Eunice aphroditois) is spectacular. They hide in their burrows until they explode upwards grabbing unsuspecting prey with a snap of their powerful jaws. The still living prey are then pulled into the sediment for consumption. Although predatory polychaetes have existed since the early Paleozoic, their bodies comprise mainly soft tissue, resulting in a very incomplete fossil record, and virtually nothing is known about their burrows and behavior beneath the seafloor. Here we use morphological, sedimentological, and geochemical data from Miocene strata in northeast Taiwan to erect a new ichnogenus, Pennichnus. This trace fossil consists of an up to 2 m long, 2–3 cm in diameter, L-shaped burrow with distinct feather-like structures around the upper shaft. A comparison of Pennichnus to biological analogs strongly suggests that this new ichnogenus is associated with ambush-predatory worms that lived about 20 million years ago.The feeding behavior of the giant ambush-predator “Bobbit worm” ( They are widely distributed in tropical to temperate shallow marine to intertidal environments in the Atlantic, Indian, and Pacific Oceans4. One of the largest eunicids, the so called “Bobbit worm” (Eunice aphroditois), has fascinated the public since its appearance in November 2017 in British Broadcasting Corporation’s “Blue Planet II”. However, because burrowing Bobbit worms only extend a minor portion of their body outside of the sediment, observations on their behavior beneath the seafloor are difficult. As a result, previous studies mainly focused on anatomy and ethology of this and other eunicids5, and little is known of their burrow morphology.Eunicid polychaetes (Family Eunicidae) are well-known for their long, segmented bodies, and strong denticulated maxillae5. They are ambush predators that feed on fish, bivalves, and other annelids7. Upon reaching sexual maturity, some Bobbit worms construct a permanent mucus-lined burrow in the seafloor8, and some use an antenna to simulate a wormlike motion for attracting prey7. When potential prey comes within striking distance, the Bobbit worm rapidly thrusts out of its burrow and grabs the prey with its eversible pharynx and sharp maxillae6. The still living animal is then dragged down into the burrow for consumption, and the desperate attempts of the prey to escape commonly leads to disturbance and collapse of the sediment around the burrow opening7.Bobbit worms can grow up to 3 m long and 2 cm in diameter10. Consequently, burrows of raptorial polychaetes could potentially occur in strata as old as the early Paleozoic. Most of the existing trace fossils produced by polychaetes, such as Planolites, Rosselia, Cylindrichnus, and Arenicolites, represent deposit-feeding, detritus-feeding, or filter-feeding behaviors, rather than predatory ones14. Trace fossils associated with predatory behavior, such as Piscichnus, are primarily produced by vertebrates including fish, rays, and marine mammals that extract prey by jetting water into the sediment18. Only a few trace fossils are described as preserving hunting behaviors of invertebrates 20, and trace fossils produced by subsurface-dwelling ambush predators have not been identified previously. Herein, we present morphological, sedimentological and geochemical data of giant L-shaped burrows in the Miocene Taliao Formation of northeast Taiwan. These data are compared to modern marine environments of the northwest Pacific and to biological analogs, and an argument is presented to suggest that the L-shaped burrows record the hunting behaviors of Miocene Bobbit worms.Polychaetes emerged early in the Cambrian, and jaws of possible raptorial worms have been described from early Paleozoic strata22. Along the Northeast Coast of Taiwan, late Oligocene to Pliocene passive margin strata consist of shallow marine to coastal swamp deposits, and record a number of transgressive–regressive cycles25; these deposits are exquisitely exposed due to a series of imbricated thrust faults  and is extensively exposed along the Northeast Coastn29 Fig. c. The lon29 Fig. . Within n29 Fig. c, large,Pennichnus are preserved in very fine- to fine-grained sandstone. They co-occur with abundant Bichordites, Ophiomorpha, Phycosiphon, Planolites, and Schaubcylindrichnus, and uncommon Macaronichnus, Teichichnus, and Skolithos. This trace-fossil assemblage is best described as a proximal Cruziana Ichnofacies32 and indicates deposition in a lower shoreface to offshore paleoenvironment. At Badouzi promontory, Pennichnus are also concentrated in very fine- to fine-grained sandstone  core scanning reveals that there is an abrupt increase in iron (Fe) in the burrow lining and the deformed sediment surrounding the burrow Fig. a. The dePennichnus were produced by mucus-secreting animals which repeatedly rebuilt the burrow opening through time. When marine invertebrates, especially soft-bodied infauna, construct their burrows, they usually secrete mucus to strengthen the burrow wall. Organic-rich compounds in the mucus then attract microbes such as sulfate-reducing bacteria that feed on the carbon leading to the formation of reducing conditions which triggers iron sulfide to precipitate in the lining37. During uplift and erosion, the diagenetically altered burrows are exposed to oxidizing pore waters, and the iron sulfide is oxidized to iron hydroxides such as hematite, limonite or goethite37; this results in elevated Fe levels in the burrow lining and disturbed zone . In contrast, low Fe concentration in the feather-like collapse structures  and Tagelus plebeius (Stout razor clam)47.These bivalves can build burrows up to 70 cm long46, and potentially produce collapse structures surrounding the actively backfilled burrows48. The maximum burrowing depth of these organisms is controlled by the strength of the substrate, which increases with depth45. The increase in sediment strength could explain the horizontal orientation of the burrows at a certain depth. However, the circular cross-section of Pennichnus is at odds with the shape of fast-burrowing bivalves, which tend to generate more almond- or keyhole-shaped burrows49. Moreover, rapid-burrowing bivalves excavate the sediment through local fluidization around their bodies44, and this commonly results in sediment collapse in their wake49. When sediment collapses into the cavity left by the bivalves, it blurs the burrow wall, which produces a burrow morphology that is very distinctive from the clear burrow wall preserved in Pennichnus. Furthermore, only few Pennichnus specimens (17.8%) display a backfill and/or inner tube, and no bivalve shells were observed within any Pennichnus even though shell material is common in the surrounding sediment. This suggests that fast-burrowing bivalves are not the trace maker.With the presence of a partial meniscate backfill and inner tube in some Pennichnus suggests that the trace maker was a large, slender organism that supported the burrow with its body, similar to how polychaetes do47. In addition, feather-like collapse structures and a disturbed zone surrounding the burrow lining indicates extensive sediment disturbance at shallow depths. These morphological features of Pennichnus are consistent with the activities of an ambush predator, and hence, we hypothesize that giant polychaetes, such as Bobbit worms, are the most probable trace makers , middle (M) and/or lower (L) part of the burrow . Specimens were ground layer-by-layer in increments < 1 mm providing serial sections through the trace fossil, and each successive layer was photographed  at the Center for Advanced Marine Core Research (CMCR), at Kochi University in Japan. The CT scanning data were then processed using OsiriX imaging software  in the EPMA lab at Institute of Earth Sciences, Academia Sinica, Taipei, Taiwan. Chemical identification was carried out using an Energy Dispersive X-ray Spectrometer (EDS: Oxford Instruments X-max80 with INCA-350), equipped on the FE-SEM. Each sample was mounted, polished, and analyzed with FE-SEM and EDS under low vacuum conditions (50 Pa), using an acceleration voltage of 15 kV and beam current of 0.12 nA. The duration of EDS counting time was 15 s for each spot.Pennichnus, two samples were studied with both an Itrax XRF Multiscanner , and an Itrax Core Scanner . Both machines are manufactured by Cox Analytical Systems.In order to measure elemental variations between different parts of 56. The density of Pennichnus specimens was quantified by dividing the number of specimens by outcrop area.Stratigraphic columns including lithology, sedimentology, ichnology and body fossils were generated for the Yehliu Sandstone Member at both Yehliu and Badouzi. For each bed, grain size, sedimentary structures and bed thicknesses were recorded. Ichnological analysis focused on identifying trace fossils and assessing the degree of bioturbation with the bioturbation index57, this study and its nomenclatural act are registered in ZooBank, the online registration system governed by ICZN, to validate the new ichnotaxon. By keying LSID (Life Sciences Identifier) in ZooBank website, associated information of this work can be viewed. The LSID for this publication is: urn:lsid:zoobank.org:pub:2B03334F-27D3-4583-B057-2B5C1E321AEF.Following the rules of International Code of Zoological Nomenclature (ICZN)Supplementary Information.


