae were used in trials , KCl (0.5 g), MgSO4·7H2O (0.4 g), CaCl2·2H2O (0.1 g), powdered skim milk (25 ml of 15%), glucose (10 g), agar (12 g) and distilled H2O (1000 ml) [The 5 mm mycelial agar disc of each isolate was transferred in triplicates into casein hydrolysis agar composed of; (KH1000 ml) , and inc4)2 SO4 (1 g), K2HPO4 (1 g), KCl (0.5 g), NaCl (5 g), and MgSO4 (0.5 g), FeSO4 (0.01 g), agar–agar (20 g), colloidal chitin (5 g) and distilled H2O (1000 ml). They were incubated at 25 °C for 10 days.Isolates were screened for chitinase activity on the chitin-agar medium according to the method suggested by Maketon et al. . The 5-m. [2PO4 1.2, MgSO4·7H2O 0.3, KH2PO4 2, CaCl2 0.25, 0.003% NaCl, 2% agar, (NH4)2SO4 at 1%, and olive oil at 2% and incubated at 25 °C for 10 days. Enzymatic index (EI) was calculated using the following formula [Isolates were screened for lipase activity according to Falony et al. . The 5 m formula :\documen7 conidia/ml. After spraying, leaves were placed onto 0.2% water agar in a petri dish and adult whiteflies were released into treated leaves (15 adults/leaf) in triplicates and incubated at 25 °C for 10 days. Similarly, the mortality of whitefly nymphs was assessed by spraying leaf discs (30 mm in diameter) containing 20 nymphs with 3 ml of conidial suspension of 1 × 107 conidia/ml. Then leaf discs were placed onto 0.2% water agar medium in a Petri dish and incubated at 25 °C for 10 days. The median of lethal time (LT50) of each isolates at 10 days of post inoculation was calculated using probit analysis.Adult and nymph whiteflies were released to tomato leaves containing sprayed residues of fungal isolates as described by Mascarin et al. . Tomato The spore production of isolates was assessed according to Mascarin et al. . To quan5–1 × 108 conidia/ml) was evaluated to estimate the average lethal concentration (LC50) values of each isolate [The multiple-dose bioassay (1 × 10 isolate . Each tr(LT50) and the lethal concentration (LC50 and LC90) values were determined with probit analysis (IBM SPSS statics 20) [Mortality data were corrected using Abbott’s formula . The cortics 20) .B. bassiana AAUMB-29 performed best in chitinase activity (EI = 3.41), whereas B. bassiana AAUMFB-77 and B. bassiana AAUEB-59 exhibited the highest lipase (EI = 4.45), and protease activity (EI = 5.44) respectively.Entomopathogenic fungi were showed significant differences in their relative enzyme activities ranging from 1.20 to 3.41 for chitinase, 1.58 to 4.45 for lipase, and 1.72 to 5.44 for protease  in percentage mortality of hs Table . The mor50) mortality values, isolates fell within the range of 3.22 to 9.26 days for B. tabaci and 3.08 to 8.16 days for T. vaporariorum nymphs  with profuse sporulation ranging from 1.3 × 105 to 6.5 × 106 on B.tabaci and 1.6 × 105 to 5.8 × 106 conidia/cadaver on T. vaporariorum.Concerning lethal time at 50% (LT5–1 × 108 conidia/ml) response evaluations, the B. bassiana AAUMB-29 revealed the lowest LC50 values of 6.8 × 104 conidia/ml on T. vaporariorum,  against T. vaporariorum.Regarding the multiple dosage  of nymphs on B. tabaci and both isolates of B. bassiana AAUMB-29 and B. bassiana AAUMFB-77 achieved 100% nymph mortality on T. vaporariorum. Several studies also confirmed the great bio-efficacy of B. bassiana and M. anisopliae against B. tabaci and T. vaporariorum, with mortality values of 86.47 to 96.61% at the concentration of 1 × 106 conidia/ml in Italy [7 conidia/ml in Mexico [In this study, the in Italy , 71 to 8n Mexico . The disality 98.% of nympn Mexico .B. bassiana AAUMFB-77 yielded the highest numbers of spores with 6.5 × 106 conidia/cadaver on B. tabaci and 5.8 × 106 conidia/cadaver on T. vaporariorum nymphs. These numbers were slightly higher than the number of spores produced; 7.9 × 105 conidia/cadaver of whitefly nymph with B. bassiana [B. bassiana (8.3 × 107 per beetle cadaver) [B. bassiana AAUMB-29 which was highly effective against the nymphs of whitefly species gave the lowest LC50 values of 6.8 × 104 conidia/ml on T. vaporariorum  . The difcadaver) . With rereported .B. bassiana and M. anisopliae indicated differences in the production of chitinase, lipase, and protease enzymes. The B. bassiana AAUMB-29, B.bassiana AAUMFB-77, and M.anisopliae AAUDM-43 were the most virulent against whitefly nymphs and adults. The whitefly nymphs were more vulnerable to infection with M. anisopliae and B. bassiana than the adult stages of the whitefly species.This particular study showed that B. bassiana and M. anisopliae against whiteflies under in-vitro conditions. A future study is required under field conditions to realize the efficiency of isolates for the development of myco-insecticide.This study is limited to the enzymatic activities and bioassay study of Additional file 1: Table S1. Sources of Beauveria bassiana and Metarhizium anisopliae isolates used in this study. Table S2. The probit analysis of lethal concentrations values of B. bassiana and M. anisopliae in multiple dose-mortality response bioassays against T. vaporariorum nymphs 10 days post-fungal application. Table S3. The probit analysis of lethal concentrations values of B. bassiana and M. anisopliae in multiple dose-mortality response bioassays against B. tabaci nymphs 10 days post-fungal application. Figure S1. Extracellular activities of entomopathogenic fungi. Lipase activity of B. bassiana AAUMFB-77 (A), protease activity of B. bassiana AAUMB-29 (B), and Chitinase activity of M. anisopliae AAUDM-43 (C). Figure S2. Cultures of selected M. anisopliae and B. bassiana isolates on potato dextrose agar media. B. bassiana AAUMB-29 (A), B. bassiana AAUMFB-77 (B), and M. anisopliae AAUDM-43 (C). Figure S3. The mortality of whitefly adults with entomopathogenic fungi on tomato leaves. The mortality of whitefly adults by B. bassiana AAUMFB-77 (A), B. bassiana AAUMB-29 (B), and M. anisopliae AAUDM-43 (C).


Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis (UC), are chronic and recurrent intestinal inflammatory disorders. Numerous studies have revealed that the nucleotide-binding domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome plays a pivotal role in the pathogenesis of IBD, and inhibition of the NLRP3 inflammasome alleviates colitis in experimental animals. Our previous study showed that C646, an inhibitor of histone acetyltransferase p300, has a protective role in dextran sulfate sodium (DSS)-induced colitis by targeting the NLRP3 inflammasome, making us further study the inhibitors of histone deacetylases (HDACs) in the treatment of colitis. In this study, we have shown that WT161, an inhibitor of HDAC6, exerts a protective role in a colitis model, blocks NLRP3 inflammasome activation, disrupts ASC speck formation, and decreases the expression of NLRP3. This study uncovers a new inhibitor of the NLRP3 inflammasome and suggests its potential application in the treatment of active IBD. Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis (UC), are chronic and recurrent intestinal inflammatory disorders . AlthougNLRP3 gene are associated with susceptibility to UC  inflammasome plays a pivotal role in the pathogenesis of IBD . The NLRty to UC , and NLRty to UC . Moreovety to UC . Thus, tPharmacological inhibition of the NLRP3 inflammasome plays a protective role in colitis . In our In this study, we found that WT161 exerts a protective role in a colitis model, blocks NLRP3 inflammasome activation, disrupts ASC speck formation, and decreases NLRP3 expression. This study uncovers a new inhibitor of the NLRP3 inflammasome and suggests its potential application in the treatment of active IBD.E. coli 0111: B4, cat. no. tlrl-3pelps), ATP (cat. no. tlrl-atpl), and nigericin (cat. no. tlrl-nig), monosodium urate (MSU) (cat. no. tlrl-msu) from InvivoGen; Protein A/G PLUS-Agarose (cat. no. sc-2003) from Santa Cruz; mouse immunoglobin IgG protein (cat. no. ab198772) from Abcam, and cell lysis buffer (CLB) (cat. no. 9803) from Cell Signaling Technology; mouse IL-1β (cat. no. 88–7013), IL-6 (cat. no. 88-7064-88) ELISA kits (Thermo Fisher).WT161 (cat. no. 1206731-57-8) from Selleck Chemicals; DSS  from MP Biomedicals; ultrapure lipopolysaccharide (LPS)  was obtained from RD System; anti–caspase-1  was from Abcam; anti-NLRP3  and ASC  were purchased from Adipogen; anti–β-actin , anti–p-p65 antibody , and anti-p65 antibody  were purchased from Cell Signaling Technology; DyLight 488–labeled secondary antibody  from InvivoGen.All procedures were performed in accordance with the Institutional Animal Care and Use Committee of the Central South University. Male C57BL/6 mice (6–8 weeks old) were purchased from Hunan SJA Laboratory 100 Animal Co. Ltd , and all animal experiments were carried out in the Department of Laboratory Animals of Central South University under SPF conditions.2O, n = 5), DSS group , low dose of WT161 treated DSS group , and high dose of WT161 treated DSS group . The mice were fed with 3% DSS or H2O in drinking water (1) weight loss (compared with day 1): 0 (≤1.0%), 1 (1.0–5.0%), 2 (5.0–10.0%), 3 (10.0–15.0%), and 4 (>15.0%); 2) stool consistency or diarrhea: 0 , 1 (soft stools), 2 (loose stools), 3 (stools attached to the anus), and 4 (watery diarrhea); 3) blood in stool: 0 (negative), 1 (weak positive), 2 (positive), 3 (strong positive), and 4 (gross blood in stool). A urine fecal occult blood test box was used to detect blood in the stool. On day 10, the mice were sacrificed, and the colon was collected for colon length measurement, colon explant culture, and histological analysis.The acute colitis model induced by DSS is widely used in studies of IBD , and theng water  for up tng water : 1) weigThe 0.3–0.5 cm distal colonic segment close to the rectum was fixed in 4% paraformaldehyde for 24 h for histological analysis. The slides were prepared and stained with H&E and examined under a Nikon ECL IPSE Ci biological microscope. The images were collected using a Nikon DS-U3 digital camera. Histological scores were evaluated according to a previously published study . Epithel2 for 24 h. Pro-inflammatory cytokines in the supernatant were detected by ELISA.The next 0.3–0.5 cm distal colonic segment (with the same weight) close to the rectum was washed with saline briefly and then washed three times with cold RPMI-1640 (Gibco) containing penicillin G (200 μg/ml) and streptomycin (200 μg/ml) to remove the residual bacteria. They were then cultured in RPMI-1640 supplemented with 10% FBS, penicillin G (200 μg/ml), and