Haematobia irritans irritans , a hematophagous external parasite of cattle, causes considerable economic losses to the livestock industry worldwide. This pest is mainly controlled with insecticides; however, horn fly populations from several countries have developed resistance to many of the products available for their control. In an attempt to better understand the adult horn fly and the development of resistance in natural populations, we used an Illumina paired-end read HiSeq and GAII approach to determine the transcriptomes of untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males from a Louisiana population of horn flies with a moderate level of pyrethroid resistance. A total of 128,769,829, 127,276,458, 67,653,920, and 64,270,124 quality-filtered Illumina reads were obtained for untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males, respectively. The de novo assemblies using CLC Genomics Workbench 8.0.1 yielded 15,699, 11,961, 2672, 7278 contigs (≥ 200 nt) for untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males, respectively. More than 56% of the assembled contigs of each data set had significant hits in the BlastX (UniProtKB/Swiss-Prot database) (E <0.001). The number of contigs in each data set with InterProScan, GO mapping, Enzyme codes and KEGG pathway annotations were: Untreated Control Adult Females – 10,331, 8770, 2963, 2183; Untreated control adult males – 8392, 7056, 2449, 1765; Permethrin-treated surviving adult males – 1992, 1609, 641, 495; Permethrin + PBO-treated killed adult males – 5561, 4463, 1628, 1211.The horn fly,  Specifications TableValue of the data•Haematobia irritans irritans.Resource for investigations of the molecular basis of insecticide resistance in the horn fly, •Provides candidate protein coding regions for the development of control strategies targeting adult flies.1https://www.ncbi.nlm.nih.gov/sra/SRP131897 or through SRA accession number SRP131897. The adult horn fly transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GGLM00000000. The version described in this paper is the first version, GGLM01000000. The overall BioProject ID is PRJNA429442 and the BioSample accessions are SAMN08355023, SAMN08355024, SAMN08355025, and SAMN08355026.RNA was isolated from unfed, newly emerged adult horn flies, including untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males. Subsequently, a single lane of 2 × 54 bp paired end RNASeq reads were obtained, de novo assembled and annotated. The raw reads are accessible at NCBI׳s SRA through the direct link 22.12, ~LD25) or permethrin  + 1% piperonyl butoxide (PBO) by the impregnated filter paper assay Adult flies were collected with aerial sweep hand nets from pastured cattle at the St. Gabriel Research Station, Saint Gabriel, Louisiana, and incubated in large inverted Erlenmeyer flasks to collect eggs that were immediately seeded into manure to allow adult fly emergence. The unfed, newly emerged flies were sexed and either immediately frozen at −80 °C for sequencing  or exposed to low doses of permethrin  and the FastRNA Pro Green Kit .2.3https://www.qiagenbioinformatics.com/) or Trimmomatic programmable-0.33 https://de.cyverse.org/de/?type=apps&app-id=8cb5c088-6b3e-11e7-a22d-008cfa5ae621&system-id=de)  followed by Sickle-quality-based-trimming_version_1.0  https://de.cyverse.org/de/?type=apps&app-id=9f5710c6-3424-11e7-9a58-008cfa5ae621&system-id=de)  and Illumina adaptor sequences and low quality bases were removed. Trimmomatic and Sickle were both run on CyVerse/Discovery Environment https://de.cyverse.org/de/?type=apps&app-id=trinity-wrangler-2.5.1u2&system-id=agave) or version 11.10.13 (https://de.cyverse.org/de/?type=apps&app-id=trinity-stmpde-11.10.13u2&system-id=agave) and SoapdenovoTrans version 1.0.3 https://de.cyverse.org/de/?type=apps&app-id=Soaptrans-1.0.3u1&system-id=agave). Both Trinity versions and SoapdenovoTrans were run on CyVerse/Discovery Environment Sequencing was performed at the National Center for Genome Research  using the standard Illumina RNAseq library preparation protocol and a single lane of the RNAseq. 2 × 54 paired-end approach. A total of 134,671,818, 132,374,494, 68,856,572, 65,427,160 paired-end Illumina raw reads were produced for untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + PBO-treated killed adult males, respectively . The rawhttps://pods.iplantcollaborative.org/wiki/display/DEapps/Compute+Contig+Statistics), rnaQUAST_1.2.0 (de novo based) https://de.cyverse.org/de/?type=apps&app-id=980dd11a-1666-11e6-9122-930ba8f23352&system-id=de) and BUSCO-v3.0  https://de.cyverse.org/de/?type=apps&app-id=7f948668-7a53-11e7-a680-008cfa5ae621&system-id=de) (The assembled transcriptomes were then compared using three tools on CyVerse/Discovery Environment m-id=de) . Assemblm-id=de)  and were-3) using Blast2GO PRO version 5.0.21 The transcriptomes were BlastX aligned against the UniProtKB/SwissProt database (E-value = 1.0 e


AbstractSinocentrus Yuan, S.brevicornis Li & Chen, sp. nov. from China, is described and illustrated. A checklist and key to species of the Sinocentrus are provided.A new species of the treehopper genus  Sinocentrus was established by Yuan , 1 r-m and 1 m-cu crossveins present, apical limbus broad, with wrinkles. Metathoracic trochanter without spines, tibia with 3 rows of cucullate setae.Large sized. Frontoclypeus distinct. Suprahumeral horns narrow, acuminate, horizontally extended laterally, width between their apex ca 0.5 to 1.0 times body length. Pronotum highly developed, with anterior part strongly inflated and evenly rounded in profile, metopidium vertical, glabrous and minutely punctate without obvious pubescence. Posterior pronotal process strongly elevated above scutellum at base, slender, elongate, evenly tapered toward apex, straight or slightly sinuate, lateral and dorsal carina well developed, apex extended beyond forewing clavus. Scutellum entirely exposed, posterior margin emarginate. Basal one-fifth of forewing with opaque sclerotization, veins M and Cu fused basally to approximately one-fifth to one-fourth of wing length then strongly divergent, veins M+Cu and R fused basally, with 1 m-cu, 2 r-m, and 1 s crossveins. Hindwing vein R branched into RCentrotinae genera by the following characters: pronotum highly developed, strongly inflated with anterior part evenly rounded, glabrous with minute punctures and no obvious pubescence, suprahumeral horns extended laterad, posterior pronotal process elevated far above scutellum, scutellum emarginate.This genus can be distinguished from other oriental China  Fig. .S.brevicornis Li & Chen, sp. nov.; China (Hainan)S.sinensis Yuan, 2002; China (Yunnan); elevation: 1600 m.Taxon classificationAnimaliaHemipteraMembracidaeLi & Chensp. nov.0F416316-4D26-546E-8578-BDD13EA0550Dhttp://zoobank.org/73BE02FB-C98E-4D45-AC88-D597817F3597Holotype: ♂, CHINA: Hainan, Bawangling, 29 April 2017, Hong-Xing Li. Paratypes: 2♀♀, same data as holotype.N = 1), female 8.9–9.3 mm (N = 2); forewing length: male 7.3 mm (N = 1), female 7.3–7.9 mm (N = 2); width between humeral angles apices: male 3.3 mm (N = 1), female 3.5–3.8 mm (N = 2); width between suprahumeral horns apices: male 4.6 mm (N = 1), female 4.6–5.2 mm (N = 2).Body length: male 8.1 mm . Humeral angles triangular with apices somewhat blunt. Suprahumeral horns short, width between horns apices nearly half length of body. Scutellum humped basally, large punctures present, longer than wide, apex extended antero-dorsally male, curved ventrally in female .brevicornis” is derived from the Latins words “brevi-” and “cornu”, referring to having the short suprahumeral horns.The word “S.sinensis Yuan, 2002, but differs from the latter in: (1) forewing veins black and apical limbus black  (2) suprahumeral horns short, width between suprahumeral horns apices shorter than body length (as long as body length in S.sinensis); (3) posterior pronotal process nearly straightly ; (4) scutellum longer than wide (wider than long in S.sinensis); (5) apex of posterior pronotal process not reaching M3+4 veins (exceeding M3+4 veins in S.sinensis).This species is similar to Taxon classificationAnimaliaHemipteraMembracidaeYuan, 2002074481DF-471C-5038-857A-48AFE38F7BD3Sinocentrussinensis Yuan, 2002: 170, by original designation.Coloration. General color reddish-brown with golden setae .While holotype was not examined, an online image of the holotype Fig.  and detaCentrotinae, Sinocentrus and treated the genus as Centrotinae, incertae sedis.In their phylogeny and genus-level revision of Sinocentrus: (1) frontoclypeus distinct (indistinct in Centrotypini); (2) posterior pronotal process elevated far above the scutellum, entirely exposed ; (3) male lateral plate with short dorsoapical lobe extending dorsally, style clasp angled ventrally; style shank with arch at central section ; (4) mesothoracic femur without ablateral and adlateral cucullate setae; metathoracic leg cucullate setae row II irregular. Although the above characteristics can suggest that the genus is related to Leptocentrini, the shape of the female second valvulae closely align S.brevicornis with the tribe Centrotypini. Given these mixed affinities, we follow Wallace and Deitz, in treating Sinocentrus as Centrotinae, incertae sedis. Proper tribal placement may be affirmed by future phylogenetic analyses of combined morphological and molecular data.We provide the following additional details on


Lower physical activity is cross-sectionally associated with greater fatigability; whether such a relationship holds for longitudinal changes in fatigability is under-studied. We examined this question in offspring  enrolled in the Long Life Family Study, a two-generation cohort enriched for exceptional longevity and their spousal controls. At Visit 2 (2014-2017), we measured self-reported physical activity (PA) with the Framingham Physical Activity Index . Perceived physical fatigability was assessed using the Pittsburgh Fatigability Scale  at Visit 2 and repeated during a follow-up contact 2.7±0.92 years later. We constructed a repeated-measures linear mixed-effect model to examine the effect of PA on longitudinal change in PFS by median age  adjusted for family structure, field center, follow-up time, sex, and self-rated health. We found a strong dose-response relationship of PFS scores across the four age/PA groups (ptrend<0.001). Specifically, older/less active (N=310) participants had the highest annual PFS increases of 0.37 points/yr (p<0.001) while those older/more active (N=340) had annual increases of 0.17 points/yr (p=0.03). Younger/less active (N=371) participants had annual PFS increases of 0.09 points/yr (p=0.008); those younger/more active (N=341) had annual decreases (improvement) of 0.18 points/yr (p<0.001). Although annual PFS changes were modest, our findings indicate physical activity attenuated age differences in these trajectories. Physical activity is emerging as a potential target for intervention aimed at reducing fatigability - an important risk factor in the disability pathway.


Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analyses. Significant antifeedant activity of 1, 3 and 10 against the beet armyworm (Spodoptera exigua) was found, indicating that these diterpenoids might also be involved in the plant defense against insect herbivores.Three new grayanane diterpenoids, pierisoids C‒E ( Pieris, Rhododendron, Kalmia, Craibiodendron and Leucothoe +; HR-ESI-MS: m/z 507.2567 [M + Na]+ ; 1H- and 13C-NMR data: see Pierisoid D (2): Colorless oils;c = 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (2.48) nm; IR (KBr) νmax: 3441, 3432, 2971, 2939, 1735, 1730, 1630, 1382, 1179, 1049, 1025 cm−1; ESI-MS: m/z 463 (88) [M + Na]+; HR-EI-MS: m/z 440.2420 ; 1H- and 13C-NMR data: see Pierisoid E (3): Colorless crystals; mp 271–272 °C;c = 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (2.29) nm; IR (KBr) νmax: 3441, 2942, 1787, 1734, 1631, 1372, 1264, 1237, 1053 cm-1; ESI-MS: m/z 661 (68) [M + Na]+; HR-EI-MS: m/z 638.2560 ; 1H- and 13C-NMR data: see Spodoptera exigua) were purchased from the Pilot-Scale Base of Bio-Pesticides, Institute of Zoology, Chinese Academy of Sciences. A modified dual-choice bioassay was performed for an antifeedant test as previously described  × 100, where C and T represent the control and treated leaf areas consumed by the insect. The insect antifeedant potency of the test compound was evaluated in terms of the EC50 value, which was determined by probit analysis for each insect species.Beet armyworms , as well as 10 known ones (4‒13), were identified from the flowers of P. formosa via their extensive 1D and 2D NMR spectroscopic data analyses. Notably, compounds 1, 3 and 10, especially 10, exhibited obvious antifeedant activity against the beet armyworm (S. exigua), suggesting that these diterpenoids were important defensive substances in P. formosa against natural enemies.Terpenoids play an important role in natural product chemistry and biology, such as antifungal and insecticidal activities . Seconda


Drosophila Hippo pathway maintains NSC quiescence, but its regulation during brain development remains unknown. Here, we show that CRL4Mahj, an evolutionarily conserved E3 ubiquitin ligase, is essential for NSC reactivation (exit from quiescence). We demonstrate that damaged DNA-binding protein 1 (DDB1) and Cullin4, two core components of Cullin4-RING ligase (CRL4), are intrinsically required for NSC reactivation. We have identified a substrate receptor of CRL4, Mahjong (Mahj), which is necessary and sufficient for NSC reactivation. Moreover, we show that CRL4Mahj forms a protein complex with Warts (Wts/large tumor suppressor [Lats]), a kinase of the Hippo signaling pathway, and Mahj promotes the ubiquitination of Wts. Our genetic analyses further support the conclusion that CRL4Mahj triggers NSC reactivation by inhibition of Wts. Given that Cullin4B mutations cause mental retardation and cerebral malformation, similar regulatory mechanisms may be applied to the human brain.The ability of neural stem cells (NSCs) to transit between quiescence and proliferation is crucial for brain development and homeostasis.  During the transition from quiescence to reactivation of neural stem cells, the E3 ubiquitin ligase CRL4Mahj promotes their reactivation by inhibiting Wts, a core kinase of Hippo signalling pathway. The ability of stem cells to switch between quiescence and proliferation is crucial for tissue homeostasis and regeneration. Most adult neural stem cells (NSCs) exist in quiescence, a reversible dormant state, without proliferation or differentiation . In respDrosophila NSCs, called neuroblasts, have emerged as a powerful model to study mechanisms underlying NSC reactivation in vivo. Most Drosophila NSCs enter quiescence at the end of embryogenesis and resume proliferation shortly after larval hatching (ALH) #44974) driven by insc-Gal4 at 24 h ALH . Similarly, 31.2%  and 26.3%  of NSCs extended a cellular process in cul4G1-3 and cul4JJ11 mutants, compared with only 6.1% of NSCs  of WT brain lobes . Moreover, at 24 h ALH upon knockdown of cul4 by two different RNAi lines driven by NSC-driver insc-Gal4, significantly more quiescent NSCs, as judged by the lack of EdU incorporation and by the presence of cellular processes, were observed  versus 12.4%  and 10.3%  PH3+ NSCs were observed in cul4RNAi1/VDRC#105668 and cul4RNAi2/VDRC#44829  and ddb1HK-2-3 , ddb15-1 , and cul4G1-3 mutant brains  were proliferating at 24 h ALH on food depleted of amino acids, as indicated by their ability to incorporate EdU , overexpression of Cul4KR resulted in a marked decrease of EdU+ NSCs to 57.2%   in control MARCM clones were EdU+, only 7.5% (n = 53) and 19.5% (n = 41) of Mira+ NSCs in cul4G1-3 and cul4JJ11 MARCM clones were EdU+ (cul4G1-3 and cul4JJ11 was completely rescued by overexpression of UAS-cul4 transgene (n = 105 and n = 69). By contrast, overexpression of UAS-Cul4KR failed to rescue the quiescent NSC phenotypes of cul4G1-3 and cul4JJ11, as indicated by 3.7% (n = 27) and 3.2% (n = 31) EdU+ NSCs, respectively  . Further control . These p mutants . At 96 here EdU+ . The quiectively . These r+ NSCs , whereas overexpression of an active form of InR (InRCA) in NSCs triggered NSC reactivation . By contrast, ddb1 knockdown dramatically suppressed this effect induced by InRCA overexpression . This result suggested that DDB1 likely functions downstream of the InR pathway during NSC reactivation.We next tested whether DDB1 is required for premature reactivation induced by overactivation of InR signaling pathway. Similar to previous reports ,11, in tDrosophila ortholog of human DCAF1/Vpr (HIV-1) Binding Protein (VprBP) #34912 and mahjRNAi2: VDRC#110669) driven by insc-Gal4 led to an increase in proportion of NSCs with a cellular process to 23.1%  and 27.1%   and 75.1%  upon mahj RNAi knockdown, whereas there were 96.6%  EdU+ NSCs in the control  in mahjRNAi1 and 9.1%  in mahjRNAi2, compared with 20.6%  in control  , as the t = 575) . Consist control . In addi control .mahj1, that was previously generated by imprecise P-element excision , depending on the combinations of constructs) displayed no PLA foci, and the rest of the cells displayed weak PLA foci (1–10 foci); on average, there were 0–0.4 ± 0.2–0.8 PLA foci per cell (n = 281) coexpressing Flag-Wts and Myc-Mahj showed PLA signals: 26.0%, 30.6%, and 41.3% of cell with strong (>20 foci), moderate (11–20 foci), and weak (1–10 foci) signals, respectively; they resulted in 16.2 ± 14.9 foci per cell (n = 176) expressing Flag-Wts and Myc-DDB1 (n = 176) showed PLA signals: 10.1%, 27.0%, and 60.7% of the cells with strong, moderate, and weak PLA signals; and on average, these cells displayed 10.7 ± 9.94 PLA foci per cell (n = 157) also indicated positive physical interaction: 11.9 ± 10.5 PLA foci per cell and 97.5% cells showed strong, moderate, and weak PLA signals of 56.7%, 26.1%, and 14.6%, respectively   . By contn of Wts . These rS2 cells .mahj depletion could aggravate NSC reactivation defects observed upon ddb1 depletion. Whereas 97.2% of control NSCs  were positive for EdU, 84.7%  of ddb1RNAi and 81.5%  of mahjRNAi controls incorporated EdU  of NSCs were EdU+  of NSCs were positive for EdU, comparable to that of the control brains   of NSCs in mahj knockdown were EdU+ at 24 h ALH, upon simultaneous knockdown of both mahj and wts by insc-Gal4, 97.1%  of NSCs incorporated EdU . These data suggest that wts knockdown significantly suppresses NSC reactivation defects observed in mahj RNAi brains. To maintain NSC quiescence, Wts phosphorylates and inactivates the transcriptional coactivator Yki to turn off expressions from Yki target genes  XE-2900) transheterozygous mutants were labeled with EdU, Dpn, and Mira. (M-N) Quantification of NSCs that are EdU+ or with process of various genotypes in (L). (O) MB NSC lineages in larval brains from WT and mahj1 at 24 h ALH were labeled with EdU, Dpn, and Dac. The enlarged views of white dotted boxes are shown to the right. Yellow arrowheads, MB NSCs surrounded by Dac-positive MB neurons. (P) At 0 h ALH, larval brains of grh-gal4>UAS-CD8-GFP were labeled with Mahj and a NSC marker (Dpn). Blue arrows, NSCs. (Q) Quantification of number of NSCs in ddb1−, cul4−, and mahj− mutant brains at 24 h ALH. Data are presented as mean ± SD. **** for P ≤ 0.0001, *** for P ≤ 0.001, ** for P ≤ 0.01, * for P ≤ 0.05, and ns for P > 0.05. Scale bars: 10 μm. The data underlying this figure can be found in Drosophila Stock Center; cul4, Cullin 4; Dac, dachshund; Dcr2, Dicer 2; ddb1, damaged DNA-binding protein 1; Dpn, Deadpan; EdU, 5-ethynyl-2′-deoxyuridine; GFP, green fluorescent protein; Mahj, Mahjong; MB, mushroom body; Mira, Miranda; ns, statistically nonsignificant; NSC, neural stem cell; PH3, phospho-Histone H3; ROD, relative of density; UAS, upstream activating sequence; VDRC, Vienna Drosophila Resource Center; WT, wild type.(A-F) At 24 h ALH, larval NSCs in control ((TIF)Click here for additional data file.S4 FigDmMahj and DmDDB1. (A) View of Mahj 1110–1441 aa  and DDB1  heterodimers. (B) Detail of predicted interaction between residues R1120 and R1123 (red stick) side chains in the HLH motif of Mahj (yellow ribbon) and DDB1 . Relevant residues are depicted in stick models: R1220 and R1123 , D1134 , and D788 . The predicted H-bonds are depicted by black dashed lines. Mahj R1120 and R1123 are corresponding to HsDCAF1 R1053 and R1056, respectively. (C) Detail of predicted interaction between R1307 and R1343 (red stick) side chains in the WD40 domain of Mahj (yellow ribbon) and DDB1 . Relevant residues are depicted in stick models: R1307 and R1342 , E1295 , and E201 . The predicted H-bonds are depicted by black dashed lines. Mahj R1307 and R1343 are corresponding to HsDCAF1 R1247, R1283, respectively. (D) A schematic diagram illustrating different Wts domains and truncated Wts proteins. (E) Wts C-terminal fragment containing kinase domain interacts with Mahj. Co-IP between Myc-Mahj and Flag-Wts or indicated truncated constructs. Anti-Myc were used for IP, followed by western blotting probed with anti-Myc, anti-Flag, or anti-Actin antibodies. Actin served as a loading control. aa, amino acid; DDB1, damaged DNA-binding protein 1; Dm, D. melanogaster; HLH, helix-loop-helix; HsDCAF1, H. sapiens DDB1-Cul4 associated factor 1; IP, immunoprecipitation; Mahj, Mahjong; Wts, Warts.(A-C) A homology model illustrating the interaction between (TIF)Click here for additional data file.S5 Fign = 272), Flag-Wts + Myc-control (n = 247), Flag-control + Myc-Mahj (n = 889), Flag-control + Myc-DDB1 (n = 284), Flag-control + Myc-Cul4 (n = 263), Flag-Wts + Myc-Mahj (n = 281), Flag-Wts + Myc-DDB1 (n = 176), and Flag-Wts + Myc-Cul4 (n = 157). The data underlying this figure can be found in (A) Co-IP between Flag-DDB1 and HA-Wts. S2 cells were cotransfected with Flag-DDB1 and HA-Wts or respective controls. Immunoprecipitation was performed using anti-Flag antibodies, and western blot was performed using anti-Flag or anti-HA antibodies. (B) A schematic illustration for PLA. (C) In situ PLA assay between Flag-Wts and either Myc-Mahj, Myc-DDB1, or Myc-Cul4. S2 cells transfected with the indicated plasmids were stained with Flag, Myc, and DAPI and detected for PLA signal (red). Cell outline was shown by DIC images. Scale bar, 10 μm. (D) Quantification graph showing the percentage of cells with PLA foci in (B). (E) Quantification for the average number of PLA foci per cell in (C). The number of cells used for quantification in  are coexpression of Flag-control + Myc-control ((TIF)Click here for additional data file.S6 FigctrlRNAi: β-galRNAi), wtsRNAi +β-galRNAi, ddb1RNAi (VDRC#44974) +β-galRNAi, and ddb1RNAi + wtsRNAi were labeled with Dpn, Mira, and EdU. (B-C) Quantification of Dpn+ Mira+ NSCs that are EdU+ (B) or with cellular process (C). In (B), control : brain lobes n = 8, total NSCs t = 517; wtsRNAi+β-galRNAi: n = 11, t = 705; ddb1RNAi+β-galRNAi: n = 10, t = 647; ddb1RNAi + wtsRNAi: n = 15, t = 955. In (C), control : n = 4, t = 320; wtsRNAi+β-galRNAi: n = 6, t = 485; ddb1RNAi+β-galRNAi: n = 6, t = 598; ddb1RNAi + wtsRNAi: n = 16, t = 1,403. (D) Larval NSCs in ctrlRNAi , YkiS168A+β-galRNAi, ddb1RNAi+ β-galRNAi, and ddb1RNAi + YkiS168A were labeled with Dpn, Mira, and EdU. (E-F) Quantification of Dpn+ Mira+ NSCs that are EdU+ (E) or with cellular process (F). In (E), control : n = 7, t = 388; YkiS168A+β-galRNAi: n = 10, t = 645; ddb1RNAi+β-galRNAi: n = 9, t = 551; ddb1RNAi + YkiS168A: n = 11, t = 809. In (F), control : n = 5, t = 429; YkiS168A+β-galRNAi: n = 4, t = 255; ddb1RNAi+β-galRNAi: n = 5, t = 357; ddb1RNAi + YkiS168A: n = 9, t = 755. (G) Larval NSCs in ctrlRNAi , wtsRNAi+β-galRNAi, cul4RNAi (VDRC#105668)+β-galRNAi, and cul4RNAi + wtsRNAi were labeled with Dpn and EdU. (H-I) Quantification of Dpn+ NSCs that are EdU+ or with Mira+ cellular process. In (H), control : n = 13, t = 1,179; wtsRNAi+β-galRNAi: n = 19, t = 1,665; cul4RNAi+β-galRNAi: n = 14, t = 1,073; cul4RNAi + wtsRNAi: n = 19, t = 1,404. In (I), control : n = 8, t = 702; wtsRNAi+β-galRNAi: n = 8, t = 696; cul4RNAi+β-galRNAi: n = 9, t = 772; cul4RNAi + wtsRNAi: n = 10, t = 847. (J) Larval NSCs in ctrlRNAi+β-galRNAi, YkiS168A+β-galRNAi, cul4RNAi+β-galRNAi, and cul4RNAi + YkiS168A were labeled with Dpn and EdU. (K-L) Quantification of Dpn+ NSCs that are EdU+ or with Mira+ cellular process. In (K), control : n = 12, t = 990; YkiS168A+β-galRNAi: n = 11, t = 917; cul4RNAi+β-galRNAi: n = 13, t = 1,056; cul4RNAi + YkiS168A: n = 15, t = 950. In (L), control : n = 7, t = 701; YkiS168A+β-galRNAi: n = 4, t = 344; cul4RNAi+β-galRNAi: n = 11, t = 974; cul4RNAi + YkiS168A: n = 10, t = 895. Driver is insc-Gal4, tub-Gal80ts (A-F), and insc-Gal4; UAS-Dcr2 (G-L). Note that to balance the number of UAS elements across different genotypes, additional control UAS line, β-galRNAi for (A-F) or CD8-GFP/BDSC#32186 for (G-L), was added to various RNAi lines, resulting in weaker phenotype in RNAi lines compared with those without additional UAS control shown earlier in this study. Yellow arrows, EdU+ NSCs. White arrows, EdU-negative NSCs. Data are presented as mean ± SD. **** for P ≤ 0.0001, *** for P ≤ 0.001, ** for P ≤ 0.01, * for P ≤ 0.05, and ns for P > 0.05. Scale bars, 10 μm. The data underlying this figure can be found in Drosophila Stock Center; CRL4, Cullin4-RING ligase; ddb1, damaged DNA-binding protein 1; Dpn, Deadpan; EdU, 5-ethynyl-2′-deoxyuridine; Mira, Miranda; ns, statistically nonsignificant; NSC, neural stem cell; RNAi, RNA interference; UAS, upstream activating sequence; VDRC, Vienna Drosophila Resource Center; Wts, Warts; Yki, Yorkie.(A) Larval NSCs in control ((TIF)Click here for additional data file.S1 TableC-term, C-terminal fragment; F, forward; FL, full length; HLH+WD40, helix-loop-helix motif and WD40 domain; N-term, N-terminal fragment; R, reverse; sc, with stop codon; wo sc, without stop codon.(XLSX)Click here for additional data file.S2 TableF, forward; R, reverse.(XLSX)Click here for additional data file.S3 TableDDB1, damaged DNA-binding protein 1; F, forward; R, reverse; RNAi, RNA interference; UAS, upstream activating sequence.(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