Chd1-8 show upon germline disruption pronounced, often developmental lethal phenotypes. Here we show that contrary to Chd1-8 disruption, Chd9–/–animals are viable, fertile and display no developmental defects or disease predisposition. Germline deletion of Chd9 only moderately affects gene expression in tissues and derived cells, whereas acute depletion in human cancer cells elicits more robust changes suggesting that CHD9 is a highly context-dependent chromatin regulator that, surprisingly, is dispensable for mouse development.Chromodomain helicase DNA-binding (CHD) chromatin remodelers regulate transcription and DNA repair. They govern cell-fate decisions during embryonic development and are often deregulated in human pathologies.  Normal organismal development requires accurate transcriptional control. This is achieved by myriad of transcription factors and chromatin regulators that act in concert to control expression of the right genes at the right time. Chromodomain helicase DNA-binding enzymes belong to the superfamily of chromatin remodelers that control nucleosomal structure and phasing to regulate gene expression and DNA repair. They contain hallmark amino-terminal double chromodomains and central SNF2 helicase-like ATPase domains, allowing nucleosome recognition and remodeling . DiffereDrosophila Kismet (KIS-L), a Trithorax family protein that counteracts gene repression by Polycomb group proteins during development [kismet loss-of-function causes specific segmentation defects and homeotic transformation [In addition to the hallmark domains, members of the subfamily III contain SANT–like and tandem brahma kismet domains (BRK), implicated in DNA  and CTCFelopment . It reguelopment  and is ielopment . Importaormation  resultinin vitro [Subfamily III members are found in both promoter and enhancer regions, regulating different stages of RNA Polymerase I and II transcription ,9. Subfain vitro .Chd7 knockout animals survive up to embryonic day 10.5 (E10.5) [Chd8 is embryonic lethal at E5.5–7.5 due to widespread p53-mediated apoptosis [Chd7 or Chd8 faithfully recapitulate features of human CHARGE syndrome [CHD enzymes appear to have non-redundant functions in murine embryonic development . Chd7 kn (E10.5) , whereaspoptosis . These esyndrome  and autisyndrome , respectCHD7 amplification and overexpression correlates with poor prognosis. In contrast, heterozygous loss of CHD9 is observed in 55% of breast cancer patients [CHD6 in bladder cancer [CHD7 in medulloblastoma [CHD8 harbors mutations in many cancer types [Subfamily III enzymes are often deregulated or mutated in human cancers. In aggressive breast cancers, patients . Low exppatients . In addir cancer , CHD7 inblastoma ,21, wherer types .CHD9  is the least characterized member of the subfamily. It was identified as transcriptional regulator in rat liver  and meseCHD9 associates with nuclear receptors and contributes to glucocorticoid receptor–mediated gene expression regulation ,25 and min vivo functions of CHD9 have remained unresolved. Here, we describe the first Chd9 knockout mice and study its contribution to development and lymphomagenesis, a tumor subtype in which insertional mutagenesis experiments suggested a potential role of CHD9.While these studies address biochemical and functional properties of CHD9, the Chd9 is ubiquitously expressed in murine tissues with the highest levels observed in brain [Chd9 knockout mice.in brain . Previouin brain , osteogein brain  and sterin brain ,25, wherin brain ,31 and hin brain . To explChd family knockouts are embryonic lethal [Chd1-8 employed gene-trap reporter constructs, the deletion of critical exons or the removal of functional domains  with two loxP sites by homologous recombination in mouse ES cells (ESC) were crossed to the β-actin-driven Cre-recombinase strain (ACTB-Cre) to obtain heterozygous Chd9 (Chd9+/–) animals , S1 Fig. animals  and back defects . The gerethality ,33,34 toethality .Chd9 gene encodes two mRNA isoforms (Chd9–/–ESCs and murine embryonic fibroblasts (MEFs)  , S2 Fig. domains , S6A FigChd9–/–mice were likely due to old age and the strain genetic background. Collectively, the survival and histopathological analysis indicate that Chd9 knockout animals are viable, fertile and display no overt phenotypes.Histopathology on small cohorts of matched littermates , homozygous knockout (n = 5)) and older , homozygous knockout (n = 6)) revealed no significant pathological changes, with mild inflammation in the liver, lung, kidney and Harderian gland being the most distinctive . WildtypChd9–/–mice could be due to activation of compensatory transcriptional programs or functional redundancy with other CHD enzymes. Additionally, CHD9 might exert its functions during embryonic development rather than in adult tissues with a concomitant greater potential for compensatory mechanisms to kick in. In adult mice Chd9 is ubiquitously expressed, with the highest levels in brain (our data and [Chd9 mRNA levels at E13.5 and E15 (Absence of overt phenotypes in data and ). During and E15 . High ex and E15 , S8 Fig.Chd9 knockout embryonic (E15) liver and brain revealed only a handful of differentially expressed genes (DEGs)  (Chd9 was the only downregulated mRNA in both tissues (Gm4322 (Chd9 expression in the developing brain (Hoxa (a7), Hoxb  and Hoxc  homeotic gene clusters was observed , recently emerging as important regulators of the developing brain [RNA–seq of FC ≥ ±1) , S3 Fig. tissues , S3 Fig.  pseudogene Gm13237 , more profound gene expression changes were seen. In total, 63 upregulated and 212 downregulated genes were observed   adult hematopoietic stem and progenitor cell population (HSPCs), although this was not statistically significant ,  and statistically significant expansion of short-term hematopoietic stem cell   of bone marrow (BM) of  0.055), . We also = 0.02) . Depleti = 0.02) . Likewisid cells , as wellid cells . Taken tEμMyc B-cell lymphomas [Chd9 locus as common integration sites in murine [CHD9 expression is reported in multiple myeloma [Chd9–/–strain with EμMyc transgenic mice hypothesizing that if CHD9 would have an oncogenic or tumor-suppressing activity this should be revealed through an acceleration or delay of tumorigenesis, respectively. However, EμMyc-driven lymphomagenesis was not affected by the Chd9 status    and the Myc mice .EμMyc Chd9 wildtype, heterozygous and knockout mice by RNA-seq. In the heterozygous tumours, 20 genes were upregulated and only leucine rich repeat neuronal 4 (Lrnn4) downregulation was observed, whereas in the knockout tumours 28 genes were up–and 8 down–regulated , Apelin receptor (APLNR) and Bridging Integrator 1 (BIN1)–are implicated in neuronal development and pathology [Lastly, we explored gene expression regulation by CHD9 in human cancer cell lines. Publicly available data sets indicate that CHD9 is overexpressed in cancers of neuronal and hematopoietic origin . Analysiblotting . Contrarepletion , S11 Figobserved . Notablyathology . This suathology .Chd9 is dispensable for embryonic development. Furthermore, knockout animals are viable, fertile, display no overt phenotypes and have a normal lifespan. We have only observed modest expansion of ST-HSC in bone marrow, however, without discernible effect on other progenitors and mature populations. Moreover, although several murine and human studies suggested a role for Chd9 in solid tumors and lymphomas, CHD9 depletion did neither accelerate nor impair EμMyc-driven lymphomagenesis. Likewise, CHD9 is not required for proliferation or oncogenic transformation of primary MEFs. Nevertheless, a complex protein of 320 kD with multiple functional domains and highly conserved in evolution should have an important role. The lack of phenotype we noted is especially remarkable given the pronounced phenotype of nearly all other CHD proteins. However, if the role of CHD9 has evolved during evolution for specific neuronal functions, that are not required or do not become apparent in the routine monitoring taking place in dedicated mouse facilities, it might be not too surprising that our analyses did not reveal such functions. Future detailed studies will be required to elucidate this.Our results reveal that, despite ubiquitous expression, The study was performed in accordance with the Dutch and European regulations on care and protection of laboratory animals. All animal experiments were approved by the local Animal Ethics Committee—Dierexperimentencommissie (DEC) of The Netherlands Cancer Institute (NKI)- (OZP ID: 05029). All animal strains used in this study were generated or obtained through The Netherlands Cancer Institute Animal Facility. The number of animals required for each of the analyses performed in this paper are indicated either in the main text, figure panels or figure legends. The comparison of survival curves between experimental animal cohorts is done using log-rank (Mantel-Cox) test using GraphPad Prism8. For all calculations of p-values, significance threshold alpha was set at 5% (p< 0.05).EμMyc mice succumb to lymphomas at average of 5 months. EμMyc;Chd9 mice were monitored for the maximum period of two years, as it was not known whether Chd9 loss would accelerate or decelerate tumorigenesis.Since no previous knowledge on CHD9 role in murine development/lifespan is available, duration of the experiment was set for the maximum period of three years as to be able to show an effect on longevity in case this would be influenced by CHD9. Previous studies show that 2, confirmed by lack of response to firm toe pinch, and necropsy was performed to identify macroscopic changes and, when deemed needed, tissue samples were processed for immunohistopathology to document microscopic abnormalities. No animals died prior to meeting the criteria for euthanasia. No anaesthesia, analgesia or special techniques were required or applied during the experiments.Animals were kept in standard husbandry conditions . Animals were monitored daily by trained animal technicians at the NKI Animal Facility. Any symptoms of disease or poor health  were considered grounds for euthanasia. Moribund animals were promptly euthanised with COChd9 locus on chromosome 8 was flanked by loxP sites on both sites of the gene in order to excise the whole coding potential of the locus upon Cre-mediated recombination. The 5’ loxP site was introduced upstream of the exon 3 (containing ATG translation initiation site), whereas at the 3’ end a second loxP site was introduced downstream of the exon 38. Targeting was performed in E14 ES IB10 cells of 129P2 genotype using the recombineering-based method for generating conditional knockout mutations [The endogenous utations . Since EChd9 conditional knockout vector are listed below:Primer sequences used for constructing the ATAAGCGGCCGCGTGAAAGCAAGCGGCTTA5’ primer A NotI-Chd9 5’ GTCAAGCTTCTGGGTGTGGTGATGTACG5’ primer B HIII-Chd9 5’ GTCAAGCTTGCGGTGTTATAGAATATGAGCG5’ primer Y HIII-Chd9 5’ TCTACTAGTGAATCACTTGGAAACACTCG5’ primer Z SpeI-Chd9 5’ ATAAGCGGCCGCCACAGAGAAACCCTGTCTGA5’ primer C NotI-Chd9 5’ GTCGAATTCAGATCTCAAGCATCTTCCTATAGTCAG5’ primer D RI/BglII-Chd9 5’ ATAGGATCCCATTCGCTTCACAGATACCT5’ primer E BamHIII-Chd9 5’ CGCGTCGACGTCGCGGTAGGTTATTACGGTATCA5’ primer F SalI-Chd9 5’ ATAAGCGGCCGCATGAACTGATCCTCGGTTACA3’ primer A NotI-Chd9 5’ GTCAAGCTTACGCCAAATGGAGTATACG3’ primer B HIII-Chd9 5’ GTCAAGCTTGAAAGGCTATCCCAGTACGT3’ primer Y HIII-Chd9 5’ TCTACTAGTACACCAAGACCAACATCATCCG3’ primer Z SpeI-Chd9 5’ ATAAGCGGCCGCTCCCTGTCTTGCCTCCTATG3’ primer C NotI-Chd9 5’ GTCGAATTCAGATCTATTGCTAATTAACGTAGATGTCG3’ primer D RI/BglII-Chd9 5’ ATAGGATCCCAAGACGGTCCTTCCATGG3’ primer E BamHI-Chd9 5’ CGCGTCGACGTCCAATCTGCAACAATATGCGTA3’ primer F SalI-Chd9 5’ The 5’ arm was retrieved from EL350 cells from BAC RP23-296  . The 3’ NotI/HindIII), 3 μL of PCR product 2 , 2 μL of MC1TK, 1 μL of 10× ligation buffer, and 1 μL of T4 DNA ligase.The retrieval vector was generated by mixing 3 μL of PCR product 1 , 3 μL of PCR product 2 , 2 μL of floxed Neo cassette , 1 μL of the retieved Bac DNA fragment NotI/SalI), 1.2 μL of 10× ligation buffer, and 1 μL of T4 DNA ligase.The First targeting. For gene targeting, 25 μg of NotI-linearized 5’ Chd9 cko-targeting vector DNA was electroporated into 10 × 106 ES cells E14 IB10 (129P2 genotype) were grown on feeder MEFs. Transfectants were selected in CM medium  with G418 (200 μg/ml). Correctly targeted clones were identified by Southern blot analysis of a HindIII digest genomic DNA with the 5′ probe . Correctly 5’ targeted clone was electroporated with the PGK-Cre/PGK-Puro plasmid in order to excise the loxP floxed neo cassette. Clones with excised neo cassette were identified by PCR analysis using primers FRW- 5’TCTCAGAGGCTGGTGTTTGTTTC; REV- 5’CACAAGAAGGTGACCCAGAA.Second targeting. The selected 5’ targeted clone was electroporated with 25 μg NotI-linearized 3’ Chd9 cko-targeting vector DNA with a Frt floxed neo cassette and a loxP sequence in the same orientation as the loxP sequence at the 5’ side of the Chd9 gene. Correctly targeted clones were identified by Southern blot analysis of a HindIII digest with the 3′ probe .Cis targeted clones. In order to detect targeted ES clones with both 5’ and 3’ loxP sequences on the same chromosome (cis targeting), double mutant ES cell clones were electroporated for transient Cre expression by PGK-Cre/PGK-Puro plasmid. Excision of the Chd9 protein-coding exons 3–38 was demonstrated by PCR using primers (Δ-PCR): FRW-5’ CCCGAGTGTGGGTTTAAATG; REV-5’ CCAATCTAACTGGGCTACCG.loxP sequences on the same chromosome flanking the Chd9 gene, were injected into C57BL/6 blastocysts, and chimeras were crossed with FVB/N mice to produce heterozygous offspring. The resulting Chd9f/+ heterozygous and subsequent Chd9f/+ homozygous mice were viable and fertile and showed a normal lifespan, indicating that the Chd9f/+ conditional allele is fully functional.Correctly targeted neomycine-resistant ES clones harbouring two Chd9 gene was excised by crossing the mice with the Flpe deleter strain (FVB background).The neo cassette at the 3’ end of the Chd9f/+ mice were crossed with Acre germline deleter strain (Beta-actin driven expression of Cre recombinase). Mice were backcrossed to FVB background for two generations and Chd9+/- males and females were bred to generate experimental cohorts.To generate heterozygous knockout allele, Chd9-/- males were crossed with females bearing EμMyc transgene [Chd9+/-; EμMyc animals, which were subsequently crossed with Chd9+/- animals to generate experimental cohorts .ransgene  (maintaiHindIII (Roche) in reaction containing 7 μL buffer B (NEB), 1.4 μL spermidine (Sigma Aldrich) and 0.7 μL BSA(Sigma) in 70 μL volume. DNA was resolved on 0.8% agarose gel at 30V overnight and transferred to Amersham Hybond N+ membrane  in SSC (Sigma). Probes were PCR amplified from genomic DNA in a reaction mix with 32P-dCTP (Perkin Elmer) using primers:  FRW: ATTTGTGCGCATGAACTGC, REV: GTCGAGCAGTAAGGCTAAAGTAA and  FRW: TGTGCATCTCCACTTTGAGC, REV: CATTAATGCATGGCCAGTTG.DNA was isolated from tails and organs using DirectPCR Lysis Reagent (Viagen Biotech). For Southern blot 10 μg of DNA was digested with 4 μL + membrane  in 20xSSC (Sigma), baked for 1.5hrs at 80°C and UV–crosslinked. Probe was PCR amplified from cDNA template in reaction mix with 32P-dCTP (Perkin Elmer) using primers: Internal-FRW: 5’ CAGACGAGGGTTCAGAGAGG, Internal-REV: 5’ TTCTTCCAGAAAGTATGTTTGAACG; and 3’ UTR- FRW: 5’CAGACGAGGGTTCAGAGAGG, 3’UTR-REV: 5’ACATCGAGTTCAGAAACGTATGG. Prehybridization (1hr) and hybridization (o/n) with the probe was done at 65°C in Church buffer. Membrane was washed consecutively in 2xSSC, 2xSSC 0.1%SDS, 0.5xSSC 0.1%SDS buffers, exposed on phosphor screen, and image was acquired on phosphorimager.Northern blot was performed using standard protocol. Briefly, RNA was isolated from frozen tissues, tumour pieces and cell pellets using TRIzol reagent (Ambion) and RNEasy columns (Qiagen). 10 μg of total RNA was resolved on agarose/formamide gel at 20mV for 6hrs; transferred to Amersham Hybond NWestern blot was performed using standard protocol. Briefly, cells were trypsinized, pelleted, washed in ice-cold PBS, lysed in RIPA buffer on ice, briefly sonicated and cleared by centrifugation. Protein was resolved on 3–8% gradient Tris-Acetate precast NuPAGE gels (Invitrogen) and transferred onto 0.45 μm Immobilon-P PVDF membrane (Merck Millipore) in 2x Tris-Glycine transfer buffer supplemented with 0.01% SDS for 16hrs at 100mA at 4°C. Membrane was blocked for 4hrs in 5% skimmed milk in TBST at 4°C. Rabbit anti-CHD9 antibody  or mouse anti-Vinculin  was incubated in blocking buffer at 1:1000 dilution o/n at 4°C. After three washes with TBST, antibody was incubated with 1:10.000 dilution of secondary goat anti-rabbit antibody coupled to horseradish peroxidase for 1hr at ambient temperature, followed by three washes in TBST. Immuno-complexes were visualised by incubation in Pierce enhanced chemiluminescent (ECL) blotting substrate (Thermofisher).MEFs were plated in 8-well glass slide chamber system (Labtek), washed in PBS, fixed in PBS/ 3.7% formaldehyde (Merck), permeabilised with PBS/ 0.1% Triton X-100 (Sigma) and blocked in 10%FBS/PBS. The rabbit anti-CHD9 antibody  at dilution 1:1000 was incubated o/n at 4°C, followed by goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (1:500); chromatin was counterstained with DAPI (Thermo Scientific), and mounted with Vectashield (Bioconnect). Images were acquired on Leica SP5 Scanning Confocal Microscope.Tissues were fixed in either 4% formaline or acidified formaldehyde . Haematoxylin and eosin (H&E) stainings and immunohistochemical procedures were performed according to standard procedures. Sections were prepared at 2 μm thickness from the paraffin blocks and stained with hematoxylin and eosin (HE) according to standard procedures. For immunohistochemistry (IHC), 4 μm -thick sections were made. Primary antibodies were obtained from the following sources: B220 , Pax5 , CD3 , Myeloperoxidase , F4/80 . Antibody staining was revealed using either diaminobenzidine or 3-amino-9-ethylcarbazole chromogen. Images were captured using a Zeiss Axioskop2 Plus microscope  and processed using AxioVision 4 software (Carl Zeiss Vision).Chd9+/- x Chd9+/- cross at day 13.5 post coitum and cultured using standard methods. Briefly, heads and organs of embryonic day 13.5 embryos were removed, and the remaining fetal tissue was minced, rinsed in PBS, and incubated for 1 hr in 0.5 ml of 0.1% trypsin/EDTA solution at 37°C. Subsequently, the cell aggregates were dissociated in DMEM supplemented with 10% FBS (GIBCO) and 1% penicillin-streptomycin (GIBCO). After removal of large clumps, the cells were plated in a 175-cm2 flask. After 2–3 days, confluent cultures were frozen down and considered as being passage 1.Mouse embryonic fibroblasts (MEFs) were isolated from the Chd9+/- x Chd9+/- cross as described before [Embryonic stem cells (ESC) were derived and cultured from the d before . ESCs we2, either in DMEM (GIBCO) supplemented with 10% FCS, 100 units per ml penicillin, 100 mg/ml streptomycin or in RPMI- 1640 (GIBCO) supplemented with 10% FCS, 100 units per ml penicillin, 100 mg/ml streptomycin. K562 and SNB19 cells were available within The Netherlands Cancer Institute, originally purchased from American Type Culture Collection (ATCC), and confirmed to be mycoplasma free.Cells were grown at 37°C, 5% COCGCTGAGTACTTCGAAATGTC, shRNA -CHD9#1: TGCATACCAGCGTACTAATAA, shRNA-CHD9#2: CCGAGCTCTCTTAGCATATTG.SNB19 cells were cultured in DMEM (Invitrogen) and K562 in RPMI-1640 (Invitrogen) media supplemented with 10% FBS and Pen/Strep (Gibco). For doxycycline induction of pLKO-tet-shRNA constructs, cells were grown in same media supplemented with 10% Tet system approved FBS (Clontech) and 100 units per ml penicillin, 100 mg/ml streptomycin. Knockdown was induced by adding 1 μg/ml doxycycline (Sigma). 48 hours upon knockdown cells were pelleted for RNA/protein isolation or plated for proliferation assay. The shRNA sequences used in this study are: shRNA-Luciferase: For proliferation assay, 25.000 MEFs were seeded in a 12-well plate, fixed in 4% PFA and stained with Crystal Violet (Merck) Ethanol solution for 10 min at RT at indicated time points. Stained cells were dissolved in 10% acetic acid, diluted 1:4 in MQ and absorbance was measured at OD590nm. Experiment was performed twice, with two knockout and wildtype MEFs seeded in triplicate. Values represent average ± SEM, two-way ANOVA was used to determine statistical significance.Proliferation assay of human SNB19 glioblastoma cells with inducible pLKO-tet-shRNA-CHD9 was done using the same protocol, with only difference that prior to seeding cells were treated with 1μg/ml doxycycline (Sigma) for 48hrs. Cells were fixed and processed at indicated time points as described above. Doxycycline was kept for duration of the whole experiment.Proliferation assay of human K562 cells was done as described above, with one major difference that cells were plated in a 6-well in triplicate and cell number was assayed daily using CASY Cell counter without replating. Doxycycline was kept for duration of the whole experiment. Values represent average ± SEM, two-way ANOVA was used to determine statistical significance.For soft agar growth analysis, MEFs were first infected with LZRS-TBX2-GFP, followed by infection with Myc, RasV12 and E1A or combinations thereof. Oncogene expression following transduction was confirmed on the Western blot. After selection, 50,000 MEFs were resuspended in 0.4% soft agar and plated on a bottom layer of 1% agar per well of a 6-well ultra-low attachment plate (Corning) in triplicate. The number of colonies growing in soft agar after two weeks of culture was quantified using ImageJ software. Experiment was performed twice, using two wildtype and knockout MEFs, data values are represented as average ± SEM, p-value calculated using two-tailed Student’s t-test.All statistical analyses of cell proliferation and transformation was done using GraphPad Prism8. For all calculations of p-values, significance threshold alpha was set at 5% (p< 0.05).Peripheral blood was collected into EDTA-coated microtubes and analyzed by FACS. Blood was depleted from red blood cells by hypotonic lysis and staining was performed with fluorochrome-labelled antibodies specific for B220, CD3, CD11b. Samples were analyzed on the LSR Fortessa cell analyzer (BD).Flow cytometric analysis was performed as described previously . BM monoBlood lymphoid. CD4-Fitc (BioLegend), CD8-PE-Cy7 (BD), B220–Pacific blue (BD), CD11b-PerCpCy5,5 (BD), CD3-APC (BioLegend); Blood myeloid and erythroid: CD3-Fitc (BD), CD19-Fitc (BD), B220-Fitc (BD), Gr1-PE (BD), F4/80-Pacific blue (BioLegend), CD11b-APC (BD);BM myeloid: Gr1-PE (BD), CD11b-PerCpCy5,5 (BD), Ter119-PECy7 (BD), F4/80–Pacific blue (BioLegend), CD19-APC (BioLegend); BM megakaryocyte: CD3-Fitc (BD), CD41-PE (BioLegend), CD11b-PerCpCy5,5 (BD), B220–Pacific blue (BD), CD19-APC (BioLegend); HSPC and CLP: Strep-APC/CY7 (Southern Biotech), cKit-APC (BD), SCA1-PacBlue (BioLegend), CD34-FITC (eBioscience), CD135-PE (BioLegend), CD150-PECy7 (BioLegend), CD127-PerCpCy5,5 (BioLegend); Hematopoietic progenitors: Strep-APC/CY7 (Southern Biotech), cKit-APC (BD), SCA1-PacBlue (BioLegend), CD34-FITC (eBioscience), CD16/32-PerCpCy5,5 (eBioscience), CD127-PE (eBioscience).BM lymphoid. IgD-Fitc (BD), CD25-PE (BioLegend), CD11b-PerCpCy5,5 (BD), IgM-PECy7 (Southern Biotech), B220-Pacific blue (BD), cKit-APC (BD); EμMyc lymphomas or human cancer cell lines (SNB19 and K562) were isolated and total RNA was extracted. Samples were prepared using TruSeq protocol, and standard sample preparation protocols and RNA-seq was performed on a Hiseq2000 machine (Illumina) at the NKI Genomics Core Facility.Tissues from individual E15 embryos , derived cells , EμMyc lymphomas), hg37 (K562 and SNB19 cells) using TopHat (v2.1.0 using bowtie 1.1.0). and number of reads per gene were measured with HTSeq count (v0.5.3). Statistical analysis of the differential expression of genes were performed using DESeq2 [Sequencing data were aligned to mm9 , mm10  was used . The x-aS1 FigChd9 allele. Cre recombinase excision removed 5’ NEO cassette to allow subsequent 3’ targeting with FRT–loxP-NEO cassette, and Flp–mediated removal in-vivo. Heterozygous mice were generated by breading with universal β-actin Cre recombinase strain, resulting in the excision of the intervening 117kb genomic DNA and removal of the coding potential of the locus (exon 3—exon 39). Southern blot hybridization in Chd9–null allele following Cre–mediated deletion. loxP sites (red triangle), FRT sites (blue triangle), Neomycin selection cassette (grey rectangle labelled Neo), Cre recombinase (Cre), Flp recombinase (Flp). HindIII restriction sites(red), genomic location of the PCR probe used for Southern blot (green rectangle). (B) Integrative Genomics Viewer (IGV) snapshot of the region surrounding the loxP site flanking exon3 (left) and region surrounding the 3’ UTR loxP site (right). The loxP sites are 117kb apart. Active transcription within the locus that gives rise to the 4.2kb mRNA species is represented by RNA–seq reads from wildtype (red) and knockout (blue) MEFs.(A) Representation of the targeting strategy whereby recombineering–generated cassettes with loxP sites were introduced sequentially by homologous recombination at the 5’ and 3’ region of the (PDF)Click here for additional data file.S2 FigEndogenous CHD9 protein is localized in distinct nuclear foci that are absent in the knockout MEFs. Background cytoplasmic signal remains visible in the knockouts. Dashed white lines indicate nuclei boundaries. , chromatin . Scale bar 10μm (white line).(PDF)Click here for additional data file.S3 FigChd9-/- and Chd9+/+ liver (A), brain (B). The DEGs with p.adj <0.05 and log2 (fold change) > ±1 are shown, Chd9 gene is highlighted in orange. Heatmap of statistically significant DEGs in Chd9-/- compared to control Chd9+/+ organs. The color scale is based on normalized read values. Scatter-plot expression profiles of each gene in the Polycomb repressive complex 1 (PRC1) and 2 (PRC2) and CHD family shown based on the log2 fold change over the average expression strength. Genes highlighted in red are found to be significant (p.adj < 0.05).(A) Liver, (B) Brain (anticlockwise): Volcano plots represent differentially expressed genes (DEGs) between (PDF)Click here for additional data file.S4 FigChd9-/- and Chd9+/+ MEFs (A), ESCs (B). The DEGs with p.adj <0.05 and log2 (fold change) > ±1 are shown, Chd9 gene is highlighted in orange. Heatmap of statistically significant DEGs in Chd9-/- compared to control Chd9+/+ organs. The color scale is based on normalized read values. Scatter-plot expression profiles of each gene in the Polycomb repressive complex 1 (PRC1) and 2 (PRC2) and CHD family shown based on the log2 fold change over the average expression strength. Genes highlighted in red are found to be significant (p.adj < 0.05).(A) MEFs, (B) ESCs (anticlockwise): Volcano plots represent differentially expressed genes (DEGs) between (PDF)Click here for additional data file.S5 FigChd9 knockout (red) and wildtype (blue) MEFs. Lower panel represents genomic location of Gm13237(ENSMUST00000122151) in the UCSC Genome Browser (mm9). (B) Western blot analysis (15% SDS–PAGE) of Hmgb1 and Hmgb2 proteins in Chd9 knockout and wildtype MEFs shows no difference in protein abundance and isoform expression. Gapdh is used as a loading control.(A) Gm13237 is a processed pseudogene (635bp), located on mouse Chromosome 4: 145,632,952–145,633,586, forward strain (mm9). It originates through genomic integration of reverse–transcribed parental gene mRNA, and bears homology to both human HMGB1 and HMGB2. Upper panel represents snapshot of Integrative Genome Viewer (IGV) RNA–seq reads of (PDF)Click here for additional data file.S6 FigChd9 wild-type (WT) and knockout (KO) thymi: Left panel, frequency of the CD4+CD8+ double positive effector memory T-cells and naive CD4+ and CD8+ single positive cells. Middle panel, frequency of the immature double–negative (DN) cell subset categories, based on their expression of CD44 and CD25 surface markers: CD44+CD25– (DNI), CD44+CD25+ (DNII), CD44–CD25+ (DN III), and CD44–CD25– (DNIV). Right panel, total count of cells in the wild–type and knockout thymi. Bellow, frequency of the CD4+ (left) and CD8+ T-cells (right) expressing TCR-β (T-cell beta) receptor.(B) Flow cytometric analysis of the Chd9 WT and KO spleens: Far left panel, frequency of the CD3+ pro-thymocytes. Left panel, frequency of the IgM/IgD double positive naive mature B-cells. Right, frequency of the CD4 and CD8 double negative, single positive and double positive T-cells. Absolute number of cells in spleens of Chd9 WT and KO animals. (C) Flow cytometric analysis of the peripheral blood populations in the Chd9 WT and KO animals: Left, frequency of the lymphoid and myeloid cells, B- and T-cells (middle) and white blood cells . Data plotted as frequency of hematopoietic cells, mean ±SD, p-value calculated using two-tailed Student’s t–test.(A) Flow cytometric analysis of the (PDF)Click here for additional data file.S7 FigChd9 mRNA levels in tissues derived from wildtype and EμMyc;Chd9-/- mice using probe spanning 3’UTR. Active transcription within the knockout Chd9 locus is revealed (labelled with *). Detected mRNA from the Chd9-/- allele migrates at the size of ~4.2kb, corresponding to the size of the remaining exons within the locus . Chd9 has two alternatively spliced mRNA isoforms . Agarose gel shows Ethidium Bromide–stained rRNA as loading control. (B) Microphotographs of a representative B-cell lymphoma (lymphoblastic lymphoma) from the EμMyc;Chd9-/- animal, showing stainings of hematoxylin-eosin (H&E), immunohistochemistry (IHC) of the B-cell markers B220 and PAX5, and IHC of general T-cell marker CD3 and leukocyte marker myeloperoxidase. All scale bars: 20mm, except for the upper right one which is 10mm. (C) Volcano plots show differentially expressed genes (DEGs) in EμMyc;Chd9+/- (left) and EμMyc;Chd9-/- (right) compared to control EμMyc;Chd9+/+ lymphomas (p.adj <0.05 and log2 (fold change) > ±1). Upregulated genes are highlighted in red, downregulated in blue. DEGs common for EμMyc;Chd9+/- and EμMyc;Chd9-/- samples are marked as black dots. Chd9 is highlighted in orange.(A) Autoradiogram of Northern blot analysis of (PDF)Click here for additional data file.S8 FigChd9 is expressed at high levels in all the samples analyzed. In Chd9 heterozygous and knockout samples, there is a marked reduction in Chd9 expression. Other family members display variable levels of expression between samples. Chd4 shows the highest expression in all the samples, whereas Chd5 is expressed at low levels in MEFs, ESC, E15 liver, and barely detectable in lymphomas.  In human K562 and SNB19 cancer cell lines CHD9 is expressed at high levels. Only CHD family members with detectable expression are annotated on the Scatter-plot. Expression profile of each gene is based on the log2 normalized read counts for all samples. (PDF)Click here for additional data file.S9 FigChd9 wildtype (WT) and two knockout (KO) clones seeded in triplicate. Right panel is quantification of the crystal violet staining by measuring absorbance at 590nm. Data indicates relative increase in OD590nm absorbance. Day 0 values are set as 1; data represented as average ± standard error mean (SEM), p-value was calculated using two-way ANOVA . (B) Anchorage–independent growth was determined by soft agar colony forming assay. Upper panel shows ImageJ colony number quantification of two independent WT and KO clones. MEFs were infected with LZRS-Tbx2-GFP virus, followed by transduction with single or combinations of indicated oncogenes . Experiment using two independent WT and KO clones seeded in triplicate was repeated twice (represented as average ± standard-error mean (SEM), p–value calculated using two-tailed Student’s t–test) (ns = not significant). Lower panel shows representative view of the wells containing soft–agar colonies of the transformed WT and KO MEFs used for ImageJ quantification in the upper panel.(A) Proliferation of MEFs assayed by crystal violet staining. Left panel shows representative example of a single well stained with crystal violet at indicated time points. Experiment was done twice using two (PDF)Click here for additional data file.S10 FigCHD9 across panel of human cancer cell lines, taken from the Broad Institute’s Cancer Cell Line Encyclopedia.Gene expression data of (PDF)Click here for additional data file.S11 Fig(A) K562 chronic myelogenous leukemia and (B) SNB19 glioblastoma: (left) Unsupervised hierarchical clustering of analyzed knockdown cells, each experiment was done in duplicate. (Right) Volcano plots represent significant differentially expressed genes (DEGs) between two shRNA hairpins targeting CHD9 and control hairpin targeting Luciferase. The DEGs with FDR < 0.05 and log2 (fold change) > ±1 are shown, 20 most significant genes are highlighted ; CHD9 gene is highlighted in orange. The DEGs are enumerated at the top of each plot. (Bellow) Proliferation curve representing relative growth of knockdown (shRNA#1 and shRNA#2 hairpins against CHD9) and control (Luciferase hairpin) cells. Cells were treated with 1μg/ml doxycycline (DOX) two days before seeding for proliferation assay. Doxycycline was kept throughout duration of the experiment. In case of K562, cell number was measured on cell counter, whereas for SNB19 glioblastoma relative cell growth was determined by crystal-violet assay. Data represented as average ± standard error mean (SEM), p-value was calculated using two-way ANOVA . Cartoon illustrates seeding strategy.(PDF)Click here for additional data file.S12 FigEach circle in the scatter plot represents a KEGG pathway, name of the pathway is denoted on the Y-axis and the enrichment score the X-axis. The size of the dots corresponds to the -log10(padj) value, indicating statistically significant KEGG categories. An enrichment score indicates over-representation (positive) or under-representation (negative) of the DEGs in a given pathway. KEGG categories mentioned in the main text are highlighted in red (over-represented), blue (under-represented).(PDF)Click here for additional data file.S13 FigThe number of the corresponding Figure in the manuscript is indicated below the image (red rectangle).(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Senna bicapsularis is an important flowering, ornamental plant. The extracts showed antioxidant activity and antibacterial properties. In addition, S. bicapsularis can be used as a model woody plants. In the present study, the complete chloroplast genome was sequenced. The result showed that the length of circular chloroplast genome was 162,744 bp, containing a large single-copy region of 91,176 bp, a small single-copy region of 18,264 bp, and two inverted repeat regions of 26,652 bp. The chloroplast genome contained 128 genes, including 83 protein-coding, eight rRNA, and 37 tRNA genes. Phylogenetic tree analysis showed that S. bicapsularis has closely relationship with Senna occidentalis, Senna tora and Senna siamea.  Senna bicapsularis (L.) Roxb. which belongs to Senna (Leguminosae) is one the important flowering, ornamental plant. Senna species are widely distributed in South American and tropical countries , and the voucher specimen (ZY190202) was deposited in Xi’an Botanical Garden Herbarium. Chloroplast genomic DNA was extracted from the fresh leaves using the modified CTAB method (Doyle and Doyle The fresh leaves of Senna tora (GenBank: NC_030193) , small single copy , and two inverted repeats . The overall GC content was 36.1%  and the chloroplast genome contained 128 genes, including 83 protein-coding, eight rRNA, and 37 tRNA genes.The total plastome length of S. bicapsularis in Leguminosae, the phylogenetic tree was constructed with MEG6  (GenBank: NC_044694) was used as an outgroup (S. bicapsularis has close relationship with S. occidentalis, S. tora, and Senna siamea.In order to investigate the phylogenetic relationship of outgroup . The res