streptomycin (200 μg/ml) at 37°C with 5% CO2.Peritoneal macrophages from healthy SPF C57BL/6J mice (6–8 weeks old) were elicited by i.p. injection of 3 ml 3% thioglycolate broth. Macrophages were harvested 3 days later by i.p. instillation and retraction of peritoneal lavage with 10 ml RPMI-1640 medium. Cells were purified by adherence and cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified incubator with 5% COPeritoneal macrophages were seeded overnight in 24-well (3–4 × 10^5/well) or six-well plates (2 × 10^6/well). After 24 h, mouse peritoneal macrophages were stimulated with LPS (100 ng/ml) for 3 h, followed by treatment with DMSO or WT161  for 30 min. Next, the cells were treated with 3% DSS for 24 h. IL-1β and IL-6 in the supernatant were detected using ELISA kits.Peritoneal macrophages were seeded overnight in 24-well (3–4 × 10^5/well) or six-well plates (2 × 10^6/well). Mouse peritoneal macrophages were primed with LPS (100 ng/ml) for 3 h, followed by treatment with DMSO or WT161  for 30 min, and then stimulated with 5 mM ATP (1 h), 10 μM nigericin (1 h), or 200 μg/ml MSU (6–8 h). To evaluate NLRP3 activation, the release of IL-1β detected by ELISA or the expression of cleaved caspase-1 p10 and cleaved IL-1β p17 detected by western blot were analyzed in the supernatant.For cell proteins, cells were lysed with CLB supplemented with a protease inhibitor cocktail and phenylmethylsulfonylfluoride (PMSF), and then the cell lysates were centrifuged at 12,000 g for 5 min at 4°C. For tissue protein analysis, the mouse colon and sodium dodecyl sulphate (SDS) lysate were lysed at a ratio of 10 mg:200 μl followed by ultrasound and grinding, centrifuged at 12,000 g for 10 min, and the supernatant was aspirated. After protein quantitative analysis, the extracts were boiled and dissolved in SDS loading buffer for western blot. Equal amounts of extracts  were separated by 10% SDS-PAGE, and then transferred onto 22-mm PVDF membranes  for immunoblot analysis. The signal intensity was detected using the ImageJ software. The data were standardized to β-actin expression levels.Peritoneal macrophages were seeded overnight in 24-well plates (3–4 × 10^5/well). The next day, the cells were treated with or without WT161 (5 μM) for 30 min, followed by LPS (100 ng/ml) for 0, 2, 4, or 8 h. After stimulation, total mRNA was extracted using the Fast 2000 kit (FASTAGEN). Then, RNA was reverse-transcribed to complementary DNA using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for RtPCR kit (TransGen) for quantitative PCR analysis. Quantitative PCR was performed using SYBR Green (Vazyme Biotech) on a LightCycler 480 (Roche Diagnostics). The data were standardized to β-actin expression levels. The relative expression changes were calculated using the 2−ΔΔCT method. Specific primers for each gene were as follows:NLRP3 forward: 5′-TGGATGGGTTTGCTGGGAT-3′,reverse: 5′-CTGCGTGTAGCGACTGTTGAG-3′;Caspasse-1 forward: 5′-ACAAGGCACGGG ACCTATG-3′,reverse: 5′-TCCCAGTCAGTCCTGGAAATG-3′;ASC forward: 5′-CTTGTCAGGGGATGAACTCAAAATT-3′,reverse: 5′-GCCATACGACTCCAG ATAGTAGC-3′;β-actin forward: 5′-AGTGTGACGTTGACATCCGT-3′,reverse: 5′-GCAGCTCAGTAACAGTCCGC-3′.Peritoneal macrophages were seeded overnight in six-well plates (2 × 10^6/well). The next day, the cells were primed with LPS (100 ng/ml) for 3 h, followed by DMSO or WT161  for 30 min, and then stimulated with 10 μM nigericin for 1 h. After stimulation, cells were rinsed in PBS and 100 μl ice-cold Triton buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100) with 0.1 mM phenylmethylsulfonylfluoride (PMSF). The cells were centrifuged at 6,000 g for 15 min at 4°C. The supernatant was resuspended in SDS loading buffer as a soluble sample. Insoluble samples (pellets) were washed twice with Triton buffer and resuspended in 200 μl Triton buffer containing 0.1 mM PMSF. Disuccinimidyl suberate (2 mM) was added to the resuspended pellets and incubated at 37°C for 30 min. The samples were then re-centrifuged at 6,000 g for 15 min at 4°C and suspended in SDS loading buffer. Both samples were then collected for western blot. The extracts (10 μg) from each sample were separated by 10% SDS-PAGE.The peritoneal macrophages were cultured overnight on chamber slides. The following day, the cells were primed with LPS (100 ng/ml) for 3 h, followed by treatment with DMSO or WT161  for 3 h, then stimulated with 10 μM nigericin or 5 mM ATP for 1 h. Cells were then fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100. After blocking with 5% BSA, cells were incubated with anti-ASC antibody (1:200) overnight at 4°C in PBS containing 5% BSA