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Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii , is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in Toxoplasma gondii has remained largely unknown without understanding its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. By contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which binds GIPLs, exhibits an enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that Δ pigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by Δ pigj early in the infection in wild-type mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with Δ pigj parasites indicate an advantage over wild-type strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcomes in the infected host.IMPORTANCEThe functional significance of sidechain modifications to the glycosylphosphatidylinositol (GPI) anchor in parasites has yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both Toxoplasmsa gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased host susceptibility to infection. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on disease outcomes. This indicates GPI sidechains are fundamental to host-parasite interactions., Competing Interests: The authors declare no conflict of interest.

Background: Asymmetric joint load is the main cause of development of ankle osteoarthritis (OA). Realignment surgery aims to transfer ankle joint load from the degenerative area toward the uninvolved area. Determination of the optimal shift is still challenging. When the degenerative area is correlated to the ankle joint mechanical axis establishing an optimal target angle for corrective surgery may become more feasible. The primary aim of our study was to investigate if the area of ankle joint activation on single-photon emission computed tomography and conventional computed tomography (SPECT/CT) imaging correlates with the mechanical ankle joint axis point (MAJAP)., Methods: In this cross-sectional study, patients 18 years or older with symptomatic asymmetric ankle OA and a hip-to-calcaneus long leg view with SPECT/CT of the affected ankle were eligible for inclusion. Primary outcome was MAJAP divided into 3 alignment categories (medial shift, neutral, lateral shift). SPECT/CT activation was determined in 8 different areas of the ankle joint. A Spearman rho correlation coefficient was calculated to investigate the relationship between the alignment categories and SPECT/CT activation in the 8 areas., Results: Forty-nine patients (mean age 58.8 [SD 10.0] years) with 52 ankles with moderate to severe asymmetric OA were included. A significantly (Spearman rho -0.379 [ P  = .006] and Spearman rho -0.279 [ P  = .045]) higher proportion of ankles with radioisotope uptake in the anteromedial ankle joint areas (zones 1 and 5) was seen in the medial shift category. A significantly (Spearman rho .312 ( P  = .025)) higher proportion of ankles with radioisotope uptake in the anterolateral ankle joint area (zone 8) was seen in the lateral shift category., Conclusion: We found in this patient group that the area of SPECT/CT uptake in asymmetric ankle OA was associated to MAJAP measured on hip-to-calcaneus weightbearing views, although the strength of the correlation is weak to moderate. Consequently, nonweightbearing metabolic SPECT/CT radiotracer uptake has the potential to help determine the area to unload in ankle joint-preserving alignment surgery., Competing Interests: Ethical ApprovalEthical approval for this study was obtained from the Martini Hospital (MEC number 2020-144). Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Disclosure forms for all authors are available online.