To investigate the diagnostic value of the interferon-γ release assay (IGRA) for detecting tuberculosis (TB) infection in patients with Behçet’s disease (BD).We retrospective analyzed the data collected from 173 BD patients hospitalized between 2010 and 2015. Ninety-nine healthy volunteers were enrolled as a control group. IGRA was performed using T-SPOT.TB. The diagnosis of active TB (ATB) was based on clinical, radiological, microbiological, histopathological information and the response to anti-TB therapy. Latent TB (LTB) infection was defined as asymptomatic patients with positive T-SPOT.TB.6 PBMC was the optimal cutoff for diagnosing ATB, with an area under the curve of 0.891. Furthermore, the median SFCs in ATB group was significantly higher than those in LTB infection  or previous TB infection . A significant discrepancy between T-SPOT.TB and tuberculin skin test was noted .TB infection was documented in 59 BD patients (34.1%). The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio of T-SPOT.TB for the diagnosis of ATB were 88.9%, 74.8%, 29.1%, 98.3%, 3.53 and 0.15, respectively. The receiver-operating-characteristic curve demonstrated that spot-forming cells (SFCs) of 70/10T-SPOT.TB, an IGRA, may assist in the diagnosis of ATB in BD patients, and the higher SFCs suggest ATB in BD patients. M.tuberculosis may act as a trigger of BD through molecular mimicking. On the other hand, defective cell-mediated immunity in BD patients may increase the susceptibility of TB [Behcet’s disease (BD) is a multi-system inflammatory disorder, characterized by recurrent oral ulcers, genital ulcers, skin lesions, and ocular involvement of unknown etiology. Notably, the clinical manifestation of orogenital ulcers and skin lesions including erythema nodosum could be mimicked by tuberculosis (TB). Additionally, active TB (ATB) is frequently observed in patients with BD, and those symptoms were relieved after anti-TB therapy , 2. BD aty of TB , and thety of TB . Therefo. Given IGRAs do not cross-react with BCG vaccination or nontuberculous mycobacteria infection, IGRAs show higher specificity than TST [Tuberculin skin test (TST), a routine screening test for latent TB (LTB) infection, has limited diagnostic value in BD patients due to cross-reactivity with the bacillus Calmette-Guérin (BCG) vaccine and nontuberculous mycobacteria as well as pathergy reaction . In addithan TST . IGRAs, than TST  and rheuthan TST . HoweverM.tuberculosis. Highly-probable TB was defined as highly suggestive clinical and radiological features for TB in combination with the appropriate response to anti-TB treatment [Medical records of the hospitalized BD patients from Peking Union Medical College Hospital between January 2010 and March 2015 were retrospectively reviewed. All patients fulfilled the International Study Group BD criteria or the new International Criteria for BD . T-SPOT.reatment . LTB infreatment .M.tuberculosis. Briefly, peripheral blood mononuclear cells were isolated from whole blood samples by density gradient separation, then stimulated with two TB specific antigens, early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), phytohaemagglutinin (PHA) (positive control) or AIM-V  (negative control) in microplate wells (2.5 × 105 per well) precoated with interferon-γ (IFN-γ) capture antibodies. The number of spot-forming cells (SFCs) was calculated after 16 to 20 h of incubation. The result was considered positive if the number of SFCs was six or more after subtracting the spot count of negative control [IGRAs were performed using T-SPOT.TB, a simplified enzyme-linked immunospot (ELISPOT) for detection of effector T cells that respond to stimulation by antigens specific for  control .TST was performed with an intradermal injection using standardized 0.1 ml (5 U)-purified protein derivative into the ventral surface of the forearm according to the Mantoux method . On the p values < 0.05 is considered statistically significant. Statistical analysis was performed by using SPSS  and the GraphPad Prism .Numerical data were expressed as mean ± standard deviation or median , and categorical data were expressed as frequencies/ percentages. The Mann-Whitney U test and Pearson chi-square test were used to compare differences between two groups. The concordance of TST and T-SPOT.TB results were assessed using κappa coefficient. . The diagnostic performance of T-SPOT.TB on ATB was evaluated by calculation of its sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), and negative likelihood ratio (NLR). The area under the receiver operating characteristic curve of the T-SPOT.TB on peripheral blood mononuclear cell (PBMC) diagnostic cutoff for ATB was calculated. A two-sided with Among the 173 BD patients performed with TB-SPOT.TB test, 114 (65.9%) were men. The mean age was 37.07 ± 14.74 years, and median disease duration of BD was 84 months (range 1–608). The most common clinical manifestation was recurrent oral ulceration (98.3%). Other common findings included fever (81.5%), genital ulcers (58.4%), skin lesions (58.4%), gastrointestinal involvement (39.3%), vascular involvement (28.3%), positive pathergy test (22.5%), ocular involvement (19%), neurologic involvement (15%), cardiac involvement (9.8%) and hematological involvement (5.2%). The median BDCAF2006 score for disease activity was 2 (range 0–5). Elevated erythrocyte sedimentation rate (ESR) and high-sensitivity C-reactive protein (hs-CRP) levels were detected in 98 (56.6%) and 118 patients (68.2%), respectively. The median ESR was 21 (range 1–140) mm/first hour and the median hs-CRP was 11.54 (range 0.12–262.77) mg/L. In the past 2 years, 113 patients (65.3%) were treated with glucocorticoids, including 13 patients (7.5%) received glucocorticoid pulse therapy. Ninety-seven patients (56.1%) were under glucocorticoids treatment when T-SPOT.TB assay was performed, and the median daily dose of corticosteroids was 40 (range 5–100) mg. Ninety-four patients received immunosuppressants, including cyclophosphamide (30.1%), leflunomide (5.2%), cyclosporin A (4.6%), azathioprine (4.6%), methotrexate (4.6%) and salazosulfapyridine (2.9%). Sixteen patients (9.2%) received two or more immunosuppressants. Biological agents were used in 12 patients (6.9%), including infliximab injection in six patients, etanercept injection in five patients, etanercept followed by infliximab in one patient , especially erythema nodosa  compare to BD-LTB infection patients.TB infection was documented in 59 BD patients 34.1%) in our study, including 18 (10.4%) ATB (BD-ATB), 29 (16.8%) LTB (BD-LTB) infection, and 12 (6.9%) patients with evidence of previous TB. Of the 18 BD-ATB cases, culture-confirmed TB and highly probable TB were diagnosed in 4 and 14 patients, respectively. Of the four culture-confirmed TB patients, pulmonary TB, tuberculous lymphadenitis, tuberculous pleuritis, tuberculous meningitis were diagnosed in one patient, respectively. Among 14 highly-probable TB, highly-probable pulmonary TB was diagnosed in 7 cases based on fever, cough, expectoration or chest pain, and CT-confirmed pulmonary cavity, infiltrating or nodules as well as % in our 6 PBMC (IQR: 108–925). Among patients with evidence of previous TB, ten patients (83.3%) had positive T-SPOT.TB with the median number of SFCs being 96/106 PBMC (IQR: 31.75–187). The median number of SFCs in BD-LTB infection patients was 68/106 PBMC (IQR: 38–208). The median number of SFCs in the BD-ATB group was higher than those in the BD-LTB infection group (p = 0.007) or those in previous TB patients (p = 0.018). The median numbers of SFCs in the BD-ATB group, BD-LTB infection group and previous TB patients were higher than those in the non-TB infection group (p = 0.000) , and the median number of spot-forming cells (SFCs) was 466/10The sensitivity, specificity, PPV and NPV of the T-SPOT.TB test for the diagnosis of ATB were 88.9% (95%CI 63.9–98.1%), 74.8% (95%CI 67.1–81.3%), 29.1% (95%CI 18–43.1%), and 98.3% (95%CI 93.4–99.7%), respectively. PLR was 3.53(95%CI 2.57–4.85) and NLR was 0.15 (95%CI 0.04–0.55).6 PBMC was the optimal cutoff for diagnosing BD-ATB, with an area under the curve of 0.891 (95%CI 0.796–0.987)  Fig. . Accordi6 PBMC (IQR:80–308). There was no significant difference in the proportion of LTB infection  and the number of SFCs detected by T-SPOT.TB  between BD patients and healthy controls.LTB infection was documented in 27(27.3%) healthy controls, and the median number of SFCs was 192/10p = 0.002).Both T-SPOT.TB and TST results were simultaneously tested in 63 patients. Among these patients, positive results of TST and T-SPOT.TB were presented in 44.4% 28/63) and 46% (29/63) patients, respectively. 30.2% (19/63) of the 63 patients had both positive TST and T-SPOT.TB, and 60.3% (38/63) had positive results on either TST or T-SPOT.TB (Table 8/63 and The sensitivity of TST for detecting ATB was 78.6% (95%CI 48.8–94.3%), which was lower than that of the T-SPOT.TB test (92.9%), but there was no significant difference between the two groups. The specificity of TST was similar with T-SPOT.TB test (Table p = 0.06). However, there was no difference in the sensitivity between parallel testing and T-SPOT.TB assay alone.We also evaluated the diagnostic values of the combination of T-SPOT.TB and TST for ATB diagnosis in these 63 patients  TB report . Patientnfection , but thenfection .M.tuberculosis-specific antigens [In recent years, IGRAs emerges as accurate diagnostic assays for screening of TB infection. IGRAs specifically detect the presence of specific effector T cells which response to antigens . Dai Y. antigens . And Theantigens . Consistantigens .6 PBMC as the cutoff, the specificity and PLR are improved for diagnosing ATB in BD patients. Although, neither T-SPOT.TB nor TST can clearly distinguish between LTB infection, ATB or history of TB infection [In this study, we evaluated T-SPOT.TB and TST for the diagnosis of ATB in BD patients. To our knowledge, this is the first study that reports the diagnostic value of T-SPOT.TB for TB in BD patients. We reported the high sensitivity (88.9%) and specificity (74.8%) of T-SPOT.TB for the diagnosis of ATB in BD patients. Compared to Dai Y’s study , our stunfection , 26, we nfection . Meanwhinfection .It has been reported that the pooled sensitivity of T-SPOT.TB was higher than that of TST for the diagnosis of ATB in culture-confirmed or non-confirmed TB . A discrMycobacterium tuberculosis antigens with no evidence of clinically ATB. Given the risk of LTB infection reactivation increases in patients receiving immunosuppressive therapy or with immune dysfunction [LTB infection is defined as the sustained immune response to function , it is ifunction –33 suggefunction –36. Alsofunction , 37, andfunction  . In ourfunction , 38. A nfunction . SecondlM.tuberculosis bacteriologically confirmed, which might introduce a high risk of bias. Fourth, given no gold standard of LTB infection is available, the precision of the diagnosis of LTB infection by T-SPOT.TB could not be assessed in this study. Nevertheless, this is the first study on the diagnostic value of T-SPOT.TB for TB in BD patients. A large-scale, multi-center study enrolled bacteriologically confirmed TB patients are warranted to confirm our findings.Our study has several limitations. First, our study is a retrospective analysis of a case series, which may have incomplete data of TST. Second, since all BD patients were enrolled from a single-center with relatively small sample sizes, and our center is a national referred center for complicated rheumatic diseases, potential selection bias of BD patients is possible. Third, it is challenging to identify active TB in BD patients based on clinical features since systemic inflammation is a shared feature of both BD and TB. The majority of our ATB patients were diagnosed according to clinical criteria rather than 6 PBMC) strongly suggested ATB. Further study is needed to investigate the diagnostic value of T-SPOT.TB for LTB infection in BD patients.T-SPOT.TB may assist in the diagnosis of ATB in BD patients, and the higher number of SFCs (> 70/10