and DyLight 488-labeled secondary antibody (1:50) for 1 h in PBS containing 5% BSA. After thorough washes, the slides were mounted with mounting medium containing DAPI. Analyses were carried out using a fluorescence microscope (Nikon Ti2-U).p < 0.05, with increasing levels of confidence displayed as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.All results are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA with Bonferroni’s test or two-way ANOVA with Bonferroni’s multiple comparisons test for the groups test in GraphPad Prism software 8.0. Statistical significance was defined as To investigate the function of WT161 in the treatment of NLRP3-related diseases, DSS-induced colitis was used. Based on previously reported pharmacokinetic properties, WT161 was formulated for i.p. administration once a day at a dose of 10 or 20 mg/kg in 3% DSS-treated mice from day 1 to day 10. Body weight, grossly bloody stools, and stool consistency were recorded daily. Compared to that of the control group, the WT161 treatment group showed improved weight loss . AccordiAberrant and excessive activation of some cytokines is involved in the pathogenesis of IBD , among w2+ and Na+ influx or by causing lysosomal rupture, respectively. WT161 reduced the release of IL-1β upon nigericin, ATP, and MSU stimulation (Considering that IL-1β mainly depends on the activation of the NLRP3 inflammasome  and the mulation . We obsemulation  upon nigUpon treatment with NLRP3 activators, NLRP3 undergoes initial self-oligomerization, after which NLRP3 recruits ASC, leading to ASC oligomerization, which is critical for subsequent caspase-1 activation. Thus, we tested ASC oligomerization to further evaluate the effect of WT161 on the activation of the NLRP3 inflammasome. Using disuccinimidyl suberate crosslinking, we found that WT161 effectively inhibited the formation of ASC polymers . This reFinally, we aimed to explore the effect of WT161 on the expression of NLRP3, ASC, and caspase-1. The priming process of NLRP3 activation is induced by various pathogen-associated molecular patterns or cytokines, leading to activation of NF-κB signaling, which induces the expression of IL-1β and NLRP3 . We treaIn this study, we found that the HDAC6 inhibitor WT161 exerts a protective effect in a DSS-induced colitis murine model and blocks NLRP3 inflammasome activation, as IL-1β release and ASC oligomerization were inhibited in the WT161-treated group. WT161 decreased the NLRP3 expression by inhibiting the NF-κB signaling. Thus, our study suggests that WT161 could be a promising agent for the treatment of active IBD.WT161 has been regarded as a remarkable anti-tumor agent for several types of cancers, including multiple myeloma , breast in vitro and in vivo. As TNF-α is the most frequent studied cytokine in mucosal inflammation, we did not further detect it. We supposed that WT161 could down-regulate TNF-α expression since it could suppress NF-κB signaling.Network of cytokines including IL-1β, TNF-α, IL-6, IL-10, IFN-γ, and IL-18, have been reported to regulate mucosal inflammation in colitis, and IL-6, TNF-α are viewed as therapeutic targets in IBD . In thisAlthough the role of the NLRP3 inflammasome in murine IBD models with NLRP3, ASC, or caspase-1 deficiency remains controversial , inhibitin vivo and in vitro needs to be further explored. In addition, Maartje et al. reported that butyrate, an inhibitor of class I HDAC, decreases the production of IL-1β in MSU-stimulated peritoneal macrophages by inhibiting NLRP3 activation (Although we speculate that WT161 modulates NF-κB signaling and inhibits the expression of NLRP3, resulting in the inhibition of NLRP3 inflammasome activation based on the results of this study, the function of WT161 as an inhibitor of HDAC6 should not be neglected. Acetylation participating in NLRP3 inflammasome activation  regulatetivation . In thistivation . Given ttivation , whetherStudies have found that HDAC1, 2, 3, 6, and 9 play important pro-inflammatory roles in the initiation and progression of IBD . In thesOverall, our data suggest that WT161 alleviated colonic inflammation in DSS-induced colitis mice as an effective inhibitor of the NLRP3 inflammasome. WT161 not only decreased NLRP3 expression by suppressing NF-κB signaling but also inhibited ASC formation. In a colitis model, WT161 ameliorated intestinal injury, suggesting its potential use in the treatment of IBD. Although further mechanisms remain to be elucidated, our studies have identified WT161 as a therapeutic agent for the treatment of NLRP3-related inflammatory diseases such as IBD.