A dynamic proteome is required for cellular adaption to changing environments including levels of O 2 , and the SKP1/CULLIN-1/F-box protein/RBX1 (SCF) family of E3 ubiquitin ligases contributes importantly to proteasome-mediated degradation. We examine, in the apicomplexan parasite Toxoplasma gondii, the influence on the interactome of SKP1 by its novel glycan attached to a hydroxyproline generated by PHYa, the likely ortholog of the HIFα PHD2 oxygen-sensor of human host cells. Strikingly, the representation of several putative F-box proteins (FBPs) is substantially reduced in PHYaΔ parasites grown in fibroblasts. One, FBXO13, is a predicted lysyl hydroxylase related to the human JmjD6 oncogene except for its F-box domain. The abundance of FBXO13, epitope-tagged at its genetic locus, was reduced in PHYaΔ parasites thus explaining its diminished presence in the SKP1 interactome. A similar effect was observed for FBXO14, a cytoplasmic protein of unknown function that may have co-evolved with PHYa in apicomplexans. Similar findings in glycosylation-mutant cells, rescue by proteasomal inhibitors, and unchanged transcript levels, suggested the involvement of the SCF in their degradation. The effect was selective, because FBXO1 was not affected by loss of PHYa. These findings are physiologically significant because the effects were phenocopied in parasites reared at 0.5% O 2 . Modest impact on steady-state SKP1 modification levels suggests that effects are mediated during a lag phase in hydroxylation of nascent SKP1. The dependence of FBP abundance on O 2 -dependent SKP1 modification likely contributes to the reduced virulence of PHYaΔ parasites owing to impaired ability to sense O 2 as an environmental signal., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The face is the initial feature used to judge gender in public spaces; it is also a source of significant gender dysphoria. Surgical techniques are available for non-cisgender male patients who desire a more masculine face by augmenting certain features to change the bony framework of the skull. Augmentation using virtually designed patient-specific polyetheretherketone implants has now become a more widely applied method in maxillofacial surgery. When designing implants for augmentation, a three-dimensional (3D) reference or template is very useful. Hence, a 3D statistical shape model was developed of a male skull shape from information from a population of 40 male patients containing the mean shape and principal components of shape variation. By overlaying the template and the patient's 3D skull model, this method identified the regions of gender dimorphism in this case to be the orbital ridge, zygomatic regions, and frontal bossing area. Based on the 3D template overlay, polyetheretherketone augmentation implants were virtually designed in close consultation with a patient to augment the aforementioned regions. The virtual statistical shape modeling template offered an objective reference, and the possibility to fully involve the patient in the treatment planning., Competing Interests: The authors have no financial interest to declare in relation to the content of this article., (Copyright © 2024 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Like most eukaryotes, the pre-metazoan social amoeba Dictyostelium depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In Dictyostelium , starvation induces a transition from unicellular feeding to a multicellular slug that responds to external signals to culminate into a fruiting body containing terminally differentiated stalk and spore cells. These transitions are subject to regulation by F-box proteins and O 2 -dependent posttranslational modifications of Skp1. Here we examine in greater depth the essential role of FbxwD and Vwa1, an intracellular vault protein inter-alpha-trypsin (VIT) and von Willebrand factor-A (vWFA) domain containing protein that was found in the FbxwD interactome by co-immunoprecipitation. Reciprocal co-IPs using gene-tagged strains confirmed the interaction and similar changes in protein levels during multicellular development suggested co-functioning. FbxwD overexpression and proteasome inhibitors did not affect Vwa1 levels suggesting a non-substrate relationship. Forced FbxwD overexpression in slug tip cells where it is normally enriched interfered with terminal cell differentiation by a mechanism that depended on its F-box and RING domains, and on Vwa1 expression itself. Whereas vwa1 -disruption alone did not affect development, overexpression of either of its three conserved domains arrested development but the effect depended on Vwa1 expression. Based on structure predictions, we propose that the Vwa1 domains exert their negative effect by artificially activating Vwa1 from an autoinhibited state, which in turn imbalances its synergistic function with FbxwD. Autoinhibition or homodimerization might be relevant to the poorly understood tumor suppressor role of the evolutionarily related VWA5A/BCSC-1 in humans., Competing Interests: 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., (Copyright © 2023 Boland, Gas-Pascual, van der Wel, Kim and West.)

Knowledge about anatomical shape variations in the pelvis is mandatory for selection, fitting, positioning, and fixation in pelvic surgery. The current knowledge on pelvic shape variation mostly relies on point-to-point measurements on 2D X-ray images and computed tomography (CT) slices. Three-dimensional region-specific assessments of pelvic morphology are scarce. Our aim was to develop a statistical shape model of the hemipelvis to assess anatomical shape variations in the hemipelvis. CT scans of 200 patients (100 male and 100 female) were used to obtain segmentations. An iterative closest point algorithm was performed to register these 3D segmentations, so a principal component analysis (PCA) could be performed, and a statistical shape model (SSM) of the hemipelvis was developed. The first 15 principal components (PCs) described 90% of the total shape variation, and the reconstruction ability of this SSM resulted in a root mean square error of 1.58 (95% CI: 1.53-1.63) mm. In summary, an SSM of the hemipelvis was developed, which describes the shape variations in a Caucasian population and is able to reconstruct an aberrant hemipelvis. Principal component analyses demonstrated that, in a general population, anatomical shape variations were mostly related to differences in the size of the pelvis (e.g., PC1 describes 68% of the total shape variation, which is attributed to size). Differences between the male and female pelvis were most pronounced in the iliac wing and pubic rami regions. These regions are often subject to injuries. Future clinical applications of our newly developed SSM may be relevant for SSM-based semi-automatic virtual reconstruction of a fractured hemipelvis as part of preoperative planning. Lastly, for companies, using our SSM might be interesting in order to assess which sizes of pelvic implants should be produced to provide proper-fitting implants for most of the population.