In this study, resistance spectrum of TA7733 was assayed by using 15 Blumeria graminis f. sp. tritici (Bgt) isolates prevalent in different regions of China. The result indicated that TA7733 was highly resistant to all tested Bgt isolates and the gene(s) on chromosome 2Mb conferred broad-spectrum resistance to powdery mildew. In order to characterize mechanism of powdery mildew resistance by identifying candidates R-genes derived from Ae. biuncialis chromosome 2Mb and develop 2Mb-specific molecular markers, we performed RNA-seq analysis on TA7733 and CS. In total we identified 7,278 unigenes that showed specific expression in TA7733 pre and post Bgt-infection when compared to CS. Of these 7,278 unigenes, 295 were annotated as putative resistance (R) genes. Comparatively analysis of R-gene sequences from TA7733 and CS and integration CS Ref Seq v1.0 were used to develop R-gene specific primers. Of 295 R-genes we identified 53 R-genes were specific to 2Mb and could be involved in powdery mildew resistance. Functional annotation of majority of the 53 R-genes encoded nucleotide binding leucine rich repeat (NLR) protein. The broad-spectrum resistance to powdery mildew in TA7733 and availability of 2Mb-derived putative candidate R-gene specific molecular markers identified in this study will lay foundations for transferring powdery mildew resistance from 2Mb to common wheat by inducing CS-Ae. biuncialis homoeologous recombination. Our study also provides useful candidates for further isolation and cloning of powdery mildew resistance gene(s) from Ae. biuncialis chromosome 2Mb.Powdery mildew is one of the most widespread diseases of wheat. The development and deployment of resistant varieties are one of the most economical and effective methods to manage this disease. Our previous study showed that the gene(s) at 2M Triticum aestivum L., 2n = 6x = 42, AABBDD), one of the most widely planted crops in the world provides 20% of the calories and 25% of its protein consumed by human [Fusarium head blight (FHB) and powdery mildew. Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases all over the world, with severe yield losses ranging from 13% to 50% [Common wheat  was more than 80 at 54 loci [Pm genes were derived from wild relatives of wheat. However, some Pm genes had been defeated by new virulent Bgt races or by races that were previously present at very low frequencies in the pathogen population [Wild relatives of common wheat contain a large number of  54 loci , of whicpulation ,11, and pulation ,13. TherAegilops biuncialis  is a tetraploid wild relative of wheat, belonging to the section Polyeides of the genus Aegilops. The Ub genome of it was derived from Ae. umbellulata , and Mb genome from the diploid Ae. comosa  [Aegilops biuncialis owns many desired agronomic traits for wheat improvement, such as resistance to yellow rust [Ae. biuncialis with wheat, develop a series of wheat-Ae. biuncialis addition lines, and transfer desired genes from Ae. biuncialis into wheat [Ae. biuncialis 2Mb disomic addition line TA7733 conferred high resistance to powdery mildew compared with its recipient parent CS [ 14, MM) . Aegiloplow rust , brown rlow rust , powderylow rust , toleranlow rust –21, highlow rust , and spelow rust . Successto wheat ,24,25. Iarent CS .Ph) genes in hexaploid wheat backgrounds [Thinopyrum intermedium by RNA-seq analysis [Pm60 from T. urartu by combining genetic mapping and RNA-seq analysis [The isolation and cloning of plant disease resistance genes had great significance for both plant disease resistance breeding and the study on molecular mechanisms of disease resistance. Map-based cloning is currently an important method to isolate novel genes. However, it is very challenging to perform fine mapping and map-based cloning of alien genes derived from wild relatives of wheat due to the strict control of homoeologous recombination by pairing homoeologous  from chromosome 2Mb, as well as further understanding of the molecular and genetic mechanisms of disease resistance conferred by Ae. biuncialis chromosome 2Mb.In this study, we report the assays of a broad-spectrum resistance gene(s) on chromosome 2Mn = 6x = 42, AABBDD), Ae. comosa TA2102 , and CS-Ae. biuncialis 2Mb disomic addition line TA7733 (2n = 44) where a pair of 2Mb chromosomes derived from Ae. biuncialis were added into CS genetic background were used in this study. All the materials were kindly provided by the Wheat Genetics Resource Center at Kansas State University, USA and maintained at the experimental station of Henan Agricultural University, China.Common wheat landrace CS  . The cytGenomic DNA (gDNA) was extracted from fresh leaves using a modified hexadecyl trimethyl ammonium bromide (CTAB) method . The conin situ hybridization (GISH) was applied to analyze the chromosomal composition of TA7733. Genomic DNA of Ae. comosa accession TA2102  and wheat CS were respectively used for probe labeling with fluorescein-12-dUTP and blocking at a ratio of 1:130 to distinguish Ae. biuncialis 2Mb chromosome. GISH was carried out as described by Liu et al. (2017) [Genomic . (2017) . Hybridiin situ hybridization (ND-FISH) [10. The first six were labeled with 6-carboxytetramethylrhodamine (TAMRA) generating red signals, and the last two being labeled with 6-carboxyfuorescein (FAM) generating green signals. All the oligonucleotides were synthesized at Sangon Biological Technology, Shanghai, China.After GISH, the hybridization signals were washed off with phosphate-buffered saline (PBS). Eight single-strand oligonucleotides were then used as probes for dual-color nondenaturing fluorescence ND-FISH) ,39. The Bgt isolates collected in Henan Province were used to evaluate the resistance of CS and CS-Ae. biuncialis 2Mb disomic addition line TA7733. Fifteen prevalent Bgt isolates collected from different regions of China were chosen to evaluate the resistance spectrum of TA7733 at the seedling stage by using CS as a susceptible control. The 15 Bgt isolates were provided by Prof. Pengtao Ma, Yantai University, China. They were all single-pustule-derived powdery mildew virulent isolates by separate artificial inoculation. The infection type (IT) were scored 7–10 days post-inoculation using a 0 to 4 rating scale [A mixture of prevailing ng scale , with 0 Bgt inoculation, the first leaves of TA7733 and CS were cut into 2 cm segments and stained with coomassie brilliant blue following Li et al. (2016) [Bgt development on the leaves.At 10 days post-. (2016)  for furtBgt isolates. Leaves at 0, 12, 24, 48 and 72 hours post-inoculation (hpi) were respectively collected, rapidly frozen in liquid nitrogen and stored at -80°C for RNA extraction.Seeds of CS and TA7733 soaking in water for 24 h at 23°C were transferred into a mixture of nutrient soil and vermiculite (1:1). Seedlings with full extended first leaf were dusted using fresh conidiophores of Total RNA of ten samples  were extracted for transcriptome sequencing. Then equal amounts of RNA samples 12–72 hpi from TA7733 and CS were mixed to generate RNA-seq sample RI and SI, respectively. RNA at 0 hpi from TA7733 and CS were accordingly represented as RNA-seq sample RC and SC. Two biological replicates were performed in this study, forming a total of eight RNA samples . The designations 1 and 2 are used to represent replicates 1 and 2, respectively. Libraries with an average insert size of 200 bp constructed from these eight samples were then sequenced using the Illumina HiSeqTM 2500 by the Beijing Genomics Institute.−5) against the NCBI non-redundant (NR) protein, SWISSPROT, gene ontology (GO), eukaryotic orthologous groups (KOG), kyoto encyclopedia of genes and genomes (KEGG) and plant resistance gene (PRG) databases.Prior to assembly, sequencing raw reads were pre-processed using a Perl script dynamic-Trim.pl to remove the adaptor sequences, low-quality sequences, low complexity sequences, short reads and empty reads. Reads data with a quality score (Qphred) ≥ 50 (Q50: ratio of an error rate of 0.01%) were then merged and input into the data assembly software Trinity for assembling into transcripts. The generated unigenes were annotated by a Blastx alignment search , 0.25 μl forward primer (10 μmol/l), 0.25 μl reverse primer (10 μmol/l), 7.5 μl Taq MasterMix  and 5 μl ddH2O. PCR cycling conditions were as follows: 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 50–66°C for 30 s, and 72°C for 1 min, followed by a final 10-min extension at 72°C. The PCR products were digested with four base-restriction enzymes. Five microliters of a restriction enzyme mixture containing 2.8 μl of ddH2O, 2.0 μl of CutSmart buffer, and 0.2 μl of an enzyme stock solution was added to 15 μl of PCR products and incubated for 3.5 h at 65°C. The PCR or restricted PCR products were separated on a 2.0% agarose gel-electrophoresis stained with ethidium bromide and visualized by UV light.R gene-specific primer sets were designed based on their transcriptome sequences to perform PCR amplification using gDNA from TA7733 and CS as templates to verify 2MAe. biuncialis chromosome 2Mb. Comparative maps of 2Mb-specific R genes were made using MapDraw software referring homoeologous chromosome locations of CS Ref Seq v1.0.Genome sequences of wheat landrace CS (CS Ref Seq v1.0) were used as references in Blastn searches to obtain position information for R genes from Ae. biuncialis 2Mb disomic addition line TA7733 by using fluorescein-labeled gDNA from M genome donor Ae. comosa as a probe and wheat CS DNA as blocker. As shown in Ae. biuncialis 2Mb chromosomes in TA7733, confirming the disomic addition of chromosome 2Mb.GISH and ND-FISH were respectively performed to confirm the chromosome composition of CS-Bgt isolates collected in Henan Province was used to inoculate seedlings with fully-extended first leaves of TA7733 and its recipient parent CS in the greenhouse. Ten days post-inoculation, the leaves of CS were covered with a large number of Bgt hyphae, with ITs of 3–4, whereas TA7733 showed only stunted spores, with ITs 0 to 1  to all the 15 Bgt isolates tested, whereas its recipient parent CS was highly susceptible (IT = 4 or 3) to all tested Bgt isolates. These results indicated that the chromosome 2Mb in TA7733 conferred broad-spectrum resistance to powdery mildew of wheat.The resistance spectrum of TA7733 was further assayed at the seedling stage by inoculation of 15 prevalent Ae. biuncialis 2Mb disomic addition line TA7733 and its recipient parent CS were respectively conducted pre and post Bgt-infection. A total of 158,953 unigenes were assembled with a total length of 198,364,757 bp. The average unigene size was 1247.95 bp ranging from 301 to 19,496 bp , 48,724 (30.65%), 40,543 (25.51%), 37,008 (23.28%), 13,414 (8.44%) and 10,969 (6.92%) annotated unigenes, respectively  unigenes were assigned to one or more GO term annotations , of whicThe KEGG database was used to systematically describe the pathway where the unigenes involved. Out of a total 158,953 annotated unigenes, 26,589 unigenes were assigned to 23 KEGG pathways . The mosAe. biuncialis chromosome 2Mb, which should be only expressed in TA7733 other than in CS. Based on pairwise comparison of unigenes of TA7733 vs CS, a total of 7,278 genes were uniquely expressed in TA7733, of which 4,382 unigenes were significantly differentially expressed post vs before Bgt-inoculation, and the remaining 2,896 unigenes had insignificantly different expression levels. In consideration of the fact that expression levels of some cloned resistance genes did show no significant difference before and after pathogen infection [b-derived R-genes involved in powdery mildew resistance.One of the objectives of this study was to explore putative R genes specific to nfection , these 2Ae. biuncialis 2Mb chromosome.To analyze the biological pathways of these 4,382 unigenes, the statistical enrichment of differentially expressed genes (DEGs) in KEGG pathways were tested using the KOBAS software. In consequence, 399 out of 4,382 DEGs were allocated to 162 KEGG pathways . The mosBased on transcriptome data analysis, 7,278 unigenes were uniquely expressed in TA7733. Only 295 of 7,278 unigenes were annotated as putative R-genes by Blastx alignment against the PRG and NCBI databases. However, of 295 R-genes sequences when blastn searched against CS Ref Seq v1.0, only 61 (20.68%) R-genes mapped to wheat homoeologous group 2, and the remaining 234 (79.32%) R-genes to none of the homoeologous group 2 chromosome of wheat.Ae. biuncialis chromosome 2Mb, a total of 61 sets of PCR primer pairs were designed based on transcriptome sequences of these R genes. PCR amplification of gDNA of CS and TA7733 confirmed 40 R genes to be specific to chromosome 2Mb, which producing unique amplification in CS-Ae. biuncialis 2Mb disomic addition line TA7733  structures, a central nucleotide-binding (NB) subdomain and a leucine-rich repeat (LRR) domain, 12 in NL class containing NBS and LRR domains, but lack of CC domain, five in class RLP which contains leucine-rich receptor-like repeat, a transmembrane region of 25AA, and a short cytoplasmic region, each one for TNL class which contains a central NB subdomain, a LRR domain, a interleukin-1 receptor (1L-1R) domain, and N class only containing NBS domain . The remAe. biuncialis chromosome 2Mb specificity with those in CS Ref Seq v1.0  on Ae. biuncialis 2Mb chromosome should be a new Pm gene(s).Development of resistant wheat varieties is the most important and environment-friendly way to control programs . Wild re group 2 , 42–44. Pm60, a map-based cloned powdery mildew gene, showed no significant differences at various time points after Bgt E09 infection based on qRT-PCR analysis [bsr-d1 in rice were not significantly up-regulated after blast infection [Bgt-resistance related candidate genes from all specifically expressed R genes in TA7733 regardless of significance of their expression level changes post vs before Bgt-infection. After PCR verification by using R gene sequence-based primer sets and integrating transcriptome sequences blastn against CS Ref Seq v1.0, we finally verified 53 R genes candidates of chromosome 2Mb specificity, which included 47 unregulated, four up-regulated and two down-regulated genes.Previous studies have generally focused on the significantly differentially expressed genes in interactions between plant and pathogens to explore disease resistance-related genes by transcriptome sequencing –47. Howenfection . So, thePm genes including Pm2 [Pm3 [Pm8 [Pm21 [Pm60 [b-specific R genes were predicted to encode CC-NBS-LRR protein. These 14 R genes should be considered as the most promising candidate genes for further isolating and cloning Pm genes carried by Ae. biuncialis chromosome 2Mb.Isolation of plant resistance (R) gene is greatly helpful to breed resistant varieties and elucidates resistance molecular mechanisms. Conventional map-based cloning proved to be a effective method to clone R genes , howeverding Pm2 , Pm3 [50Pm3 [Pm8 , Pm21 [2m8 [Pm21 ,52 and P21 [Pm60 , have beD. villosum 6V#4S-specifc markers using transcriptome data [Ae. longissima chromosome-specific markers by RNA-seq [D. villosum#4 based on transcriptome data [Ae. biuncialis chromosome 2Mb. These markers will be useful to assist the transfer resistance gene(s) from 2Mb into common wheat by inducing CS-Ae. biuncialis 2Mb homoeologous recombination for wheat disease breeding in the future.GISH is a popular visual method to identify alien chromosome or chromatin in wheat background. Whereas GISH is expensive and time-consuming especially used to screen a large population derived from distant crossing between wheat and its wild relatives . In contome data . Wang et RNA-seq . Li et aome data . Furtherome data . Therefoome data ,58. In tAe. biuncialis chromosome 2Mb was verified to be board-spectrum in this study. It could be a valuable disease-resistance resource for wheat breeding programs. Fifty-three disease resistance gene candidates of 2Mb specificity, which were selected based on transcriptome sequencing analyses, will be greatly helpful to further isolate and clone Pm gene(s) derived from chromosome 2Mb and provide the insights into molecular mechanism of 2Mb-conferred powdery mildew resistance. Furthermore, 53 R gene sequence-based functional molecular markers of 2Mb specificity in this study will facilitate the transfer of resistance gene(s) from 2Mb to common wheat by inducing CS-Ae. biuncialis homoeologous recombination.In summary, powdery mildew resistance gene(s) on S1 Fig(M) 100 bp DNA Ladder.  CS.  TA7733. (A) CL119404Contig1. (B) CL88277Contig1. (C) CL82670Contig1. (D) 82789Contig1. (E) CL82700Contig1. (F) CL85355Contig1. (G) CL66003Contig1. (H) CL89405Contig1. (I) CL106750Contig1. (J) CL119216Contig1. (K) CL19981Contig2. (L) CL93721Contig1. (M) CL84424Contig1. (N) CL88613Contig1. (O) CL91022Contig1. (P) 96221Contig1. (Q) comp19533_c0_seq1_6. (R) CL85258Contig1. (S) CL113949Contig1. (T) CL86521Contig1. (U) CL105879Contig1. (V) CL90029Contig1. (W) CL84846Contig2. (X) CL87530Contig1. (Y) CL29910Contig1. (Z) CL92547Contig1. (AA) CL75219Contig1. (AB) CL108886Contig1. (AC) CL113652Contig1. (AD) CL80063Contig1. (AE) CL89447Contig1. (AF) CL93169Contig1. (AG) CL114224Contig1. (AH) CL116612Contig1. (AI) CL67241Contig1. (AJ) CL119539Contig1. (AK) CL90483Contig1. (AL) CL91742Contig1. (AM) comp84147_c0_seq1_6. (AN) CL124Contig7. (AO) CL100654Contig1. (AP) CL104996Contig1. (AQ) CL107524Contig1. (AR) CL107607Contig1. (AS) CL465Contig5. (AT) CL66266Contig1. (AU) CL72629Contig1. (AV) CL75868Contig1. (AW) CL86319Contig1. (AX) CL79458Contig1. (AY) comp121700_c0_seq1_5. (AZ) comp80277_c0_seq1_7. (BA) comp93868_c0_seq1_7.(TIF)Click here for additional data file.S1 Raw_imageAegilops biuncialis 2Mb disomic addition line TA77333; 3, common wheat CS; 4, CS-Ae. biuncialis 2Mb disomic addition line TA77333. (A) CL119404Contig1. (B) CL88277Contig1. (C) CL82670Contig1. (D) 82789Contig1. (E) CL82700Contig1. (F) CL85355Contig1. (G) CL66003Contig1. (H) CL89405Contig1. (I) CL106750Contig1. (J) CL119216Contig1. (K) CL19981Contig2. (L) CL93721Contig1. (M) CL84424Contig1. (N) CL88613Contig1. (O) CL91022Contig1. (P) 96221Contig1. (Q) comp19533_c0_seq1_6. (R) CL85258Contig1. (S) CL113949Contig1. (T) CL86521Contig1. (U) CL105879Contig1. (V) CL90029Contig1. (W) CL84846Contig2. (X) CL87530Contig1. (Y) CL29910Contig1. (Z) CL92547Contig1. (AA) CL75219Contig1. (AB) CL108886Contig1. (AC) CL113652Contig1. (AD) CL80063Contig1. (AE) CL89447Contig1. (AF) CL93169Contig1. (AG) CL114224Contig1. (AH) CL116612Contig1. (AI) CL67241Contig1. (AJ) CL119539Contig1. (AK) CL90483Contig1. (AL) CL91742Contig1. (AM) comp84147_c0_seq1_6. (AN) CL124Contig7. (AO) CL100654Contig1. (AP) CL104996Contig1. (AQ) CL107524Contig1. (AR) CL107607Contig1. (AS) CL465Contig5. (AT) CL66266Contig1. (AU) CL72629Contig1. (AV) CL75868Contig1. (AW) CL86319Contig1. (AX) CL79458Contig1. (AY) comp121700_c0_seq1_5. (AZ) comp80277_c0_seq1_7. (BA) comp93868_c0_seq1_7.Lanes: M, 100 bp Ladder DNA Marker; 1, common wheat CS; 2, CS-(PDF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.