The X-ray crystal and molecular structures, DFT study and Hirshfeld surface analysis of a novel spirobicyclic diselenide are reported. 70H70N8O4Se2, is a spiro bicyclic diselenide, made up of two [SeC6H4CH=N—N(CO)C6H4(C)C6H3NEt2(O)C6H3NEt2] units related by a twofold crystallographic symmetry element bisecting the diselenide bond. The compound crystallizes in a non-centrosymmetric polar space group  and the structure was refined as an inversion twin. The two diethyl amine groups and their attached phenyl groups of the xanthene ring are disordered over two orientations, with occupancies of 0.664 (19)/0.336 (19) and 0.665 (11)/0.335 (11), respectively. The dihedral angles between the mean planes of the central isoindoline and the phenyl rings are 26.8 (2) and 2.5 (4)°, respectively. The mean plane of the central xanthene ring forms dihedral angles of 2.0 (5), 8.8 (5), 1.7 (5) and 7.9 (6)° with the peripheral phenyl rings. The isoindoline and xanthene rings subtend a dihedral angle of 89.8 (2)°. The molecular conformation is stabilized by an intramolecular C—H⋯O hydrogen bond generating an S(6) ring motif. In the crystal, molecules are linked by C—H⋯O hydrogen bonds together with C—H⋯π (ring) interactions, forming a three-dimensional network. A Hirshfeld surface analysis of the crystal structure indicates that the most important contributions to the crystal packing are from H⋯H (68.1%), C⋯H/H⋯C (21.2%) and O⋯H/H⋯O (8.7%) contacts. The optimized structure calculated using density functional theory (DFT) at the B3LYP/6 – 31 G(d) level is compared with the experimentally determined molecular structure in the solid state. The HOMO–LUMO behaviour was used to determine the energy gap and the molecular electrostatic potential (MEP) of the compound was investigated.The title compound, C Herein we report the crystal structure, DFT and Hirshfeld surface analysis of 2,2′′--(diselanediyl)bis]bis(methaneylylidene)}bis(azaneylylidene))bis-3-one], isolated from the condensation of rhodamine B hydrazide with bis(o-formylphenyl)diselenide.Diaryl diselenides and aryl selenolates have been previously used as ligand precursors for the synthesis of transition-metal complexes , and the structure was refined as an inversion twin .The title compound Fig. 1, a rare c axis is presented in Fig. 2D⋯A distances of 3.391 (10) and 3.447 (10) Å, respectively  between neighbouring molecules (Table 1D⋯A distance of 2.940 (9) Å leading to an S(6) ring motif . Bright-red spots on top and bottom of the HS near N3 indicate an intermolecular C—H⋯π (ring) interaction involving H25B of the C25 methyl group and a benzene ring .Hirshfeld surface (HS) calculations  geometry-optimized molecular orbital calculation (c), hardness (h), ionization enthalpy (IE), dipole moment (m), electron gain enthalpy (EE), electrophilicity (ω) and softness (s), are collated in Table 4h and s is to evaluate both the reactivity and stability.Calculated molecular orbital energies (eV) for the surfaces of the frontier molecular orbitals of the title compound are shown in Fig. 5E = ELUMO − EHOMO] of the molecule is 3.7536 eV, and the frontier molecular orbital energies, EHOMO and ELUMO, are −5.0048 and −1.2512 eV, respectively.As shown in Fig. 5The molecular electrostatic potential (MEP) map Fig. 6 was calcet al., 2016et al., 2012ca 81° and those of CIDXET and CIDXUJ diselenide diselenide  in ethanol (30 mL) over approximately 45 minutes in a dropping funnel. The solution mixture was stirred further for 4 h at room temperature. After cooling, the solid was filtered and washed three times with cold ethanol. Pale-yellow crystals of the title compound suitable for single-crystal X-ray diffraction study were obtained from chloroform/pentane (1:1 mixture), yield 0.461 g, 81%, m.p. 519 K  = 3387, 2967, 1613, 1514, 1218, 1117, 753. 1H NMR : 1.15 , 3.33 , 6.29 , 6.43 , 6.46 , 7.09 , 7.19 , 7.43 , 7.92 , 8.60 . 13C NMR : 12.7 (NCH2CH3), 44.5 (NCH2CH3), 66.1 (spiro carbon), 98.2, 104.7, 108.2, 123.1, 123.9, 130.9, 132.6, 149.1, 151.7, 154.0, 166.3 (C=O).The title compound was obtained by the condensation of rhodamine B hydrazide  I. DOI: 10.1107/S2056989021013189/zl5020Isup3.hklStructure factors: contains datablock(s) I. DOI: 2127961CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


In vivo studies showed that Gαi3 shRNA AAV (adeno-associated viruses) intratumoral injection largely inhibited the growth of subcutaneous xenografts of primary OS cells. Moreover, the growth of the Gαi3-knockout primary OS xenografts was much slower than that of OS xenografts with empty vector. In Gαi3-depleted OS xenografts tissues, Gαi3 downregulation and Akt-mTOR inactivation were detected. Taken together, overexpressed Gαi3 mediates RTK-Akt signaling to drive OS progression.Sustained activation of multiple receptor tyrosine kinases (RTKs) simultaneously is vital for tumorigenesis and progression of osteosarcoma (OS). Gαi proteins recruitment to various RTKs mediates downstream oncogenic signaling activation. The expression, functions and underlying mechanisms of Gαi3 in human OS were examined. Expression of Gαi3 is robustly elevated in human OS tissues and is correlated with a poor overall survival. In patient-derived primary OS cells and immortalized lines (MG63 and U2OS), Gαi3 depletion, by shRNA and CRISPR/Cas9 strategies, robustly suppressed cell viability, proliferation and migration, while provoking G1-S arrest and apoptosis activation. Conversely, Gαi3 overexpressing ectopically can accelerate proliferation and migration of OS cells. In OS cells, Gαi3 immunoprecipitated with VEGFR2, FGFR, PGDFR and EGFR, mediating downstream cascade transduction. Akt-mTOR activation in primary OS cells was potently inhibited by Gαi3 shRNA, knockout or dominant negative mutation, but augmented after Gαi3 overexpression.  