As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2′s ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)–Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors. The importance of the ubiquitin ligase murine double mutant 2 (MDM2) as a key negative regulator of the tumour suppressor protein p53 has been studied extensively. MDM2 keeps p53′s activity low under normal conditions, and since p53 induces the expression of MDM2, both proteins are kept at low concentrations.in vivo mouse studies with ligase defective MDM2 resulted in the same lethality as MDM2 knockout experiments.MDM2 regulates p53 by two distinct mechanisms. First, it binds p53 through its N-terminal p53-binding domain, which blocks the transcriptional activity of p53. Second, it promotes the proteasomal degradation of p53 by recruiting Ub conjugating enzyme (E2) thioesterified with ubiquitin  via its RING domain and mediates ubiquitin (Ub) transfer from E2 to p53,MDM2 has been an attractive anti-cancer drug target, especially in tumours where p53 is wild-type with abnormal expression of MDM2.18The RING domain of MDM2 has been discussed as an alternative target to stabilize p53.Here, we show that the aggregation of the MDM2 RING domain is species dependent. By comparing the aggregation behaviour of the MDM2 RING domain from several species followed by systematic alteration of human RING domain sequence, we identify a single point mutation that exclusively yields dimeric protein. Structural and biochemical analyses show that the mutation does not affect E2~Ub binding and the ligase activity. These results identify a suitable strategy for expression and purification of recombinant human MDM2 RING domain for structural studies and should enable future structure-guided design of small molecules that target the RING domain.E. coli expression in our prior structural study, this approach offers low reproducibility and is not practical. In contrast, the human MDM2-MDMX RING domain heterodimer predominantly expressed as a dimer and could readily be purified for structural studies.Xenopus tropicalis (western clawed frog) and Danio rerio (zebrafish), which showed a comparably high sequence variation between themselves and human MDM2. We expressed and purified the MDM2 RING domain from these species and the human counterpart  with an N-terminal cleavable GST-tag and applied the cleaved protein on a size-exclusion chromatography (SEC) column, where protein molecular weight markers were used to estimate whether the cleaved MDM2 RING domain was dimeric (~16 kDa) or aggregated  ), indicating that the protein was heavily aggregated  without significant precipitation.Human MDM2 RING domain is prone to aggregate upon cleavage from a fusion taggregated  consistef and MDM2z. We obtained crystals for MDM2z and MDM2f, which diffracted to 2.87 and 2.27 Å, respectively , where all secondary structure elements and the coordination of zinc ions are conserved (h), which are located N-terminal to the RING domain and partly involved in the formation of a 310-helix in MDM2h. Likewise, the asymmetric unit of the MDM2f crystal contains two molecules of MDM2f, which form a dimer. Similar to MDM2z, there was no electron density for residues located N-terminal to the RING domain   was shown to be important for E2~Ub recruitment. In particular, N433 is involved in a hydrogen bond network with the second MDM2h protomer and the donor Ub.f and MDM2z, it is unclear how this would impact on their ability to recruit E2~Ub for catalysis. Before further examination of the relationship between structure, sequence and protein aggregation, we wanted to ensure that both MDM2f and MDM2z are competent E3s. To assess the ability of MDM2f and MDM2z to bind E2~Ub, we performed binding analyses using Surface Plasmon Resonance (SPR). Stable E2–Ub conjugate was obtained by mutating UbcH5B’s catalytic C85 to lysine followed by covalent conjugation of the C-terminus of Ub to the catalytic lysine to form a stable isopeptide linkage that mimics the thioester linkage.f and MDM2z bind UbcH5B–Ub with a comparable binding affinity, albeit 2.5-fold reduced compared to MDM2h  MDM2 RING domain eluted mainly at ~ 0.6 CV consistent of a dimer, and only a small fraction eluted as aggregate. In contrast, a substantial fraction of Otolemur galettii  MDM2 RING domain aggregated  was stable upon removal of the GST-tag and the SEC elution profile showed reduced aggregation with bulk of MDM2hGT eluting at ~0.6 CV consistent of a dimer.It is unclear to which extent the disordered N-terminal region preceding their RING domains or sequence variations in the RING domain contribute to the reduced aggregation. In order to get a better understanding of MDM2 RING domain aggregation, we selected a few species that were closer to MDM2h’s G443, or the corresponding residue in MDM2f, T434, is solvent exposed and located within a loop. None of the available crystal structures could explain why a glycine at this position would cause the MDM2 RING domain to aggregate and that a threonine would prevent it. The corresponding residue in MDM2 from zebrafish and opossum, which both predominantly form stable dimers, is serine, suggesting that the polar side chain at this site could reduce aggregation. Since the G443T substitution produced a stable MDM2h dimer, we wanted to understand its impact on MDM2h’s structure and function. We purified MDM2hGT on a large scale with the aim to crystallize it in complex with UbcH5B–Ub. The SEC elution profile (f (h-UbcH5B–Ub complex (PDB: 6SQO). Both unit cells contain a single MDM2hGT dimer bound to two UbcH5B–Ub molecules .10-helices potentially respond differently to S429-phosphoregulation compared to MDM2h. Various phosphorylation sites near the RING domain have been identified10-helices but might also serve as a phosphorylation site itself and thereby alter E2~Ub recruitment.Ancestral MDM2 was shown to bind and ubiquitinate human p53 in cells.h, which could be eliminated by a single point mutation G443T. As the structures of MDM2h and MDM2hGT are indistinguishable, it remains unclear why the wild-type aggregates. It is noteworthy that in the human MDM2-MDMX heterodimer G443 is present in MDM2 but we did not observe aggregation, suggesting that the symmetric nature of MDM2h homodimer might contribute to the initiation of this process. The formation of supramolecular assemblies has been observed for other RING E3s,We identified G443 as a single residue being responsible for the pronounced aggregation of MDM2Escherichia coli BL21(DE3) GOLD. Cells were grown in LB medium to OD600 = 0.6–1.0 at 37 °C, induced with 0.2 mM Isopropyl-β-D-1-thiogalactopyranoside and protein expression was conducted for 16–20 h at 20 °C. Cells were resuspended and lysed in the corresponding wash buffer of the first purification step supplemented with 2.5 mM phenylmethylsulfonyl fluoride. UBA1, UbcH5B, Ub and MDM2 were purified as described previously.MDM2 constructs were cloned into pGEX-4 T1 and expressed with an N-terminal GST-tag followed by a TEV cleavage site, human UBA1 was cloned into pET21d with an N-terminal 6xHis-tag,Crystals were grown at 19 °C using sitting drop vapour diffusion technique (drop volume: 0.4 μL) and where required optimized by hanging drop vapour diffusion techniques (drop volume: 2.0 μL). All proteins were mixed with reservoir solution at 1:1 ratio.z (3 mg/mL) crystals were obtained in condition containing 0.1 M imidazole, pH 6.5, 0.12 M monosaccharides, 37.5 % (w/v) MPD_P1K_P3350  and flash-frozen in the same condition.MDM2f (5–9 mg/mL) crystals were obtained in condition containing 0.1 M PCTP, pH 7.0, 25 % (w/v) PEG 1500  and flash-frozen in the same condition containing 30 % (v/v) MPD.MDM2f-UbcH5B–Ub crystals were obtained by mixing MDM2f (5–9 mg/mL) and UbcH5B–Ub (15 mg/mL) at 1:1 molar ratio in condition containing 0.1 M HEPES, pH 7.0, 10 % (w/v) PEG 20,000 and flash-frozen in the same condition containing 25 % (v/v) ethylene glycol.MDM2hGT-UbcH5B–Ub crystals were obtained by mixing MDM2hGT (11 mg/mL) and UbcH5B–Ub (15 mg/m) at 1:1 ratio. Crystal form 1 was obtained in condition containing 0.1 M Tris, pH 8.0, 0.075 M NaOAc, 0.1 M NaCl, 15 % (w/v) PEG Smear Medium and flash-frozen in the same condition containing 25 % (v/v) ethylene glycol. Crystal form 2 was obtained in condition containing 0.1 M HEPES, pH 7.5, 0.2 M (NH4)NO3, 20 % (w/v) PEG Smear Broad. Both crystal forms were flash-frozen in the same condition containing 25 % (v/v) ethylene glycol.MDM2f-UbcH5B–Ub), autoPROC (MDM2z)Diffraction data were collected at beamlines I03, I04 and I04-1, Diamond Light Source (DLS). Data were processed by automated XDSSPR binding experiments were performed at 25 °C on a Biacore T200 instrument using a CM-5 chip  with coupled anti-GST antibody as described previously.2, 5 mM ATP at 23 °C. The reaction was started by adding GST-MDM2 variants  and stopped at the indicated time point with 4X LDS Loading dye containing 400 mM DTT followed by separation on SDS-PAGE. The ubiquitinated products were visualized with an Odyssey CLx Imaging System (LI-COR Biosciences) and staining with coomassie. Parentheses indicated final concentration of proteins in the reaction.UbcH5B (5 μM) was pre-charged for 20 min with UBA1 (0.2 μM) and fluorescently-labeled Ub (70 μM) in 50 mM Tris, pH 7.6, 50 mM NaCl, 5 mM MgClGST-tagged MDM2 variants were concentrated to 3 mg/mL and incubated with 1:10 TEV protease for 16–20 h at 4 °C to cleavage the GST-tag. Cleavage was confirmed by SDS-PAGE. The soluble fraction of the cleaved products was applied on a Superdex 75 Increase 10/300 SEC column  and the elution profiles were analyzed by comparison with molecular weight markers (Sigma-Aldrich) and by SDS-PAGE.PDB: 7AH2, 7AHY, 7AHZ, 7AI0 and 7AI1z), 7AHY (MDM2f), 7AHZ (MDM2f-UbcH5B–Ub), 7AI0 , 7AI1 .Atomic coordinates and structure factors are deposited in the Protein Data Bank with ascension codes 7AH2 (MDM2Helge M. Magnussen: Conceptualization, Methodology, Investigation. Danny T. Huang: Conceptualization, Supervision, Funding acquisition.