The aim of this study was to investigate the value of 3D Statistical Shape Modelling for orthognathic surgery planning. The goal was to objectify shape variations in the orthognathic population and differences between male and female patients by means of a statistical shape modelling method. Pre-operative CBCT scans of patients for whom 3D Virtual Surgical Plans (3D VSP) were developed at the University Medical Center Groningen between 2019 and 2020 were included. Automatic segmentation algorithms were used to create 3D models of the mandibles, and the statistical shape model was built through principal component analysis. Unpaired t -tests were performed to compare the principal components of the male and female models. A total of 194 patients (130 females and 64 males) were included. The mandibular shape could be visually described by the first five principal components: (1) The height of the mandibular ramus and condyles, (2) the variation in the gonial angle of the mandible, (3) the width of the ramus and the anterior/posterior projection of the chin, (4) the lateral projection of the mandible's angle, and (5) the lateral slope of the ramus and the inter-condylar distance. The statistical test showed significant differences between male and female mandibular shapes in 10 principal components. This study demonstrates the feasibility of using statistical shape modelling to inform physicians about mandible shape variations and relevant differences between male and female mandibles. The information obtained from this study could be used to quantify masculine and feminine mandibular shape aspects and to improve surgical planning for mandibular shape manipulations.

O-GlcNAcylation is a prominent modification of nuclear and cytoplasmic proteins in animals and plants and is mediated by a single O-GlcNAc transferase (OGT). Spindly (Spy), a paralog of OGT first discovered in higher plants, has an ortholog in the apicomplexan parasite Toxoplasma gondii, and both enzymes are now recognized as O-fucosyltransferases (OFTs). Here we investigate the evolution of spy-like genes and experimentally confirm OFT activity in the social amoeba Dictyostelium-a protist that is more related to fungi and metazoa. Immunofluorescence probing with the fucose-specific Aleuria aurantia lectin (AAL) and biochemical cell fractionation combined with western blotting suggested the occurrence of nucleocytoplasmic fucosylation. The absence of reactivity in mutants deleted in spy or gmd (unable to synthesize GDP-Fuc) suggested monofucosylation mediated by Spy. Genetic ablation of the modE locus, previously predicted to encode a GDP-fucose transporter, confirmed its necessity for fucosylation in the secretory pathway but not for the nucleocytoplasmic proteins. Affinity capture of these proteins combined with mass spectrometry confirmed monofucosylation of Ser and Thr residues of several known nucleocytoplasmic proteins. As in Toxoplasma, the Spy OFT was required for optimal proliferation of Dictyostelium under laboratory conditions. These findings support a new phylogenetic analysis of OGT and OFT evolution that indicates their occurrence in the last eukaryotic common ancestor but mostly complementary presence in its eukaryotic descendants with the notable exception that both occur in red algae and plants. Their generally exclusive expression, high degree of conservation, and shared monoglycosylation targets suggest overlapping roles in physiological regulation., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Detection of lymph node metastases in cervical cancer patients is important for guiding treatment decisions, however accuracies of current detection methods are limited. We evaluated associations of abnormal glycosylation, represented by Tn and STn antigens on mucin (MUC) proteins, in primary tumor specimens with lymph node metastasis or recurrence of cervical cancer patients., Methods: Surgical specimens were prospectively collected from 139 patients with locally-advanced cervical cancer undergoing lymphadenectomy enrolled in a nation-wide clinical trial (NCT00460356). Of these patients, 133 had primary cervix tumor, 67 had pelvic lymph node (PLN) and 28 had para-aortic lymph node (PALN) specimens. Fixed tissue serial sections were immunohistochemically