For the advanced osteosarcoma (OS) patients with metastatic, recurrent or therapy-resistant OS, the prognosis is poor et al., have shown that Gαi proteins are important for the agonist-induced cAMP production in osteosarcoma cells that were derived from rat et al., showed that pertussis toxin can inhibit bradykinin-induced Ca2+ mobilization in MG63 OS cells The inhibitory guanine nucleotide regulatory proteins, Gαi proteins, consists of three subunits, including Gαi1, Gαi2 and Gαi3 Our group has identified an essential role of Gαi proteins in transducing signals for multiple RTKs The protocols of the present study were reviewed and approved by the Ethics Committee of Soochow University and were in according to the principle of Helsinki declaration.LY294002 was from Sigma Aldrich . The antibodies were described in our previous studies The immortalized OS lines, MG63 and U2OS, as well as the established hFOB1.19 osteoblastic cells were cultivated as described Protein detection by Western blotting, RNA assays by qRT-PCR and co-immunoprecipitation (co-IP) examining protein-protein interaction were extensively described early in vitro cell infection were described early in vivo studies, the Gαi3 shRNA sequence or shC sequence was sub-cloned into an established adenoviral vector, adeno-associated virus 9/AAV9 construct (Genechem). Through Lipofectamine 3000 the construct was thereafter transfected to HEK-293T cells, and the shRNA-expressing AAV virus was generated and was injected to xenograft tumors.The lentivirus-encoded Gαi3 shRNAs and Gαi3 KO screening was carried out and thereafter the Gαi3 KO cells were eventually established. The control OS cells were with a lenti-CRISPR/Cas-9 empty vector with non-sense small guide RNA (“Cas9-C”).OS cells were transfected with Cas9-expressing construct (Genechem) by Lipofectamine 3000  to establish stable cells. Next, the lenti-CRISPR/Cas-9 Gαi3 KO construct Gαi3 overexpression and dominant negative mutation, stable cells establishment and verification were described in our previous studies OS cells were infected with the constitutively-active Akt1 -expressing adenovirus  were added and transfected to cultured OS cells. After 48h, cells were cultured and selected in puromycin-containing medium for another 96h. Akt1/2 silencing was always examined.The cell viability detection by cell counting kit-8 (CCK-8), the EdU nuclear staining assaying of cell proliferation, propidium iodide (PI)-FACS, “Transwell” assays were carried out using the previously described protocols Apoptosis-related assays, including the TUNEL nuclear staining, 7-AAD and Annexin V double staining, caspase-3/-9 activities measurement, and ELISA testing the cellular ssDNA (single strand DNA) contents were described early s.c.) injected to the nude mice /2] and animal body weights were weekly recorded. Soochow University's Ethics Committee and Institutional Animal Care and Use Committee (IACUC) reviewed and approved the protocols of animal studies.The primary pOS1 cells (five million cells in every mouse) were subcutaneously  was first consulted to analyze Gαi3 transcripts in human sarcoma tissues. Total 264 samples (HTSeq-FPKM) were collected, including the two normal specimens and the 262 sarcoma specimens. As shown, Gαi3 transcripts in the retrieved sarcoma tissue specimens were higher than those in the retrieved normal tissue specimens . The Kaplan-Meier survival, Figure 1B, verified that high Gαi3 expression in sarcoma patients was correlated with a poor prognosis . Subgroup analysis by different clinical features demonstrated that Gαi3-high expression was associated with a poor prognosis in male sarcoma patients , age≤60 , Tumor depth: deep .The Cancer Genome Atlas (TCGA) database (available on the public domain 1F showed that Gαi3 mRNA in the OS tissue specimens (“T”) was more than six-fold higher than that in the adjacent normal bone tissues (“N”). Gαi3 protein expression was tested as well. In the OS tissues of four representative OS patients (Patient #1/#2/#3/#4), protein expression of Gαi3 was significantly elevated . When combining all blotting results of the ten sets tissues, Gαi3 protein was found to be significantly upregulated in the OS tissues . The immunohistochemistry (IHC) staining assay results, in Figure 1I, verified Gαi3 protein elevation in OS tumor tissues , While low expression of Gαi3 was detected in the adjacent normal tissue specimens .Next, we tested Gαi3 expression in local OS tissues. As previously described  Gαi3 mRNA in different primary and immortalized OS cells was dramatically higher than that in hFOB1.19 human osteoblastic cells and patient-derived human osteoblasts (“pOsteoblasts”) . In addition, Gαi3 protein upregulation was also shown in different primary and established OS cells . Taken together, in OS tissues and cells Gαi3 is upregulated.The expression of Gαi3 in different human OS cells was examined. Patient-derived primary OS cells , as well as the immortalized cells (MG63 and U2OS lines), were cultured. The expression ofGαi3 mRNA expression decreased over 90-95% in sh-Gαi3-expressing stable pOS-1 cells . Expression of Gαi1 and Gαi2 mRNA was unaffected . Gαi3 protein silencing was detected in the stable pOS-1 cells with Gαi3 shRNAs. Gαi1 and Gαi2 protein expression was again unchanged .pOS-1 primary cells were separately transfected with two different Gαi3 lentiviral shRNAs, sh-Gαi3-seq1/sh-Gαi3-seq2 2C). Moreover, Gαi3 shRNA potently inhibited pOS-1 cell proliferation, as the ratio of EdU positively stained nuclei was robustly decreased in sh-Gαi3-expressing pOS-1 cells . The PI-FACS assays were employed to test cell cycle progression. The ratio of G1-phase cells was significantly increased in pOS-1 cells expressing Gαi3 shRNA , where S-phase pOS-1 cell percentage was decreased . These results implied that the designated Gαi3 shRNAs induced G1-S arrest in primary OS cells. The “Transwell” results demonstrated that Gαi3 shRNA dramatically inhibited pOS-1 cell in vitro migration . The scramble control shRNA, or shC, did not significantly alter Gαi1/2/3 expression  and affect OS cellular behaviors .CCK-8 assays showed that Gαi3 shRNA potently decreased pOS-1 cell viability . Gαi3 shRNA largely suppressed CCK-8 viability , proliferation  and migration  in pOS2 primary cells and established lines.Next, OS cells that were derived from another OS patient, pOS2 1J-K) was tested as well. hFOB1.19 osteoblastic cell line or patient-derived primary osteoblasts (pOsteoblasts) were stably transduced with the lentiviral sh-Gαi3-seq1, causing dramatic Gαi3 mRNA downregulation . Interestingly, in hFOB1.19 cells and pOsteoblasts, Gαi3 shRNA failed to significantly inhibit CCK-8 viability  and cell proliferation , supporting a cancer cell-specific effect by Gαi3 shRNA.The potential effect of Gαi3 shRNA on the Gαi3-low human osteoblasts  were augmented in sh-Gαi3-expressing stable pOS-1 cells. Figure 3C showed that Gαi3 shRNA induced cleavages of caspase-3, PARP and caspase-9 in pOS-1 primary cells. Confirming increased DNA breaks in pOS-1 cells with Gαi3 shRNA, we showed that ssDNA contents were dramatically increased . Moreover, FACS assay results in Figure 3E showed that Gαi3 silencing increased the pOS-1 cell number with Annexin V-7-AAD double positive staining, confirming apoptosis activation. As expected, shC did not provoke caspase-apoptosis activation in the primary pOS-1 cells .Since Gαi3 silencing induced viability reduction, growth arrest, proliferation inhibition, we therefore tested its potential function on apoptosis in OS cells. Results showed that the relative activities of caspase-3 and caspase-9  similarly augmented the relative caspase-3 activity . Apoptosis induction was observed as well in the sh-Gαi3-seq1-expressing pOS-2 cells and immortalized cell lines, evidenced by the significantly increased Annexin V-7-AAD double staining . Conversely, in the osteoblastic cell line hFOB1.19 and pOsteoblasts, shRNA-induced silencing Gαi3  failed to significantly induce apoptosis .In pOS-2 cells and immortalized lines (MG63 and U2OS), Gαi3 shRNA  depletion was detected. Gαi3 KO largely decreased CCK-8 viability  and inhibited cell proliferation  and migration  in pOS-1 cells. On the contrast, caspase-3 and caspase-9 activities  were augmented in ko-Gαi3 pOS-1 cells, where caspase-3, PARP and caspase-9 cleavages were induced . In addition, apoptosis induction was detected in the ko-Gαi3 pOS-1 cells, as the ratio of TUNEL-positively stained nuclei was increased significantly .Next, a previously-described CRISPR/Cas9-Gαi3-KO-puro construct 4F). As shown, Gαi3 KO inhibited cell proliferation  and in vitro migration  in pOS-2 and established OS cells. Increased TUNEL-positive nuclei ratio confirmed apoptosis activation in ko-Gαi3 pOS-2 cells and immortalized lines . Together, Gαi3 KO, by the CRISPR/Cas9 strategy, resulted in profound anti-OS cell activity.The Gαi3-KO construct was employed to knockout Gαi3 in primary pOS-2 cells and immortalized lines (MG63 and U2OS), and stable cells established (“ko-Gαi3”) after screening  were robustly elevated in the OE-Gαi3 pOS-1 cells. Gαi3 overexpression accelerated pOS-1 cell proliferation  and in vitro migration . In pOS-2 cells and the immortalized lines (MG63 and U2OS), ectopic overexpression of Gαi3 using the same construct  enhanced cell proliferation  and in vitro migration . Therefore, these results again supported the key cancer-promoting function of Gαi3 in OS cells.Next, a Gαi3-expressing lentiviral construct  and immortalized U2OS cells. Moreover, the association between Gαi3 and multiple RTKs  was detected in the human OS tissues from three representative patients . When testing downstream Akt-mTOR signaling, we showed that levels of phosphorylated-Akt (Ser-473) and phosphorylated-S6K (Thr-389) were dramatically decreased in pOS-1 cells bearing Gαi3 shRNAs . Moreover, CRISPR/Cas9-induced Gαi3 KO  largely inhibited Akt-S6K phosphorylations in pOS-1 cells . Notably, RTKs  expression and phosphorylation were unaffected by Gαi3 shRNA (sh-Gαi3-seq1) or Gαi3 KO . Conversely, ectopic overexpression of Gαi3  significantly increased Akt-S6K activation in pOS-1 cells .Our previous studies have shown that Gαi proteins associated with several oncogenic RTKs , mediating downstream signaling activation 6F). Expression of RTKs was however unchanged . Importantly, dnGαi3 largely inhibited Akt-S6K phosphorylations in pOS-1 primary cells . The dnGαi3 largely suppressed pOS-1 cell in vitro proliferation and migration, examined through the nuclear EdU staining  and “Transwell”  assays, respectively.To block Gαi3-RTKs association, the lentiviral dominant negative (dn) Gαi3 mutant construct was stably transduced into pOS-1 cells. The dnGαi3 mutant will replace the conserved Gly (G) residue with the Thr (T) residue in the G3 box, thereby preventing Gαi1/3 interaction with the associated proteins 7A). Significantly, Ad-caAkt1 restored proliferation  and in vitro migration  of koGαi3 pOS-1 cells. Furthermore, shRNA-induced silencing of Akt1/2  blocked Akt-S6K phosphorylations  and mimicked Gαi3 depletion-induced actions, suppressing pOS-1 cell proliferation  and in vitro migration . Significantly, re-introducing the Gαi3 shRNA lentivirus or the Gαi3-expresing construct  was