IV dendritic arborization (c4da) neurons of Drosophila larvae specifically prune their dendrites at the onset of metamorphosis in an ecdysone‐dependent manner. Dendrite pruning requires local cytoskeleton remodeling, and the actin‐severing enzyme Mical is an important ecdysone target. In a screen for pruning factors, we identified the protein phosphatase 2 A (PP2A). PP2A interacts genetically with the actin‐severing enzymes Mical and cofilin as well as other actin regulators during pruning. Moreover, Drosophila cofilin undergoes a change in localization at the onset of metamorphosis indicative of a change in actin dynamics. This change is abolished both upon loss of Mical and PP2A. We conclude that PP2A regulates actin dynamics during dendrite pruning.Large‐scale pruning, the developmentally regulated degeneration of axons or dendrites, is an important specificity mechanism during neuronal circuit formation. The peripheral sensory class  PP2A is required for pruning, the regulated elimination of neurites during neuronal development, by promoting the disassembly of actin structures.The protein phosphatase  Drosophila, the so‐called class IV dendritic arborization (c4da) neurons specifically prune their long and branched larval dendrites at the onset of the pupal phase by a degenerative mechanism Neurite pruning, the physiological degeneration of axons or dendrites during development, is an important specificity mechanism during neuronal circuit formation ization cda neuronization cda neuronization cda neuronmicrotubule star/mts), and one of several regulatory B subunits. In addition to Wdb, the B subunit Twins (Tws) has also been linked to actin regulation during imaginal disk development While the roles and importance of microtubule regulation during pruning are emerging, relatively little is known about actin regulation during the process. While it is clear that the actin‐severing enzyme Mical is induced in c4da neurons at the onset of the pupal stage and is required for dendrite pruning In this study, we identify PP2A as a regulator of c4da neuron dendrite pruning. In a survey of phosphoregulators of dendrite pruning, we found that loss of the scaffold subunit PP2A‐29B caused strong dendrite pruning defects. The catalytic subunit mts and—in a somewhat redundant manner, the B subunits Wdb and Tws are also required for this process, indicating the involvement of a specific PP2A holoenzyme. During dendrite pruning, PP2A displays genetic interactions with Mical, the actin disassembly factor cofilin and other actin regulators, indicating that PP2A acts via regulated actin disassembly during the process.ppk‐GAL4 and visualized c4da neurons by coexpression of membrane‐bound GFP. Control c4da neurons expressing Orco dsRNA (against Odorant receptor co‐receptor which is not expressed in c4da neurons) have long and branched dendrites at the third‐instar larval stage , caused a decrease in branching and dendritic field coverage at the third‐instar larval stage , PR72/CG4733, Well rounded (Wrd), and Widerborst (Wdb). While the expression of an sgRNA construct directed against CG4733 under ppk‐GAL4 did not cause dendrite pruning defects at 18 h APF, an sgRNA construct against Tws resulted in mild pruning defects  involved in dendrite pruning could ameliorate the dendrite pruning defects caused by this treatment. lves Fig A–E. We n‐29B Fig F–J, suggPP2A‐29B dsRNA displayed varicosities at 18 h APF that were larger and often occurred at distal dendrite tips. Sometimes, they developed small filopodia‐like protrusions , was itself required for c4da neuron dendrite pruning. MARCM clones homozygous for the cofilin mutant allele tsrN121 did not display dendrite pruning defects at 18 h APF . GFP::tsr localized to multiple punctate, sometimes elongated structures in the soma and dendrites of c4da neurons during the larval stage . At 6 h APF, most GFP::tsr punctae in c4da neurons had vanished and GFP::tsr now displayed a largely dispersed distribution , or abelson kinase , did not cause dendrite pruning defects, overexpression of a constitutively active form of protein kinase A (PKA mc*) caused strong and fully penetrant c4da neuron dendrite pruning defects Fig . HoweverTaken together, our results suggest that PP2A regulates one or several aspects of actin dynamics and/or cortical actin organization that are required for dendrite pruning.Drosophila c4da neurons during metamorphosis. Our data indicate that a PP2A holoenzyme containing either the Wdb or Tws regulatory subunit is involved in pruning regulation. Our data strongly link PP2A to actin regulation during dendrite pruning. In particular, our genetic interaction studies show that the pruning defects caused by loss of PP2A can be suppressed by overexpression of the actin‐severing enzyme Mical. Mical is induced by ecdysone at the onset of the pupal stage and is required for c4da neuron dendrite pruning. While our data and an accompanying study by Yu and colleagues In this study, we identified an important role for the protein phosphatase PP2A during large‐scale dendrite pruning of While our genetic data suggest that PP2A is not involved in the temporal regulation of microtubule disassembly via the Par‐1/tau pathway Fig , the parppk‐GAL4 insertions on the second and third chromosomes SOP‐FLPnsyb‐GAL4R57C10GE16781FRT40A. Other fly lines were UAS‐Mical KOFRT G13, tsrN121 (BL #9109), FRT 82B, wdb14 (BL #53712) 210/CyO (BL #25404). UAS‐dsRNA lines were as follows: PP2A‐29B (BL #29384), Wrd (BL #30512), Tws (VDRC# 34340), chicadee (VDRC #102759), moesin (VDRC #37917). Orco dsRNA (BL #31278) was used as control. UAS‐dsRNA lines were used with UAS‐dicer2 TEDAll crosses were done at 25°C under standard conditions. For expression in c4da neurons, we used sRNA BL #278 was urd BL #302, Tws . UAS‐GFP::tsr was expressed under ppk‐GAL4 and imaged live in ether‐anaesthetized third‐instar larvae or at 6 h APF. All images were analyzed using Fiji For analysis of pruning defects, pupae were dissected out of the pupal case at 18 h APF and analyzed live using a Zeiss LSM 710 confocal microscope. For Pupal body wall filets were dissected quickly in PBS and fixed in PBS containing 4% formaldehyde for 20 minutes. Chicken anti‐GFP antibodies were from Aves labs, respectively. Other antibodies were guinea pig anti‐Sox14 Pruning phenotypes in Figs For quantification of immunofluorescence experiments, c4da neuron staining intensities were measured in Fiji and normalized to c1da neuron staining intensities in the same image. These data were analyzed using Wilcoxon's test.The number of GFP::tsr punctae was mean ± s.d. and analyzed using Wilcoxon's test. For dendritic field coverage analysis, dendritic fields were defined by segment border denticles (in the anterior/posterior direction), the dorsal midline, and the middle between dorsal and lateroventral c4da neuron dendrites . Actual coverage was calculated by connecting outer dendrite terminals with each other as in Ref. NW and SR designed and conceived the project and interpreted the data. NW and SR performed experiments. NW, UG, and SR contributed reagents. SR wrote the manuscript.The authors declare that they have no conflict of interest.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.


More recent observations warrant revision of the classic view and help to explain some otherwise puzzling aspects of excitotoxic cell injury. Studies using pharmacological and genetic approaches show that superoxide produced by NMDAr activation originates primarily from NADPH oxidase rather than from mitochondria. As NADPH oxidase is localized to the plasma membrane, this also provides an explanation for the extracellular release of superoxide and cell-to-cell “spread” of excitotoxic injury observed in vitro and in vivo. The signaling pathway linking NMDAr to NADPH oxidase involves Ca2+ influx, phosphoinositol-3-kinase, and protein kinase Cζ, and interventions at any of these steps can prevent superoxide production and excitotoxic injury. Ca2+ influx specifically through NMDAr is normally required to induce excitotoxicity, through a mechanism presumed to involve privileged Ca2+ access to local signaling domains. However, experiments using selective blockade of the NMDAr ion channel and artificial reconstitution of Ca2+ by other routes indicate that the special effects of NMDAr activation are attributable instead to concurrent non-ionotropic NMDAr signaling by agonist binding to NMDAr. The non-ionotropic signaling driving NADPH oxidase activation is mediated in part by phosphoinositol-3-kinase binding to the C-terminal domain of GluN2B receptor subunits. These more recently identified aspects of excitotoxicity expand our appreciation of the complexity of excitotoxic processes and suggest novel approaches for limiting neuronal injury.Excitotoxicity is classically attributed to Ca However, recent studies have identified several additional complexities that challenge this classical view and identify novel ways to suppress excitotoxic neuronal death. These complexities arise from interactions between superoxide and nitric oxide, the sources of superoxide formation, and the newly appreciated role of non-ionotropic NMDA receptor (NMDAr) signaling.The term “excitotoxicity” was first used in reference to rapid neuronal death caused by glutamate receptor activation . The ter2–), by contrast, is polar, largely lipid impermeable, and has much shorter half-life and mean diffusion distance. Nitric oxide is generated in a subset of neurons by neuronal nitric oxide synthase. It is considered a reactive oxygen species (ROS) because it contains an unpaired electron, but it is not intrinsically a highly reactive molecule in biological systems. Nevertheless, studies using both pharmacological and genetic abrogation of nitric oxide production showed near-complete suppression of excitotoxic neuronal death (An intriguing aspect of excitotoxic cell death is that it requires the combined effects of two independently generated “gaseous” reactive oxygen species: nitric oxide and superoxide. Nitric oxide (NO) is non-polar, lipid permeable, and has a relatively long half-life and diffusion distance in brain . Superoxal death , 1996.–)  . Peroxyn–) . Though –) . Increas–) . Only wi+ by electrons from mitochondrial dehydrogenases or electron transport complexes . NOX is a membrane-associated enzyme that transfers an electron from NADPH on one side of the membrane to molecular oxygen on the other side, forming O2– . NOX was2– . NOX is  subunit . Electro subunit .phox subunit . Accordingly, NMDAr-induced superoxide production and cell death can be blocked with the PI3K inhibitor wortmannin, and this blockade can be circumvented with either exogenous phosphoinositol-3-phosphate or with a constitutively active form of PKCζ  was originally designed to block NMDAr-induced nitric oxide production by displacing GluN2B from the PSD95, with which neuronal nitric oxide synthase is associated . Measure2+ dependent remains unresolved. Other Ca2+-dependent NMDAr signaling events have been shown to be mediated by calcium/calmodulin-dependent protein kinases  do not produce nitric oxide production, superoxide production, or excitotoxic cell death , thus suggesting a mechanism coupling non-ionotropic signaling to NOX2 activation.“Non-ionotropic” receptor signaling refers to signal transduction events triggered by transmembrane conformational changes rather than by ion flux. Non-ionotropic signaling by NMDAr was first identified by Westbrook and colleagues, who found that agonist binding independent of ion flux could downregulate GluN1/GluN2A receptor function through a process involving dephosphorylation of GluN1 tyrosine residues . Subsequmulation . Crucial+ influx . Ligand +, Ca2+, and other ions and molecules (2+ and Na+ influx essentially amplify the elevations caused by ion flux through the NMDAr itself. A synthetic peptide that prevents pannexin-1 channel activation by preventing its phosphorylation by Src kinase attenuates NMDAr-induced Ca2+ elevations and neuronal death (A second and apparently independent role of metabotropic signaling in excitotoxicity involves pannexin channel opening. When activated, pannexin ion channels become permeable to Naolecules . Thompsoal death , 2016. T2+, superoxide, and nitric oxide in excitotoxicity have stood the test of time, but subsequent observations have identified complexities that both expand our understanding of this process and open additional questions. Among these complexities is that elevations in either superoxide or nitric oxide levels can drive production of peroxynitrite, such that it is rarely possible to ascertain which one of these ROS is driving excitotoxic injury. It has also been demonstrated that superoxide production induced by NMDAr stimulation is generated primarily by NOX2, rather than mitochondria, but it remains uncertain whether this superoxide signal may be amplified by resultant mitochondrial dysfunction. The identification of NOX2 as the primary source of superoxide and provides an explanation for the extracellular actions of superoxide in excitoxicity, and also indicates the existence of a signaling cascade linking NMDAr to NADPH oxidase. This signaling cascade has been shown to include Ca2+ influx, PI3K activation, and PKCζ activation, but the nature of the Ca2+-dependent step remains to be established. Notably, Ca2+ influx alone is not sufficient to induce superoxide formation or excitotoxicity, and recent studies suggest that these processes also require concomitant non-ionotropic signaling induced by agonist binding to NMDAr.The early observations that identified key roles for CaRS and JW both contributed to the literature research and writing for this review. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The phenolic profile was assessed by Reverse Phase–High-Performance Liquid Chromatography–Diode Array Detector (RP-HPLC-DAD). The antioxidant activity of the bread extracts was determined by 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), diphenyl-1-picrylhidrazyl radical (DPPH), and ferric reducing antioxidant power (FRAP) methodologies. Acorn flour can be considered a good source of bioactive compounds and antioxidants in GF bread. Acorn flour showed good technological properties in GF baking, improving bread nutritional and sensory characteristics.Polyphenols are important bioactive compounds whose regular ingestion has shown different positive impacts in health. Celiac patients have nutritional deficiencies, bringing many problems to their health. Thus, it is important to develop gluten-free (GF) products, such as bread, with nutritional benefits. The acorn is the fruit of holm oak and cork oak, being an underexploited resource nowadays. Its nutritional and functional characteristics are remarkable: rich in unsaturated fatty acids and fiber, vitamin E, chlorophylls, carotenoids, phenolic compounds, and antioxidant properties. The purpose of this study was to assess the use of acorn flour as a bioactive compounds source and natural GF ingredient for baking GF bread. Bread loaves were prepared with buckwheat, rice, acorn flour, and potato starch. Two levels of acorn flour (23% and 35% of the flour mixture) were tested. The physical, nutritional, and sensory characteristics of the bread were analysed, as well as the composition of phenolic compounds: total phenols,  Bioactive compounds characterisation has been a subject of several studies. According to Biesalski et al. , “bioactCeliac disease (CD) is an autoimmune disorder of the small intestine that occurs in individuals with a genetic predisposal when gluten is ingested, causing intestinal mucosa inflammation ,5,6. It Gluten is responsible for the viscoelastic characteristics of the dough; hence, baking without gluten presents a technological challenge. For this reason, the production of gluten-free bread (GFB) leads to the loss of many physical qualities in terms of texture, crumb, crust, and colour, mouth feel, flavour, specific volume, staling rate, and shelf life ,18,19. TQuercus rotundifolia and Q. ilex) and Cork oak (Q. suber). Both Quercus species have origin in Southern Europe and are abundant in Portugal, Spain, France, and Italy [Acorn is the fruit of Holm oak  when 23% of acorn flour was incorporated, in comparison with the control. However, when the amount of acorn flour increased to 35%, the volume decreased again, presenting similar values to the control. These results are in agreement with Korus et al. [The bread’s volume increased significantly  changes between both percentages of acorn flour and the control. These results are in accordance with Turkut et al. [In relation to bake loss, there were no significant  with the increase of acorn flour incorporation, showing a reduction in whiteness and a rise in the browning index, which represents the darkness of the bread. The increase in crumb darkness was significant (p < 0.05) and progressive in comparison with the control when the acorn level increased from 23% to 35%. In general, GFB has a lighter colour in comparison with wheat breads [According to different authors, food colour is one of the most important attributes regarding consumer choices, and bread is no exception ,47. In Ft breads ,18, so pt breads , who alsThe colour of the bread is influenced by its ingredients’ colours, as well as by the reactions that occur throughout the baking process ,38. Whilp < 0.05) rise with acorn incorporation, which means a predominance of red over green. In relation to the b* parameter, it is possible to see that the control bread presents a significantly (p < 0.05) lower value, when comparing with the two levels of acorn flour breads. This means a more intensive yellow than blue colour in acorn flour breads’ crumb, although no significant differences were found between them (p > 0.05). Similar results were obtained by Skendi et al. [In relation to the a* parameter of bread crumb, it is possible to observe a significant  with the introduction of acorn flour when compared with the control.Concerning the crust, both the a* and b* parameters did not present significant statistical differences  = 21.22; ΔE*(control − A23%) = 12.72; ΔE*(A23% − A35%) = 8.78. Thus, it is possible to understand three totally different colours between the crumb of breads in study. When comparing bread’s crust colours, the obtained results were: ΔE*(control − A35%) = 12.18; ΔE*(control − A23%) = 14.36; ΔE*(A23% − A35%) = 2.53. Therefore, it is possible to observe that the crust colour is different between control bread and acorn flour breads. Nevertheless, between both acorn flour breads, as we can see from E* value indicates. Acorn flour contributes to improving the GFB colour, thus making it more appealing to consumers.Comparing bread’s crumb colours, the obtained results were: Δw), both acorn flour levels of incorporation presented significant differences (p < 0.05) from the control, showing lower values, but with no differences between them. The mean values were respectively: C 0.965, A23% 0.949, and A35% 0.955. The acorn flour breads produced in this study revealed low values of aw, when compared with other authors. Hager et al. [w (0.969). The study of Encina-Zelada et al. [Regarding water activity  when compared with the control, whereas with a 35% incorporation, the firmness dropped. Thus, showing the same characteristic we have seen previously with volume, Korus et al. [Together with colour, texture is also a very important quality bread parameter, greatly influencing consumers’ choices . Consides et al.  obtainedp > 0.05) between the control and A23%, but A35% showed a significant (p < 0.05) reduction in cohesiveness in comparison with the two other samples.Concerning cohesiveness, there were no significant differences  changes when replacing buckwheat flour by 23% and 35% of acorn flour. Buckwheat has about 12% of protein [With regard to the protein of the analysed breads, there were no significant  between the control and 23% acorn flour.As far as ash is concerned, there was a significant decrease in 23% acorn flour incorporation, when comparing with the control bread, but the higher level (35%) of acorn flour in the bread did not show significant differences (p > 0.05) variations were revealed between the control and the breads produced, with both acorn flour levels of incorporation. Korus et al. [Regarding carbohydrates and moisture, no significant  with both acorn flour incorporation levels, compared to the control bread. This was predictable, owing to acorn flour’s higher content of lipids, which was about 8.5%, while buckwheat has around 3.4% according to the manufacturer’s information, which are similar to authors that have studied, respectively, these two ingredients [Concerning the lipid content in our GFB, it increased significantly  in the two levels of acorn flour incorporation, in comparison with control bread. Significant differences between them were found. In contrary, the total polyunsaturated FA decreased significantly (p < 0.05), with the reduction of buckwheat flour in breads A23% and A35%. Maggio and Orecchio [p > 0.05) between control, A23%, and A35% bread samples.As for fatty acids (FA) profile, both total saturated FA and monounsaturated FA have increased significantly  between the three developed breads. Potassium (K) levels increased significantly (p < 0.05) from the control to both acorn flour contents. Magnesium (Mg) showed the opposite behaviour, decreasing significantly (p < 0.05) from the control to A35%. Nevertheless, the bread with 23% acorn flour incorporation serves 30 mg Mg/100 g of fresh bread. For instance, according to European Food Safety Association (EFSA) [p < 0.05) reduction from the control bread to A35%. Zinc (Zn) did not present differences (p > 0.05) between the three types of bread. Finally, manganese (Mn) had a significant (p < 0.05) climb when acorn flour was added to bread formulation.The mineral composition of breads is reported in n (EFSA) , adult fWith regard to minerals, it is also important to explain its reduction from the buckwheat flour control bread to both acorn flour breads. Bilgiçli and İbanoğlu  studied These results go towards our objective of improving the GFB nutritional profile with acorn flour, particularly because GFB is usually described as having poorer nutritional characteristics when compared to its gluten-containing counterparts.From a nutritional perspective, Polimac et al. , concludThe results for bioactive compounds and antioxidant activity of the GFB produced in the present study are summarised in Ortho-diphenols content (ODC), A35% presented significantly (p < 0.05) higher values than A23%. These results show the influence of a progressive incorporation of acorn flour in the bread’s formulation. Flavonoids content (FlC) also increased with acorn flour addition, but only with a 35% level of acorn flour was FlC significantly different (p < 0.05) from the control bread. Concerning antioxidant capacity, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and diphenyl-1-picrylhidrazyl radical (DPPH) did not show significant differences between the bread with 23%, and 35% of acorn flour incorporation. ferric reducing antioxidant power (FRAP) assay methodology results illustrated a significantly higher value in the 35% level of acorn flour when comparing with 23% acorn flour, as well as with control bread.According to Vinha et al. , likewisDifferent studies about antioxidants and polyphenols in buckwheat GF bread and wheat-containing bread have shown the highest values regarding this pseudocereal when compared with amaranth and quinoa, or even with wheat bread ,58. DespAnother important aspect to refer to is the influence of baking conditions, time, and temperature on antioxidant activity and bioactive compounds, since changes may occur during the breadmaking process ,59. AlthIn comparison with other research studies, Parsaei et al.  studied Another study carried out by Korus et al. , concernHowever, a comparison could not be performed between our study and all the other ones mentioned in this article because the extractions conditions, analysis methods, and unit measurements were different. Torres et al. , in a reConcerning ODC and FlC, as far as we know, no study on GFB with acorn flour has been performed, neither regarding radical scavenging capacity. Acorn flour addition enriched significantly GFB regarding TPC, ODC and antioxidant capacity.Once the Reverse Phase–High-Performance Liquid Chromatography–Diode Array Detector (RP-HPLC-DAD) provided more specific information about the phenolic profile, the identification and quantification of GFB phenolics was performed; the results are presented in The obtained results are in accordance with the performed colorimetric methods, in which the acorn flour breads presented also a higher content of phenolic compounds when compared with control bread.p < 0.05) between them. In fact, the level of both compounds increased with the incorporation of acorn flour in the bread, and also when the level of this flour was higher, oscillating approximately between 0.48 and 5.29 mg g−1 for gallic acid and between 0.77 and 10.13 mg g−1 for syringic acid.The analysis revealed the presence of gallic acid and syringic acid in all bread samples, being significantly different (p < 0.05) between the samples A23% and A35%, except for rutin, where no significant differences between both acorn bread samples were