Scyphiphora hydrophyllacea Gaertn. collected in Hainan Province of China resulted in the isolation of a new iridoid, scyphiphin C (1) and a known iridoid glycoside, shanzhiside methyl ester (2). Their structures were elucidated by a study of their physical and spectral data.Chemical investigation of the ethanol extract of the aerial parts of  Scyphiphora hydrophyllacea Gaertn. F. (Rubiaceae), one of the mangrove plants, is distributed from south to southeast Asia, Caroline Islands, Australia, and New Caledonia  to give 26 fractions. Fraction 16 (5.02 g) was subjected to column chromatography over silica gel eluted with light petroleum-EtOAc (4:6) to afford nine further fractions. Sub-fraction 4 (624.2 mg) was fractionated by column chromatography (Sephadex LH-20) eluted with 95 % EtOH and then rechromatographed on a silica-gel column with light petroleum-EtOAc (3:7) to afford compound 1 (18.7 mg). Fraction 18 (10.06 g) was subjected to vacuum liquid column chromatography over RP-18 eluted with MeOH-H2O  gradually to afford eight further fractions. Sub-fraction 1 (2.18 g) was purified by silica gel CC eluted with CHCl3-MeOH (9:1) to afford compound 2 (290.2 mg).The dried, milled aerial parts of Scyphiphin C (1): White amorphous powder,  = - 13.4 . IR (KBr): n = 3875, 3858, 3605, 3308, 2941, 2247, 1482, 865 cm-1. HR-ESI-MS (positive): m/z = 269.0998 . 1H and 13C-NMR: see Shanzhigenin methyl ester (2): White amorphous powder,  = - 380.0 . IR (KBr): n = 3922, 3886, 3779, 3654, 3700, 3543, 3112, 3081, 2326, 1583, 891 cm-1. HR-ESI-MS (positive): m/z = 429.1380 .Scyphiphora hydrophyllaceaGaertn., a new iridoid scyphiphin C (1) and a known iridoid glucoside shanzhigenin methyl ester (2) were isolated. Their structures were established on the basis of spectroscopic evidence.As a part of our chemical investigation on


Open Biol.7, 170165. (Published online 13 September 2017). (doi:10.1098/rsob.170165)A correction is required to The correct version of table 2 is as follows:


Flammulina rossica (wood-rotting basidiomycete) genome was performed to identify its carbohydrate-active enzymes (CAZymes). De novo genome assembly (31 kmer) revealed a total length of 35,646,506 bp (49.79% GC content). In total, 12,588 gene models of F. rossica were predicted using an ab initio gene prediction tool (AUGUSTUS). Orthologous analysis with other fungal species revealed that 7433 groups contained at least one F. rossica gene. Additionally, 12,033 (95.6%) of 12,588 genes for F. rossica proteins had orthologs among the Dikarya, and F. rossica contained 12 species-specific genes. CAZyme annotation in the F. rossica genome revealed 511 genes predicted to encode CAZymes including 102 auxiliary activities, 236 glycoside hydrolases, 94 glycosyltransferases, 19 polysaccharide lyases, 56 carbohydrate esterases, and 21 carbohydrate binding-modules. Among the 511 genes, several genes were predicted to simultaneously encode two different CAZymes such as glycoside hydrolases (GH) as well as carbohydrate-binding module (CBM). The genome information of F. rossica offers opportunities to understand the wood-degrading machinery of this fungus and will be useful for biotechnological and industrial applications.Next-generation sequencing (NGS) of the  Flammulina rossica  was first identified in 1999 by Redhead and Petersen E) , suggesting that these GHs can be secreted (The GH16 family consists of agarase (EC 3.2.1.81), lichenase (EC 3.2.1.73), κ-carrageenase (EC 3.2.1.83), xyloglucan xyloglucosyltransferase (EC 2.4.1.207), endo-β-1,3-glucanase (EC 3.2.1.39), endo-β-1,3-1,4-glucanase (EC 3.2.1.6), and endo-β-galactosidase (EC 3.2.1.103), and most of these enzymes contain conserved motifs such as Glu-Xaa-Asp-Xaa-(Xaa)-Glu (EXDX[X]E). The first and last glutamic acid (E) residues of the conserved motif are characterized as a nucleophile and Brønsted acid/base, respectively, and play an important role in the catalytic activity of GH16 family enzymes ,52,53. AXDX[X]E) . Althougsecreted . F. rossica also contains a series of genes related to cellulase (27 genes), xylanase (6 genes), and chitinase (23 genes) in its genome sequence  . Cellulasequence . Cellobis genome .F. rossica with more than 200 genes encoding various GHs showed strong potential for diverse applications, such as biotechnology and industry.Simultaneous actions of several GHs are necessary to effectively degrade plant cell wall complexes composed of cellulose and xylan. Recently, genome sequencing of various bacterial and fungal species has reviewed the various activities of GHs on cellulose, chitin, and xylan degradation and their potential for biotechnological applications and industrial degradation of biopolymers ,59,60,61F. rossica genome based on three different databases  searches (PLs EC 4.2.2.-) cleave polymer chains of polysaccharides, essential cellular components of all living organisms, through a β-elimination mechanism to produce unsaturated polysaccharides ,63. PLs .2.2.- clsearches . Among tsearches . In addisearches . PL20 wasearches  64]..F. rossid-galacturonate) or pectin  is an acidic polysaccharide in plant cell walls [F. rossica and other fungal species. However, according to this study, genes encoding PL families 2 and 10 were not found in F. rossica and in other fungal species genomes  but not in other fungal species  are classified into AA families, which are mainly involved in the depolymerization of non-carbohydrate structural components (lignin) of plants . These Asequence . Among tectively . Interes species . In addiomycetes .3-His-Xaa4-Met/Leu/Phe [18-Glu that interacts with the FAD cofactor [F. rossica was also found to contain 24 genes predicted to encode AA3, and 17 genes contained the β-α-β dinucleotide binding motif in their amino acid sequences  has conserved motifs (copper binding motifs) within its amino acid sequence such as His-Xaa-His, His-Xaa-His-Gly, His-Xaa-Xaa-His-Xaa-His, and His-Cys-His-Xaa/Leu/Phe . Our rescofactor ,71,72. Fequences . These rAmino acid sequence within a carbohydrate-active enzyme with carbohydrate-binding activity is known as CBM ,76. CBM F. rossica genome based on three different database  searches. CBM family 13 was prominent, and six families, including CBM18, -20, -22, -35, -48, -63, and -67, were represented by only one CBM in the F. rossica genome  genes that simultaneously encode CBM suggest that CBM is required for efficient substrate degradation , trisaccharide (cellotriose), and oligosaccharide (cellotetraose) to ethanol at similar recovery rates to glucose and to convert glucose to ethanol at a similar level as S. cerevisiae [F. velutipes is suitable for consolidated bioprocessing (CBP), which is considered an effective process for bioethanol production from lignocellulosic biomass [F. velutipes, the closest white rot fungus to F. rossica, is a very attractive model for bioethanol production due to the numerous genes associated with lignocellulolytic enzymes such as CAZymes [F. rossica genome to identify genes involved in lignocellulose degradation. As described above, various CAZyme genes were identified in the F. rossica genome including 102 auxiliary activities, 236 glycoside hydrolases, 94 glycosyltransferases, 19 polysaccharide lyases, 56 carbohydrate esterases, and 21 carbohydrate binding-modules. Although further studies on CAZyme genes are needed, this study suggests that F. rossica has great potential for future production of biomaterials such as bioenergy.This study aimed to improve the understanding of lignocellulolytic machinery in revisiae ,80. This biomass ,82,83. P


Multiple medication use within one year is associated with increased fall injury risk in older adults. However, chronically using multiple medications and treated fall injury have rarely been explored, particularly in cohort studies linked with claims data. We examined using >5 medications in 2 or more consecutive years (chronic medication use) as a risk factor for treated fall injury in 1,898 community-dwelling adults  with linked Medicare Fee-For-Service (FFS) claims from the Health, Aging and Body Composition Study since 1997/98 clinic visit. Incident fall injury (N=546) was the first claim from 1998/99 clinic visit to 12/31/08 with an ICD-9 fall code and non-fracture injury code, or fracture code with/without a fall code. Stepwise Cox models with a time-varying predictor of chronic medication use before fall injury or censoring (N=414) vs. not using >5 medications at the same time (N=1008) were adjusted for baseline demographics, lifestyle factors, fall history, quadriceps strength, cardiovascular disease (CVD), diabetes, sensory nerve impairment, and kidney function. Fall injury risk increased for chronic medication users (37%) vs. non-users (29%) (HR=1.25[1.00-1.57]), though was attenuated after adjustment for CVD and diabetes (HR=1.18[0.93-1.51]). Sensitivity analyses excluding fall-risk-increasing drugs (FRIDs) from medication counts (HR=1.32[0.54-3.20]), or including those using >5 medications non-chronically (N=365) in referent groups (HR=1.22[0.96-1.55]) had consistent findings. Unmeasured comorbidity differences may confound associations of chronic medication use and treated fall injury risk in older adults with Medicare FFS. Considering both chronic diseases and medication use in fall risk assessments is needed.


Stenotrophomonas maltophilia is a prevalent nosocomial pathogen with multidrug resistance. Here, we describe the complete genome of S. maltophilia myophage Moby, which shares characteristics with Enterobacteria phage T4 and is closely related to Stenotrophomonas phage IME-SM1. Moby has a 159,365-bp genome with 271 predicted protein-coding genes and 24 predicted tRNAs. Stenotrophomonas maltophilia is a prevalent nosocomial pathogen with multidrug resistance. Here, we describe the complete genome of S. maltophilia myophage Moby, which shares characteristics with Enterobacteria phage T4 and is closely related to Stenotrophomonas phage IME-SM1. Moby has a 159,365-bp genome with 271 predicted protein-coding genes and 24 predicted tRNAs. Stenotrophomonas maltophilia is a multidrug-resistant Gram-negative bacterium with rising prevalence as a nosocomial pathogen  was grown aerobically at 30°C in nutrient broth or agar (BD), and the phage was propagated by the soft-agar overlay method (www.bioinformatics.babraham.ac.uk/projects/fastqc), and trimming was performed using FastX Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/). Assembly into a single contig with 673.7-fold coverage was performed using SPAdes v3.5.0 , and, unless otherwise stated, were executed using default parameters (Moby was isolated from filtered (0.2-μm pore size) wastewater collected in Bryan, TX. The y method . Phage Dy method . An Illust ATCC 1807 was gs v3.5.0 . Rho-inds v3.5.0 . Gene fus v3.5.0 –15. Stru v3.5.0 –. progresost ATCC 807 was grameters . Phage mrameters .pac-type headful DNA packaging mechanism used by T4-like phages (Stenotrophomonas phage IME-SM1 (GenBank accession no. KR560069), an unclassified myovirus. No introns were identified in Moby, including in the predicted thymidylate synthase and nrdB genes, which contain introns in phage T4 (GenBank accession no. NC_000866)  .MN095772, BioProject accession no. PRJNA222858, SRA accession no. SRR8893605, and BioSample accession no. SAMN11414490.The genome sequence and associated data for phage Moby were deposited under GenBank accession no.