unable to further influence cell proliferation  and migration  in Akt1/2-silenced pOS-1 cells. Moreover, in the Gαi3-overexpressed pOS-1 cells (OE-Gαi3-sL1), treatment with LY294002, a PI3K-Akt inhibitor 7H) and inhibited cell proliferation  and in vitro cell migration . Thus, Akt-mTOR inhibition should be responsible for Gαi3 depletion-induced anti-OS cell activity.To support that Akt-mTOR inhibition was the main mechanism of Gαi3 depletion-caused anti-OS cell activity, we expressed the constitutively active Akt1 (caAkt1) s.c.) injected to the nude mice. Within 20 days of cell inoculation, the subcutaneous pOS-1 xenograft tumors were established and each tumor volume was close to 100 mm3 (“Day-0”). The xenograft-bearing mice were thereafter assigned into three different groups randomly, with six mice in every group (n = 6). Afterwards, the mice were intratumorally injected daily with the AAV-packed Gαi3 shRNAs  or AAV-packed control shRNA (AAV-shC), for 12 consecutive days. Figure 8A, recording tumor growth, demonstrated that the growth of pOS-1 xenograft tumors was robustly mitigated after AAV-sh-Gαi3 injection. The volumes of AAV-sh-Gαi3-injected pOS-1 xenografts were dramatically lower than those of AAV-shC-injected pOS-1 xenografts . The estimated daily tumor growth (see previous studies 8B). At Day-42, all the animals were anaesthetized and decapitated, and pOS-1 xenografts carefully isolated and weighted. The pOS-1 tumors with AAV-sh-Gαi3 injection were significantly lighter than pOS-1 tumors with the control shRNA virus injection . Among the three mice groups there was no significant difference in the mice body weights .At last, pOS-1 cells were subcutaneously , where Gαi3 protein downregulation as well as p-Akt and p-S6K inhibition were detected . Supporting apoptosis activation, we showed that cleaved-caspsae-3/cleaved-PARP levels were augmented in Gαi3-silenced pOS-1 xenograft tissues . Thus, intratumoral injection of AAV-packed Gαi3 shRNA suppressed Akt-mTOR activation and provoked apoptosis in pOS-1 xenografts.At experimental Day-7 and Day-14, 3h after virus injection, one tumor of each group was carefully isolated, and total six tumors were obtained. 4) were injected to the nude mice, forming subcutaneous xenografts. After 20 days, tumor recordings were started (“Day-0”). As shown ko-Gαi3 pOS-1 xenograft growth was slower than the Ca9-C xenografts , while animal body weights were indifferent . At experimental Day-7, we carefully isolated one tumor xenograft per group. Gαi3 mRNA and protein  expression was completely depleted in ko-Gαi3 pOS-1 xenograft tissues, where p-Akt was decreased . The cleaved-caspsae-3 and the cleaved-PARP levels were increased in Gαi3-KO xenograft tissues , supporting apoptosis induction in vivo.In addition the ko-Gαi3 pOS-1 cells and the Ca9-C control cells  depletion activated protein kinase A (PKA)-Akt-nuclear factor (NF)-κB signaling to promote OS cell growth et al. demonstrated that GPR110 (G protein-coupled receptor 110) silencing inhibited OS cell growth The GPCR superfamily is composed of the immense structural and functional different proteins, participating in various biological processes and signals in the bone Importantly, studies have reported that Gαi-coupled GPCRs, including Apelin receptors After showing the essential role of Gαi 1/3 in activation of oncogenic signalings by RTKs, we previously explored Gαi 1/3 in different human cancers. Gαi 1 and Gαi 3 expression is elevated in glioma, correlating with tumor stage Gαi3 are significantly upregulated in sarcoma tissues, and high-Gαi3 expression in sarcoma correlating with the poor overall survival. Gαi3 elevation was observed in local OS tissues as well as in different immortalized and primary OS cells, while low expression was observed in cancer-surrounding normal bone tissues and in immortalized and primary osteoblasts. Functional studies showed that in different OS cells, Gαi3 depletion, by shRNA or CRISPR/Cas9 strategies, robustly suppressed cell survival, proliferation and cell migration, and provoking G1-S arrest and apoptosis. Contrarily, ectopic Gαi3 overexpression can further accelerate OS cell growth. In vivo, Gαi3 shRNA AAV intratumoral injection potently suppressed the growth of the patient-derived OS xenografts in nude mice. Moreover, the growth of primary OS xenografts of the Gαi3 KO cells was largely suppressed.Gαi3 should be a vital gene for OS progression. TCGA database shows that transcripts of We have previously discovered that Gαi1/3 association with multiple RTKs was required for downstream signaling activation. For instance, Gαi1/3 are key proteins in mediating VEGF-induced VEGFR2 signaling In vivo, Akt-S6K phosphorylations were decreased in Gαi3-depleted OS xenografts tissues. Importantly, disrupting Gαi3-RTKs association, through dnGαi3, largely inhibited Akt-S6K activation and OS cell proliferation and migration. Restoring Akt-S6K activation, by caAkt1, rescued proliferation and migration of Gαi3-KO OS cells. Conversely, mimicking Gαi3 depletion-induced actions, Akt1/2 silencing inhibited OS cell proliferation and migration. Significantly, exogenously altering Gαi3 expression failed to affect proliferation and migration in Akt1/2-silenced cells. Therefore, Gαi3-driven OS cell growth was possibly due to mediating RTKs-Akt signaling.The present study implied that Gαi3-driven OS cell growth was primarily through mediating Akt-mTOR cascade activation. In OS cell and tissues Gαi3 associated with RTKs , essential for downstream Akt-mTOR activation. In OS cells Akt-S6K activation was robustly suppressed by Gαi3 shRNA/KO, but augmented after Gαi3 overexpression. Over three-fifths of bone sarcoma are OS