stained for Tn, STn, MUC1 or MUC4. Neuraminidase was used to validate Tn versus STn antibody specificity. Stain scores were compared with clinical characteristics., Results: Primary tumor STn expression above the median was associated with negative PLN status (p-value: 0.0387; odds ratio 0.439, 95% CI: 0.206 to 0.935). PLN had higher STn compared to primary tumor, while primary tumor had higher MUC1 compared to PALN, and MUC4 compared to PALN or PLN (p = 0.017, p = 0.011, p = 0.016 and p < 0.001, respectively). Tn and STn expression correlated in primary tumor, PALN, and PLN, Tn and MUC1 expression correlated in primary tumors only (Spearman correlation coefficient [r] = 0.301, r = 0.686, r = 0.603 and r = 0.249, respectively)., Conclusions: STn antigen expression in primary cervical tumors is a candidate biomarker for guiding treatment decisions and for mechanistic involvement in PLN metastases., Competing Interests: Declaration of Competing Interest Dr. Wei Deng, Dr. Christopher West, Dr. Rajani Rai, Dr. Rachel Conrad, Mrs. Hanke van der Wel, Dr. Sanam Husain, Dr. Mae Zakhour, Dr. Amanda Jackson and Dr. Doris Benbrook have no conflicts of interest to disclose. Dr. Michael Gold reports receiving honoraria from ASCCP as well as serving as a past member of the Board as well as serving as Secretary, Treasurer, Vice-President, and President for ASCCP. Dr. Moore reports personal fees and other from Astra Zeneca, grants, personal fees and other from Genentech/Roche, grants, personal fees and other from Immunogen, grants, personal fees and other from GSK/Tesaro, other from Pfizer, personal fees from Aravive, personal fees from VBL Therapeutics, personal fees and other from Onco Med, grants and other from Lilly, personal fees from Eisai, personal fees from Vavotar, personal fees from Abbvie, personal fees from Tarveda, personal fees from Myriad, personal fees from Rubius, personal fees from Elevar, personal fees from Merck, personal fees from Mersana, personal fees from Sorrento, personal fees from OncXerna, personal fees from Alkemeres, personal fees from blueprint pharmaceuticals, personal fees from Mereo, personal fees from IMab, outside the submitted work; and serves as the Associate Director for GOG Partners and a GOG Foundation Board of Directors member. Dr. Nick Spirtos would like to disclose receiving research funding to his Institution from AbbVie, AstraZeneca, Genentech/Roche, Clovis Oncology, and Seattle Genetics. With regard to Patents, Royalties, and Other Intellectual Property, Dr. Spirtos wishes to disclose Application No. PCT/US 2019/19465 Cannabis based therapeutic and method of use Application No. Title Country Status Filed Date Application No. Patent Ref. No.199236–701,611/EP Title: CANNABIS BASED THERAPEUTIC AND METHOD OF USE Country: European Patent Status: Published 2/25/2019 19,710,540.6 Patent Ref. No.199236–701,691/PCT-BR Title: CANNABIS BASED THERAPEUTIC AND METHOD OF USE Country Brazil Status: Application 2/25/2019 1,120,200,170,232 Patent Ref. No.199236–701,831/PCT-US Title: CANNABIS BASED THERAPEUTIC AND METHOD OF USE Country United States of America Status: Published 2/25/2019 16/971,781 Patent Ref. No.199236–701,891/HK Title: CANNABIS BASED THERAPEUTIC AND METHOD OF USE Country Hong Kong Status: Published 6/25/2021 62,021,033,676.9 Patent Ref. No. Title Country Status Filed Date Application No. Patent Ref. No.199237–701,601/PCT Title: COMPOSITIONS COMPRISING CANNABIDIOL AND FLAVANONES Country: Patent Cooperation Treaty Status: Application 7/1/2021 PCT/US21/40115 Patent Ref. No.199237–701,691/BR Title: COMPOSITIONS COMPRISING CANNABIDIOL AND FLAVANONES Country: Brazil Status: Application 11/19/2020 1,020,200,236,644 Patent Ref. No.199237–7,019,761/UY Title: COMPOSITIONS COMPRISING AND FLAVANONES Country: Uruguay Status: Application 11/20/2020 38,965. Dr. Cara Mathews reports receiving funding from the NCI to her institution. Dr. Mathews also reports funding from Syros, Decophera, Astellas Pharma, Tesaro/GSK, Seattle Genetics, Regeneron, Moderna, Laekna Therapeutics, outside of the submitted work. In addition, Dr. Mathews reports receiving support from GSK and Seattle Genetics to attend investigator meetings and served on an Advisory Board for IMAB Biopharma., (Copyright © 2023 Elsevier Inc. All rights reserved.)