Triticum aestivum L.) through development and cultivation of superior genotypes incorporating yield-related agronomic and physiological traits derived from genetically diverse and complementary genetic pool. Despite significant breeding progress, yield levels in wheat have remained relatively low and stagnant under marginal growing environments. There is a need for genetic improvement of wheat using yield-promoting morpho-physiological attributes and desired genotypes under the target production environments to meet the demand for food and feed. This review presents breeding progress in wheat for yield gains using agronomic and physiological traits. Further, the paper discusses globally available wheat genetic resources to identify and select promising genotypes possessing useful agronomic and physiological traits to enhance water, nutrient-, and radiation-use efficiency to improve grain yield potential and tolerance to abiotic stresses . Finally, the paper highlights quantitative trait loci (QTL) linked to agronomic and physiological traits to aid breeding of high-performing wheat genotypes.Enhanced grain yield has been achieved in bread wheat ( Triticum aestivum L., 2n = 6x = 42) is the world’s third important staple food crop after maize (Zea mays) and rice (Oryza sativa). It is the most widely grown crop globally  are discussed. Finally, the paper highlights QTL linked to agronomic and physiological traits to aid breeding of high-performing wheat genotypes.Grain yield response in wheat is influenced by several agronomic and physiological traits . AgronomIndia, Russia, China, and Kazakhstan are currently the leading wheat producers with approximately 30, 27, 24, and 12 million hectares devoted to wheat production, respectively. In terms of total production, China is the world’s leading wheat producer with approximately 131 million tons per year . Other c-1 yr-1), Chile (246 kg ha-1 yr-1), France (123 kg ha-1 yr-1), and Mexico (41.77 kg ha-1 yr-1), whereas relatively lower genetic progress were reported in Spain (24 kg ha-1 yr-1), Australia (25 kg ha-1 yr-1), and Siberia (15.3 kg ha-1 yr-1). Annual yield gains in Egypt, India, and Pakistan were estimated at 27.4 (0.55%), 21.4 (0.62%), 111.6 (1.13%), 32.5 (0.83%), and 18.5 kg ha-1 yr-1 (0.5%), respectively (-1 (0.65%) (Wheat yield gains across the major wheat producing countries are presented in ectively . Genetic (0.65%) . Countri (0.65%) .Genetic progress is relatively lower under low-yielding environments compared to high-yielding environments . TherefoGenetic gains in grain yield have been attributed to development and deployment of high-yielding wheat genotypes with improved agronomic and physiological traits related with high yield potential . For exaGrain yield in wheat is influenced by several agronomic traits  which haVrn-A1, Vrn-B1, and Vrn-D1 regulate flowering and maturity in wheat . The effect of Vrn loci on heading and maturity, and grain yield potential are ranked as follows: Vrn-A1 < Vrn-B1 < Vrn- D1  among Chinese wheat cultivars followed by Vrn-A1 , grain yield (23 and 40%), and HI (31 and 50%) from tall and dwarf genotypes, respectively. Canadian spring wheat carrying dominant allele of Vrn-B1, photo-period insensitive allele of Ppd-D1, and height reducing allele Rht-1 produced shorter plants and higher grain yield , Rht2 (Rht-D1b), Rht-D1c, and Rht8  but resulted in reduced grain number per spike and increased 1,000–kernel weight, above-ground biomass and grain yield in wheat  have negligible effects on biomass production, whereas some  can increase above-ground biomass, kernel weight, and grain yield  and 0.3% yr-1 for TKW among Chinese and American wheat genotypes, respectively. Similarly, -1 among Brazilian wheat genotypes. TKW is reportedly linear with moderate to high correlation with grain yield (Grain yield improvement has been significantly associated with increased thousand kernel weight (TKW) . On the in yield  suggestiin yield , rather -1 y-1 (0.44% yr-1) between 1980 and 2003 attributed to high number of kernels spike-1 (0.24 kernels spike-1 yr-1) in Spain. Similarly, -2 yr-1 through breeding and selection spanning over 50 years in Iran. Grain number among Brazilian wheat genotypes was increased by 77.89 grains yr-1. In China grain number per spike varied between 26 for landraces developed in 1941 to 38 for improved wheat genotypes released between 2007 and 2011 (The number of grains per spike has been identified as an important trait for improving grain yield . Yield gand 2011 . Among wand 2011 . Althougand 2011 . Therefoand 2011 . The reland 2011 . On the and 2011 .-1 was derived from ‘Zhoumai 16’ x ‘Liken 4’. Zhoumai 16 was developed from ‘Yumai 21’ x ‘Zhou 8425B’ whereby Zhou 8425B is characterized by large spikes and high TKW (Spike fertility (SF) is a grain yield component that influences the increase in the number of kernels per spike . For inshigh TKW , demonstOther spike characteristics useful for breeding include SL, SPS, and SC . The numTin1 tiller inhibition gene can increase grain number per spike  and/or anthesis date genes (e.g. Ppd1) resulted in improvement of WSC. In addition, wheat genotypes with high WSC produce more fertile tillers, reduced days to anthesis, increased biomass, grain number, and grain yield than genotypes with low WSC  possess useful source of alleles for enhancing drought tolerance and improving yield and its component traits (Aegilops tauschii) with durum wheat (T. durum Desf.) (T. turgidum L.) is also identified as a useful genetic resource for wheat breeding possessing functional genes that surpass the early maturity effect caused by the early flowering allele Ppd-A1a found in T. turgidum L.   . Tetraplamidale) . Wild emamidale) . Wheat gamidale) . Furtheramidale) .QTLs QTn.ipk-5D, QTn.ipk-2D, QTn.ipk-3B, and QTn.ipk-1B which are associated with productive tiller number (QFlt.dms-2D, QFlt.dms-5B, QFlt.dms-2D, QFlt.dms-7A, and QFlt.dms-6B.2 are linked to days to flowering; whereas, QTLs QMat.dms-2D, QMat.dms-2D, QMat.dms-7A.2, and QMat.dms-4A.1 are associated with days to maturity (QTL mapping of agronomic and physiological traits is important for marker-assisted breeding in wheat . Agronomr number . QTL QFlmaturity . About 4maturity . New QTLmaturity . QTLs fomaturity Table 3 maturity . QTL’s fmaturity . The idematurity .2 concentration were co-located with QTL for grain yield and/or yield components (mponents . QTL whimponents . Such plmponents . Marker-mponents . Generalmponents , their imponents . TherefoIn conclusion, genetic improvement can be achieved by either direct selection for primary traits such as grain yield or indirectly through selection of secondary traits related to higher grain yield potential. Breeding high-yielding genotypes incorporating yield-promoting agronomic and physiological traits has accelerated yield gains in wheat. As a result, further grain yield improvement will likely be achieved through in/direct selection targeting yield-related agronomic and physiological attributes. Furthermore, QTL associated with agronomic and physiological traits linked to grain yield are useful for marker-assisted selection of high-performing wheat genotypes.All authors listed have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brassica rapa, an important vegetable cultivated worldwide.Genome-wide maps of histone modifications have been obtained for several plant species. However, most studies focus on model systems and do not enforce FAIR data management principles. Here we study the H3K27me3 epigenome and associated transcriptome of B. rapa leaves and inflorescences. To analyze these data we developed a Reproducible Epigenomic Analysis pipeline using Galaxy and Jupyter, packaged into Docker images to facilitate transparency and reuse. We found that H3K27me3 covers roughly one-third of all B. rapa protein-coding genes and its presence correlates with low transcript levels. The comparative analysis between leaves and inflorescences suggested that the expression of various floral regulatory genes during development depends on H3K27me3. To demonstrate the importance of H3K27me3 for B. rapa development, we characterized a mutant line deficient in the H3K27 methyltransferase activity. We found that braA.clf mutant plants presented pleiotropic alterations, e.g., curly leaves due to increased expression and reduced H3K27me3 levels at AGAMOUS-like loci.We performed H3K27me3 chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis by 3′-end RNA sequencing from B. rapa development. Our work reveals the epigenomic landscape of H3K27me3 in B. rapa and provides novel genomics datasets and bioinformatics analytical resources. We anticipate that this work will lead the way to further epigenomic studies in the complex genome of Brassica crops.We characterized the epigenetic mark H3K27me3 at genome-wide levels and provide genetic evidence for its relevance in  The epigenome comprises alternative chromatin states that can affect gene activity . These iArabidopsis thaliana (hereinafter referred to as Arabidopsis) the core PRC2 subunits are well conserved and H3K27me3 is crucial for plant development . Ca. CaB. raRRID:SCR_006989) [P < 0.05; and Plant GO slim ontology type); data were visualized reduced in complexity and redundant GO terms using REViGO  [Gene Ontology analysis was performed using agriGO v2.0   (Fisher _005825)  with defB. rapa v3.0 gene models was obtained from Zhang et al. [B. rapa genome v3.0 was obtained by blasting coding sequences against B. rapa genome v1.5  and Arabidopsis .Functional annotation of g et al. . Custom B. rapa AGAMOUS genes, we found that BraA.AG.a (BraA01g010430.3C) gene structure annotation was not correct in the recent B. rapa genome v3.0. We curated BraA.AG.a gene structure using AUGUSTUS  [B. rapa genome V1.5) gene information at the B. rapa database [During our analyses of H3K27me3 levels on _008417)  and Bra0database .Project name: Epigenomics Workflow on Galaxy and Jupyterhttps://github.com/wilkinsonlab/epigenomics_pipelineProject home page: Operating systems: Platform independentProgramming languages: Python, R, BashOther requirements: noneLicense: MITRRID:SCR_017544https://github.com/wilkinsonlab/epigenomics_pipeline; this is associated with a Zenodo release to match the configuration used in this publication [RRID:SCR_017544 and biotools: Epigenomics_Workflow_on_Galaxy_and_Jupyter at SciCrunch and bio.tools databases, respectively. Compiled Docker images are available at https://hub.docker.com/u/mpaya. All supporting data are available in the GigaScience GigaDB database [ChIP-seq and RNA-seq data sets supporting the results of this article are available at NCBI SRA under the accession number PRJNA542357. Latest versions of the components of the REA pipeline, and instructions to deploy the Galaxy/Jupyter containers and run the analysis, can be found in the GitHub repository lication . The REAdatabase .Figure S1: Differences between epic2 and MACS2 peak-calling algorithmsB. rapa inflorescencesFigure S2: ChIP-seq analysis of H3K27me3 regions in B. rapa inflorescencesFigure S3: Comparisons of H3K27me3 ChIP-seq signal in B. rapa H3K27 methyltransferase-coding genesFigure S4: Transcript levels of BraA.clf-1 mutant phenotypesFigure S5: B. rapa plant sampling materialsFigure S6: Pictures of Figure S7. ChIP-seq sample-sample correlation testsFigure S8. RNA-seq sample-sample correlation testsTable S1. Alignment statisticsTable S2. Comparison of Bowtie2 and BWA performanceB. rapaTable S3. H3K27 trimethylated regions identified in B. rapa leavesTable S4. List of H3K27me3 peaks in B. rapa inflorescencesTable S5. List of H3K27me3 peaks in Table S6. 3′RNA-seq resultsTable S7. List of H3K27me3 differentially marked regionsTable S8. H3K27me3-regulated genesTable S9. Primer listgiz147_GIGA-D-19-00256_Original_SubmissionClick here for additional data file.giz147_GIGA-D-19-00256_Revision_1Click here for additional data file.giz147_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giz147_Reviewer_1_Report_Original_SubmissionDiep Ganguly -- 8/28/2019 ReviewedClick here for additional data file.giz147_Reviewer_1_Report_Revision_1Diep Ganguly -- 11/7/2019 ReviewedClick here for additional data file.giz147_Reviewer_2_Report_Original_SubmissionJingyu Zhang -- 9/1/2019 ReviewedClick here for additional data file.giz147_Supplemental_FilesClick here for additional data file.M: log2 fold-change of the normalized H3K27me3 read densities in leaves relative to inflorescences calculated by MAnorm; MACS: Model-based Analysis of ChIP-Seq; mRNA: messenger RNA; NCBI: National Center for Biotechnology Information; PRC2: Polycomb repressive complex 2; REA: Reproducible Epigenomic Analysis; RNA-seq: RNA sequencing; RT-qPCR: real-time quantitative reverse transcription PCR; s.d: standard deviation; SEA: singular enrichment analysis; SICER: Spatial Clustering for Identification of ChIP-Enriched Regions; SRA: Sequence Read Archive.3′RNA-seq: 3′-end mRNA high-throughput sequencing; AG: AGAMOUS; bp: base pairs; cDNA: complementary DNA; ChIP: chromatin immunoprecipitation; ChIP-seq: ChIP followed by high-throughput sequencing; ChIP-qPCR: ChIP followed by real-time quantitative PCR; CLF: CURLY LEAF; DAG: days after germination; DEG: differentially expressed genes; FAIR: findability, accessibility, interoperability, and reusability; FC: fold change; FDR: false discovery rate; GO: gene ontology; H3K27me3: histone H3 lysine 27 trimethylation; IGV: integrative genomics viewer; kb: kilobase pairs; LOWESS: Locally Weighted Scatterplot Smoothing; The authors declare that they have no competing interests.This work was supported by grants RTI2018-097749-B-100, BIO2015–68031-R and RYC-2013–14689 to P.C., and BES-2016–078939 fellowship to L.P.V. from the Spanish Ministerio de Economia y Competitividad ; and by the "Severo Ochoa Program for Centres of Excellence in R&D” from the Agencia Estatal de Investigación of Spain, grant SEV-2016-0672 (2017–2021) to the CBGP. M.P.M. was supported by a Postdoctoral contract associated with the Severo Ochoa Program.P.C. conceived the work; M.P.M. performed computational biology analyses; L.P.V. performed initial bioinformatic analysis and all experimental research. P.M.U. and D.L.A. performed 3′RNA-seq library preparation and sequencing. M.W. contributed analytical tools and metadata generation. P.C. and M.P.M. wrote the first draft of the manuscript, which was completed with the assistance of L.P.V. and M.W. All authors approved the final version of the article.


Scientific Reports 10.1038/s41598-019-50208-x, published online 26 September 2019Correction to: The Acknowledgements section in this Article is incomplete.“We thank Michael Miller at W.S.I. for discussion.”should read:“We thank Michael Miller at W.S.I. for discussion. We gratefully acknowledge Dr. Tyler Maxwell for his contribution to Figure S11c.”


The publisher apologizes for the error.The fifth author is listed incorrectly in the citation. The correct citation is: Ljones K, Ness HO, Solvang-Garten K, Gaustad SE, Høydal MA (2017) Acute exhaustive aerobic exercise training impair cardiomyocyte function and calcium handling in Sprague-Dawley rats. PLoS ONE 12(3): e0173449.