found. For all compounds, the bread with 35% of acorn flour incorporation showed a higher concentration of phenolic compounds. Acorn flour is a major contribution for the increase of phenolic content in the breads A23% and A35% when compared with the control bread. Cantos et al. [Catechin, bezoinc acid unidentified, ellagic acid, rutin, and both unidentified flavonols were only present in acorn flour breads. The concentration of each compound showed significant differences , as a gelling agent, hydroxipropyl methylcellulose, HPMC , sugar, sunflower oil, salt, and water. All ingredients were provided free of charge from their principal suppliers, except for sugar, yeast, salt, and oil which were obtained locally.Ingredients used for the bread formulation were buckwheat flour  , rice flour  , potato starch  , acorn flour from Holm oak  , dried yeast  with the following results: buckwheat flour, 12.35%; rice flour, 11.08%; potato starch, 18.10%; and acorn flour, 8.03%. The water absorption in each flour blend was adjusted in a micro-doughLAB 2800  to achieve an optimum dough consistency, using a procedure previously optimised [Starting with a previously studied GF bread recipe  based onptimised . The forControl (C): 46.0 g buckwheat flour, 31.0 g rice flour, 23.0 g potato starch, 65.0% of water absorption (14% moisture basis) and the rest of the ingredients, per 100 g of flours, were 5.5 g sunflower oil, 4.6 g HPMC, 2.8 g dried yeast, 2.8 g sugar, 1.8 g salt. The water content was chosen by testing different water hydrations to produce a control bread with good quality, based on bread volume and crumb firmness (preliminary assays not presented). The peak value of torque of the optimised control formulation was used as a reference (93 mN.m).Acorn A23%: 23.0 g buckwheat flour, 31.0 g rice flour, 23.0 g potato starch, 23.0 g acorn flour, 63.0% of water absorption (14% moisture basis), and the rest of the ingredients, per 100 g of flours, were 5.5 g sunflower oil, 4.6 g HPMC, 2.8 g dried yeast, 2.8 g sugar, 1.8 g salt. Water absorption was adjusted using the torque reference value ±4%.Acorn A35%: 12.0 g buckwheat flour, 31.0 rice flour, 23.0 g potato starch, 35.0 g acorn flour, 62.0% of water absorption (14% moisture basis), and the rest of the ingredients, per 100 g of flours, were 5.5 g sunflower oil, 4.6 g HPMC, 2.8 g dried yeast, 2.8 g sugar, 1.8 g salt. Water absorption was adjusted using the torque reference value ±4%.Ingredients were mixed in a Thermo processor equipment , firstly to activate the yeast by adding water, yeast, and sugar for 2 min at 27 °C, at velocity 1. Then, the rest of the ingredients were added and mixed for 10 min in a dough mixing program (wheat ear symbol). The dough (400 g) was poured into an aluminium baking tin, previously greased with oil, and placed in a fermentation chamber Arianna XLT133  for 50 min at 37 °C. After fermenting, the dough was baked in an electric oven Johnson A60  for 50 min at 180 °C. Finally, the bread was removed from the tin and left to cool to room temperature for 2 h before analysis.Three loaves of bread of each formulation were prepared, and all the analyses were performed in triplicate: three measurements were taken from each slice individually from three independent slices from each loaf. The mean values and standard deviation obtained from each determination were recorded.The volume of the bread was measured using the rapeseed displacement method. The same weight of ingredients, in relation to 300 g of flours, was used to prepare all the breads .bbW is the weight of the dough before baking, and abW is the weight after baking and cooling.The bread bake loss was determined using the following expression :(2)Bake The crust and crumb colours were determined using a colorimeter Croma-Meter CR 400 , through the CIE L*a*b* system  using the following parameters: L*—lightness variable ; a**—intensity of green (−60 < a* < 0) or red (0 < a* < +60); and b**—intensity of blue (−60 < b* < 0) or yellow (0 < b* < +60).w) was measured using a HygroPalm-HP23 . Samples were prepared blending the crumbs from the interior of the bread slices.Water activity  was performed 2 and 24 h after baking, with a texturometer TA.XT.plus . First, 20 mm bread slices were used to perform the double puncture test (“two-bite test”). Test conditions were the following: 1 mm/s of crosshead speed, 8 mm of penetration distance, and 5 s of waiting time, with a 25 mm diameter acrylic cylindrical probe. Firmness and cohesiveness were considered the main representative texture parameters obtained from TPA to characterise the bread, as has been previously used by other authors .All nutritional analyses were carried out in triplicate and were performed after drying and grinding the bread samples.Total protein content was determined using the Kjeldhal method based on ISO 20483:2006 . The totTotal ash content was also determined gravimetrically, incinerating samples at 550 °C in a muffle furnace .Bread moisture content was measured gravimetrically using an automatic moisture analyser PMB 202  until a constant weight at 130 °C was reached.Total fat content was determined by the technique used for cereals and derived products, using the Portuguese standard method NP 4168 (1991) . This meFatty acids profile was determined using the method ISO 5509:2000  with somTM iCap Series 7000 . A sample of 0.25 g was digested with a mixture of nitric acid and hydrochloric acid (ratio 1:3) at 105 °C. The mixture was cooled down and when room temperature was reached, it was filtered and diluted to 50 mL with distilled water. The extracts were subsequently analysed for Na, K, Ca, Mg, P, S, Zn, and Mn.Mineral content was determined using acid digestion followed by absorbance reading in an Inductively Coupled Plasma Optical-Emission Spectrometry (ICP-OES) Thermo ScientificThe bioactive compound content was analysed using spectrophotometric methodologies as described by Gouvinhas et al.  and MachRadical scavenging activity was assessed using the methodologies of ABTS●+ and DPPH● performed as described by Teixeira-Guedes et al.  and DomíReagents manufacturers: sodium nitrate, aluminum chloride, and sodium hydroxide, all extra pure (>99%), saline water (0.9% NaCl), and methanol were acquired from Merck . Folin–Ciocalteu’s reagent, 3,4,5-trihydroxybenzoic acid , acetic acid, both extra pure (>99%), and sodium hydroxide were purchased from Panreac . Sodium molybdate (99.5%) was obtained from Chem-Lab . The compounds 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS+), 2,2-diphenyl-1-picrylhidrazyl radical (DPPH), potassium phosphate, catechin, potassium persulfate, sodium acetate, 2,4,6-Tripyridyl-s-Triazine (TPTZ iron reagent), acetic acid, hydrochloric acid, and iron(III) chloride were obtained from Sigma-Aldrich . Additionally, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from Fluka Chemika .All authentic standards of phenolic compounds used in the chromatographic analysis, including syringic acid (≥99%), rutin (quercetin-3-rutinoside) (≥94%), ellagic acid (≥95%), and luteolin (≥98%) were purchased from Chem-Lab . Gallic acid (>97.5%) and catechin (≥98%) were acquired from Sigma Aldrich .n = 3) for each sample.All analyses were performed using 96-well micro plates  and a microplate reader Infinite M200  and were evaluated in triplicate (v/v)). Samples were stirred for 30 min at room temperature and then centrifuged at 5000 rpm for 15 min at 4 °C to separate the supernatant. The final volume was made up to 5 mL with methanol/distilled water . The procedure was repeated three times.To extract the phenolic compounds from the bread samples, 40 mg of each bread already in the form of dried powder were prepared and added to 1.5 mL of extracting solvent  .Ortho-diphenols in bread were determined by adding 40 μL of sodium molibdate to 160 μL of samples properly diluted. Mixtures were vortexed and kept protected from light, at room temperature, for 15 min. Absorbance was measured at 375 nm, and the ortho-diphenols content was quantified using gallic acid as standard. The results were expressed in milligrams equivalent of gallic acid per gram of dry weight (mg GAE/g DW) [AE/g DW) .−1). After 5 min, 28 μL of aluminium chloride (100 g L−1) was added, and the mixture was left to react for 6 min. Then, 120 μL of sodium hydroxide (1 M) was added to the mixture, and before reading the absorbance at 510 nm, the microplate was shaken for 30 s. Flavonoid content was quantified using catechin as standard, and the results were expressed in milligrams equivalent of catechin per gram of dry weight (mg CE/g DW) [Flavonoids content in bread was measured as follows: 24 μL of sample was mixed with 28 μL of sodium nitrite (50 g LCE/g DW) .To evaluate ABTS radical inhibition, 12 μL of sample or standard was placed in the microplate followed by 188 μL of ABTS●+ working solution. The plate was allowed to rest in the dark for 30 min at room temperature, and the absorbance was read at 734 nm. The inhibition of ABTS●+ radicals was calculated using the following formula:The antioxidant activity of the extracts was determined by interpolation of the calibration curve for Trolox, and the results were expressed in mmol Trolox per gram of dry weight (mmol Trolox/ g DW) .DPPH antioxidant capacity assessment was carried out adding 10 μL of sample or Trolox standard and 190 μL of the DPPH to each well of the microplate. The mixture was incubated in the dark at room temperature for 30 min, and absorbance was measured at 520 nm. The inhibition of free radical DPPH● was calculated using the formula previously presented; meanwhile, the ABTS●+ DPPH● scavenging capacity of the samples was determined by interpolation of the calibration curve for Trolox, and the results were expressed in mmol Trolox per gram of dry weight (mmol Trolox/ g DW) .To measure ferric reducing antioxidant power (FRAP), 20 μL of sample was placed in each well of the microplate followed by adding 280 μL of FRAP working solution. After incubating the reaction for 30 min at 37 °C in the dark, absorbance was read at 593 nm. Trolox was used as standard, and the results were expressed in mmol Trolox per gram of dry weight (mmol Trolox/g DW) .2O/HCOOH , and solvent B, CH3CN/HCOOH , together with a non-polar stationary phase. A linear gradient scheme was used, with the following characteristics: : , , , , , , and . At 55 min, return to 5% of B to stabilise and prepare the column for the next sample. All of the analysis was performed at room temperature (25 °C) with a flow rate of 1.0 mL/min. The injection volume of the samples was 20 μL. All samples were injected in triplicate. The equipment consisted of an LC pump (SRVYR-LPUMP), an auto-sampler (SRVYR-AS), and a photodiode array detector (SRVYR-PDA5) in series  [The phenolic profile of bread samples was assessed by Reverse Phase–High-Performance Liquid Chromatography–Diode Array Detector (RP-HPLC-DAD), using a C18 column , using the method of Leal et al. . The metMA, USA) .−1 DW).All the compounds were identified at 280 nm with the standards of: gallic acid, catechin, syringic acid, ellagic acid, rutin, and luteolin. For the quantification of unidentified flavanols, the standard of rutin was used. The results were expressed in milligrams per gram of dry weight . Experimental data were compared using analysis of variance (one-way ANOVA), and Tukey’s test was used to evaluate mean differences at a confidence level of 95%, with significant differences considered to be This study highlights acorn characteristics and enhances its benefits. Acorn flour is a good source of bioactive compounds and antioxidants for the production of GFB. All of the bioactive compounds analysed presented higher levels with the incorporation of acorn flour in GFB, with the only exception of flavonoids. Further investigation on anti-nutrients is needed in order to analyse if eventually any negative effects of acorn flour can be found.With regard to sensorial characteristics, the addition of acorn flour positively influenced most of these parameters , making the bread darker and more appealing in appearance.Our study revealed that the 23% incorporation level of acorn flour was the most preferred in terms of sensory evaluation. Therefore, the use of acorn flour provides nutrients and bioactive compounds, allowing the enrichment of GFB, and at the same time revealing attractive sensory characteristics. Furthermore, it is possible to assert the value of acorn flour through its use in GFB.Interestingly, by rediscovering the value of such an important ingredient in the past, we are moving towards innovation. In addition, acorn is an underexploited resource that can be reintroduced in the food chain with several benefits.