E3-SCF (Skp1/cullin-1/F-box protein) polyubiquitin ligases activate the proteasomal degradation of over a thousand proteins, but the evolutionary diversification of the F-box protein (FBP) family of substrate receptor subunits has challenged their elucidation in protists. Here, we expand the FBP candidate list in the social amoeba Dictyostelium and show that the Skp1 interactome is highly remodeled as cells transition from growth to multicellular development. Importantly, a subset of candidate FBPs was less represented when the posttranslational hydroxylation and glycosylation of Skp1 was abrogated by deletion of the O 2 -sensing Skp1 prolyl hydroxylase PhyA. A role for this Skp1 modification for SCF activity was indicated by partial rescue of development, which normally depends on high O 2 and PhyA, of phyA-KO cells by proteasomal inhibitors. Further examination of two FBPs, FbxwD and the Jumonji C protein JcdI, suggested that Skp1 was substituted by other factors in phyA-KO cells. Although a double-KO of jcdI and its paralog jcdH did not affect development, overexpression of JcdI increased its sensitivity to O 2 . JcdI, a nonheme dioxygenase shown to have physiological O 2 dependence, is conserved across protists with its F-box and other domains, and is related to the human oncogene JmjD6. Sensitization of JcdI-overexpression cells to O 2 depended on its dioxygenase activity and other domains, but not its F-box, which may however be the mediator of its reduced levels in WT relative to Skp1 modification mutant cells. The findings suggest that activation of JcdI by O 2 is tempered by homeostatic downregulation via PhyA and association with Skp1., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Success of valgus-type supramalleolar osteotomy (SMOT) depends on adequate correction of malalignment, which can be hard to achieve with current 2-dimensional (2D) planning and operative techniques. A personalized digital 3-dimensional (3D) workflow to virtually plan and perform a 2-step 3D-guided medial opening (MO) SMOT has the potential to improve precision of correction., Methods: Computed tomography (CT)-based Proplan medical 3D models were made to virtually plan the desired MO SMOT, and exported to 3-Matic medical to develop patient-specific 2-step cutting and wedge guides. Workflow accuracy was tested in this limited clinical pilot study (3 patients) by comparing the virtual planned position of the osteotomized distal tibial fragment with the 1-year post-MO SMOT configuration. Two millimeters or less translation deviation in every plane was defined as accurate., Results: Primary outcome analysis of the osteotomized distal tibial fragment deviation showed a median translation in all planes of 0.7 (range 0-8.2) mm (interquartile range 1.55) with an excellent interrater reliability of the measurements (intraclass correlation coefficient 0.998). There was a strong reduction in ankle pain as reflected by an increase of the AOFAS-AH score and decrease of NRS pain score with an unrestricted hindfoot motion 1 year after surgery., Conclusion: 3D virtually planned bone cutting and wedge guides is a promising approach associated with minimal postoperative deviation from the desired correction in medial opening supramalleolar osteotomy.

Medical imaging techniques, such as (cone beam) computed tomography and magnetic resonance imaging, have proven to be a valuable component for oral and maxillofacial surgery (OMFS). Accurate segmentation of the mandible from head and neck (H&N) scans is an important step in order to build a personalized 3D digital mandible model for 3D printing and treatment planning of OMFS. Segmented mandible structures are used to effectively visualize the mandible volumes and to evaluate particular mandible properties quantitatively. However, mandible segmentation is always challenging for both clinicians and researchers, due to complex structures and higher attenuation materials, such as teeth (filling) or metal implants that easily lead to high noise and strong artifacts during scanning. Moreover, the size and shape of the mandible vary to a large extent between individuals. Therefore, mandible segmentation is a tedious and time-consuming task and requires adequate training to be performed properly. With the advancement of computer vision approaches, researchers have developed several algorithms to automatically segment the mandible during the last two decades. The objective of this review was to present the available fully (semi)automatic segmentation methods of the mandible published in different scientific articles. This review provides a vivid description of the scientific advancements to clinicians and researchers in this field to help develop novel automatic methods for clinical applications.

Accurate segmentation of the mandible from cone-beam computed tomography (CBCT) scans is an important step for building a personalized 3D digital mandible model for maxillofacial surgery and orthodontic treatment planning because of the low radiation dose and short scanning duration. CBCT images, however, exhibit lower contrast and higher levels of noise and artifacts due to extremely low radiation in comparison with the conventional computed tomography (CT), which makes automatic mandible segmentation from CBCT data challenging. In this work, we propose a novel coarse-to-fine segmentation framework based on 3D convolutional neural network and recurrent SegUnet for mandible segmentation in CBCT scans. Specifically, the mandible segmentation is decomposed into two stages: localization of the mandible-like region by rough segmentation and further accurate segmentation of the mandible details. The method was evaluated using a dental CBCT dataset. In addition, we evaluated the proposed method and compared it with state-of-the-art methods in two CT datasets. The experiments indicate that the proposed algorithm can provide more accurate and robust segmentation results for different imaging techniques in comparison with the state-of-the-art models with respect to these three datasets.