Scientific Reports 10.1038/s41598-019-49575-2, published online 17 October 2019Correction to: This Article contains errors in Reference 46 which was incorrectly given as:Odorico, J. S. et al. Extracellular matrix scaffold and hydrogel derived from decellularized and delipidized human pancreas. Sci. Rep. 8, 1–16 (2018)The correct reference is listed below as Reference


Luminal B cancers show much worse outcomes compared to luminal A. This present study aims to screen key lncRNAs and mRNAs correlated with luminal-B breast cancer.cis nearby-targeted DEmRNAs of DElncRNAs. DElncRNA-DEmRNA co-expression networks were performed. The mRNA and lncRNA expression profiles were downloaded from The Cancer Genome Atlas (TCGA) database to validate the expression patterns of selected DEmRNAs and DElncRNAs.Luminal-B breast cancer tissue samples and adjacent tissue samples were obtained from 4 patients with luminal-B breast cancer. To obtain differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) between luminal-B breast cancer tumor tissues and adjacent tissues, RNA-sequencing and bioinformatics analysis were performed. Functional annotation of DEmRNAs and protein-protein interaction networks (PPI) construction were performed. DEmRNAs transcribed within a 100 kb window up- or down-stream of DElncRNAs were searched, which were defined as A total of 1178 DEmRNAs and 273 DElncRNAs between luminal-B breast cancer tumor tissues and adjacent tissues were obtained. Hematopoietic cell lineage, Cytokine-cytokine receptor interaction, Cell adhesion molecules (CAMs) and Primary immunodeficiency were significantly enriched KEGG pathways in luminal-B breast cancer. FN1, EGFR, JAK3, TUBB3 and PTPRC were five hub proteins of the PPI networks. A total of 99 DElncRNAs-nearby-targeted DEmRNA pairs and 1878 DElncRNA-DEmRNA co-expression pairs were obtained. Gene expression results validated in TCGA database were consistent with our RNA-sequencing results, generally.This study determined key genes and lncRNAs involved in luminal-B breast cancer, which expected to present a new avenue for the diagnosis and treatment of luminal-B breast cancer. Breast cancer is the leading cause of cancer-related death in women, both overall and in less developed countries . It is aWith advances in high-throughput technology, it is discovered that human transcriptome mainly consists of non-coding RNAs (ncRNAs) with limited or no protein-coding capacity , 10. Loncis nearby-targeted DEmRNAs of DElncRNAs and construction of DElncRNA-DEmRNA co-expression networks were performed. In this light, we expect this study could represent a new avenue to improve the understanding of the pathogenesis and be helpful for treatment of luminal B breast cancer.In this study, differentially expressed lncRNAs (DElncRNA) and mRNAs (DEmRNAs) in tumor tissues of patients with luminal-B breast cancer were identified by RNA-sequencing. Subsequently, protein-protein interaction (PPI) networks of DEmRNAs were conducted. Identification of Luminal-B breast cancer tissue samples and adjacent tissue samples were obtained from 4 patients with luminal-B breast cancer in People’s Hospital of Deyang City, which were free of treatment. The detailed characteristics of patients are displayed in Table http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), the FASTQ sequence data were acquired from the RNA-sequencing data. Read QC tool in FastQC version 0.11.4 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used for the quality control of FASTQ data with Q > 30. Trimming of raw data was performed with cutadapt version 1.16 (http://cutadapt.readthedocs.io). Reads with low quality  were removed to obtain the clean reads.According to the manufacturer’s protocol, RNA was extracted with PAXgene blood RNA kit . With Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit), the concentration, integrity and RNA integrity number (RIN) values of RNA were assessed. Sequencing was performed based on the Illumina Hiseq X-ten platform  with PE150 bp sequencing mode. The sequencing was done with paired-ends and 10G depth. With Base Calling version 0.11.4 (ftp://ftp.ncbi.nlm.nih.gov/genomes/Homo_sapiens), HISAT2 version 2.1.0 (https://ccb.jhu.edu/software/hisat2/index.shtml) was applied. Expression of mRNAs and lncRNAs was normalized and outputted with StringTie version 1.3.3b (http://ccb.jhu.edu/software/stringtie/). Fragments per Kilobase of exon per million fragments mapped (FPKM) of lncRNAs and mRNAs were calculated with StringTie. With edgeR version 3.24 (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html), both DEmRNAs and DElncRNAs were obtained with |log2FC| > 1 and p-value < 0.05. By using R package “pheatmap”, hierarchical clustering analysis of DElncRNAs and DEmRNAs were conducted.In order to align the clean reads with the human reference genome, Ensemble GRCh38.p7 (http://metascape.org/gp/index.html). A value of p < 0.05 was set as the cut-off for significance.Functional annotation, including Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses was performed with Metascape (http://www.uniprot.org/database/DB-0184). Then, PPI networks were visualized with Cytoscape software .Top 100 up- and down-regulated DEmRNAs were scanned with the BioGrid .A total of 1178 DEmRNAs (666 up-regulated and 512 down-regulated DEmRNAs) and 273 DElncRNAs (181 up-regulated and 92 down-regulated DElncRNAs) were obtained. The top 10 up- and down-regulated DEmRNAs and DElncRNAs were exhibited in Table p = 1.14E-50), cytokine-mediated signaling pathway (p = 5.74E-44) and cytokine production (p = 8.78E-38) were significantly enriched GO terms in luminal-B breast cancer , Cytokine-cytokine receptor interaction (p = 8.19E-21), Cell adhesion molecules (CAMs) (p = 3.61E-18) and Primary immunodeficiency (p = 1.17E-14) were significantly enriched KEGG pathways in luminal-B breast cancer , EGFR (degree = 14), JAK3 (degree = 11), TUBB3 (degree = 11) and PTPRC (degree = 10) were five hub proteins of the PPI networks , regulation of cell-cell adhesion (p = 7.86E-12), cytokine binding (p = 3.41E-07), T cell selection (p = 7.75E-07) and negative regulation of secretion (p = 1.38E-06) were significantly enriched GO terms , Cytokine-cytokine receptor interaction (p = 2.81E-03), Primary immunodeficiency (p = 7.71E-03), AMPK signaling pathway (p = 9.30E-03) and Wnt signaling pathway (p = 1.46E-02) were significantly enriched KEGG pathways , cytokine-mediated signaling pathway (p = 9.35E-28), cytokine production (p = 1.68E-27), leukocyte migration (p = 3.20E-22) and alpha-beta T cell activation (p = 4.89E-20) were significantly enriched GO terms , Hematopoietic cell lineage (p = 9.66E-16), Primary immunodeficiency (p = 2.85E-15), Th1 and Th2 cell differentiation (p = 3.38E-13) and Cell adhesion molecules (CAMs) (p = 1.66E-12) were significantly enriched KEGG pathways  . During Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved lncRNA, and its over-expression in multiple cancerous tissues has been linked to the proliferation and metastasis of tumor cells. It was first identified as being up-regulated in lung tumors, and a prognostic marker for metastasis and patient survival in non-small cell lung cancer (NSCLC), specifically in early stages of lung adenocarcinoma . Subsequ+ T cells promotes metastasis in luminal breast cancer  is primarily expressed in heart and fetal brain tissue . DysreguThe Wilms’ tumor 1 (WT1) was first cloned in 1990 as a suppressor in Wilms’ tumor, which was located at chromosome 11p13 . WT1 genWilms tumor 1 Antisense RNA (WT1-AS) is located upstream of the WT1 gene, and these two genes are bi-directionally transcribed from the same promoter region. Down-regulation of WT1-AS was related to a poorer prognosis in ovarian clear cell adenocarcinoma . Lv et aIn conclusion, a total of 1178 DEmRNAs and 273 DElncRNAs between luminal-B breast cancer tumor tissues and adjacent tissues were obtained. We discussed and emphasized the importance role of three DElncRNA-DEmRNA pairs, including MALAT1-S100A7, MIAT-CCL5 and WT1-AS-WT1, involved in luminal B breast cancer, which expected to provide new insight into understanding the mechanism underlying pathogenesis of luminal B breast cancer. The small sample size was a limitation of our study. Although the validation results in TCGA database indicated that our RNA-sequencing results were generally reliable, larger cohorts of patients and further experimental validation studies are needed to conduct to verify this conclusion.


Oriens lacunosum-moleculare (O-LM) interneurons constitute 40% of hippocampal interneurons expressing Somatostatin (SST). Recent evidence has indicated a dual origin for these cells in the medial and caudal ganglionic eminences (MGE and CGE), with expression of Htr3a as a distinguishing factor. This is strikingly different from cortical SST interneurons that have a single origin within the MGE/preoptic area (POA). We reassessed the origin of hippocampal SST interneurons using a range of genetic lineage-tracing mice combined with single-cell transcriptomic analysis. We find a common origin for all hippocampal SST interneurons in NKX2-1-expressing progenitors of the telencephalic neuroepithelium and an MGE/POA-like transcriptomic signature for all SST clusters. This suggests that functional heterogeneity within the SST CA1 population cannot be attributed to a differential MGE/CGE genetic origin.  •+ progenitors generate all hippocampal CA1 somatostatin (SST) interneuronsNKX2-1•+ cells generated in the absence of NKX2-1 do not migrate to the hippocampusSST•LHX6 expression maintained in MGE-derived cortical and hippocampal CA1 interneurons•Htr3a is detected in MGE-derived hippocampal CA1 interneuronsLow expression of  In this article, Kessaris and colleagues combine genetic lineage tracing and single-cell transcriptomic analysis to identify the embryonic origin of hippocampal CA1 Somatostatin interneurons. They report a common origin in the medial ganglionic eminence/preoptic area from neuroepithelial precursors expressing NKX2-1. The pri the CGE . The pre the CGE .oriens lacunosum-moleculare (O-LM) interneurons, which have their cell bodies within stratum oriens (s.o.) and represent 40% of all SST interneurons in the hippocampus  mapping of Sst interneurons in the developing mouse brain suggested that most, if not all, telencephalic Sst cells originate in the diagonal area (or anterior entopeduncular area [AEP]) and not the pallidal neuroepithelium  (Recent ithelium . This su (E13.5) .Lhx6 to label MGE-derived interneurons in the cortex and hippocampus   B–1F. Aro30 (P30) B and 1F.30 (P30)  or from 30 (P30) A (Fogart30 (P30) . Analysi30 (P30) C and 1F,30 (P30) D and 1F.with GFP E and 1F.rneurons .Figure 1Sst interneurons in the developing mouse brain suggested that most, if not all, telencephalic Sst cells originate in the AEP  and that failure to label 30% of cortical interneurons in Nkx2-1-Cre mice may have been caused by lack of NKX2-1 expression in the AEP at E13.5  embryos for the presence of SST-positive cells. At E13.5, most Sst cells were missing from the telencephalon in Nkx2-1 KO embryos compared with controls  ∼30% of SST cells with typical O-LM characteristics were labeled with GFP in Htr3a-GFP mice; (2) a proportion of s.o. SST O-LM cells were labeled with GFP in Mash1-CreER;RCE transgenic mice following tamoxifen administration; and (3) there was only ∼10% overlap in expression of tdTomato and GFP in Nkx2-1-Cre;tdTOM;Htr3a-GFP mice 1Wdr (MGI:3761164) ((Nkx6-2-icre)1Kess (MGI:4355562) (JAX 027798), Tg(Lhx6-icre)1Kess (MGI:4355717) (JAX 026555) (tm1(EGFP/cre)Cjt (MGI:3053959) (JAX 005622) (tm2.1(cre)Zjh (MGI:4838416) (JAX 013044) (KI (JAX 006148) (KI reporter mice (JAX 004077)  , Tg(Nkx6 026555) , Shhtm1( 005622) , Ssttm2. 013044) , R26R-YF 006148) , and R26 004077)  have beed allele . All aniLhx6- and Sst DIG-labeled probes were generated from a plasmid (kind gift from V. Pachnis) and IMAGE clone 4218815 (Source Biosciences), respectively.Tissue processing, immunohistochemistry, and ISH were carried out as previously described . Primary+ cells per animal were counted in order to generate the data shown in Images were captured using a Hamamatsu C4742-95 camera on a Zeiss Axioplan fluorescence microscope and Digital Pixel software. Confocal images were captured on a Leica CTR6500 confocal microscope or a Zeiss LSM880 with Airyscan. ISH images were captured on a Zeiss Axio Scan.Z1 scanner. Image composites were assembled using Microsoft ICE software  and processed with Adobe Photoshop CS6 . Figures were generated in Adobe Illustrator CS6 (Adobe Systems). Quantification was performed as described previously . Three ahttps://github.com/cortexlab/Transcriptomics. To visualize different cell subtypes, we used the negative binomial t-stochastic neighbor-embedding (nbtSNE) algorithm as previously described (The single-cell data presented were obtained by reanalyzing our previously published datasets . The codescribed .Conceptualization, Z.A. and N. Kessaris; Methodology, N. Kessaris and K.D.H.; Investigation, Z.A., L.M., N. Ktena, K.D.H., and N. Kessaris; Writing – Original Draft, Z.A. and N. Kessaris; Writing – Review & Editing, Z.A., L.M., K.D.H., and N. Kessaris; Funding Acquisition, K.D.H. and N. Kessaris.