Accurate mandible segmentation is significant in the field of maxillofacial surgery to guide clinical diagnosis and treatment and develop appropriate surgical plans. In particular, cone-beam computed tomography (CBCT) images with metal parts, such as those used in oral and maxillofacial surgery (OMFS), often have susceptibilities when metal artifacts are present such as weak and blurred boundaries caused by a high-attenuation material and a low radiation dose in image acquisition. To overcome this problem, this paper proposes a novel deep learning-based approach (SASeg) for automated mandible segmentation that perceives overall mandible anatomical knowledge. SASeg utilizes a prior shape feature extractor (PSFE) module based on a mean mandible shape, and recurrent connections maintain the continuity structure of the mandible. The effectiveness of the proposed network is substantiated on a dental CBCT dataset from orthodontic treatment containing 59 patients. The experiments show that the proposed SASeg can be easily used to improve the prediction accuracy in a dental CBCT dataset corrupted by metal artifacts. In addition, the experimental results on the PDDCA dataset demonstrate that, compared with the state-of-the-art mandible segmentation models, our proposed SASeg can achieve better segmentation performance.

Once considered unusual, nucleocytoplasmic glycosylation is now recognized as a conserved feature of eukaryotes. While in animals, O-GlcNAc transferase (OGT) modifies thousands of intracellular proteins, the human pathogen Toxoplasma gondii transfers a different sugar, fucose, to proteins involved in transcription, mRNA processing, and signaling. Knockout experiments showed that TgSPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclear O-fucosylation. Here we verify that TgSPY is the nucleocytoplasmic O-fucosyltransferase (OFT) by 1) complementation with TgSPY-MYC 3 , 2) its functional dependence on amino acids critical for OGT activity, and 3) its ability to O-fucosylate itself and a model substrate and to specifically hydrolyze GDP-Fuc. While many of the endogenous proteins modified by O-Fuc are important for tachyzoite fitness, O-fucosylation by TgSPY is not essential. Growth of Δspy tachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically expressed proteins normally modified with O-fucose. Intact TgSPY-MYC 3 localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δspy tachyzoites of a luciferase-expressing type II strain exhibited infection kinetics in mice similar to wild-type but increased persistence in the chronic brain phase, potentially due to an imbalance of regulatory protein levels. The modest changes in parasite fitness in vitro and in mice, despite profound effects on reporter protein accumulation, and the characteristic punctate localization of O-fucosylated proteins suggest that TgSPY controls the levels of proteins to be held in reserve for response to novel stresses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O 2 regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium , influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma , the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1 Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of nonpolar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, which emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Mandalasi et al.)

Skp1 is an adapter that links F-box proteins to cullin-1 in the Skp1/cullin-1/F-box (SCF) protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent protein degradation. Skp1 from the amoebozoan Dictyostelium forms a stable homodimer in vitro with a K d of 2.5 μM as determined by sedimentation velocity studies yet is monomeric in crystal complexes with F-box proteins. To investigate the molecular basis for the difference, we determined the solution NMR structure of a doubly truncated Skp1 homodimer (Skp1ΔΔ). The solution structure of the Skp1ΔΔ dimer reveals a 2-fold symmetry with an interface that buries ∼750 Å 2 of predominantly hydrophobic surface. The dimer interface overlaps with subsite 1 of the F-box interaction area, explaining why only the Skp1 monomer binds F-box proteins (FBPs). To confirm the model, Rosetta was used to predict amino acid substitutions that might disrupt the dimer interface, and the F97E substitution was chosen to potentially minimize interference with F-box interactions. A nearly full-length version of Skp1 with this substitution (Skp1ΔF97E) behaved as a stable monomer at concentrations of ≤500 μM and actively bound a model FBP, mammalian Fbs1, which suggests that the dimeric state is not required for Skp1 to carry out a basic biochemical function. Finally, Skp1ΔF97E is expected to serve as a monomer model for high-resolution NMR studies previously hindered by dimerization.

Skp1 is hydroxylated by an O2-dependent prolyl hydroxylase (PhyA) that contributes to O2-sensing in the social amoeba Dictyostelium and the mammalian pathogen Toxoplasma gondii. HO-Skp1 is subject to glycosylation and the resulting pentasaccharide affects Skp1 conformation in a way that influences association of Skp1 with F-box proteins, and potentially the assembly of E3(SCF) ubiquitin ligase complexes that mediate the polyubiquitination of target proteins that are degraded in the 26S-proteasome. To investigate the conservation and specificity of these modifications, we analyzed proteins from the oomycete Pythium ultimum, an important crop plant pathogen. Putative coding sequences for Pythium's predicted PhyA and first glycosyltransferase in the predicted five-enzyme pathway, a GlcNAc-transferase (Gnt1), predict a bifunctional enzyme (Phgt) that, when expressed in Dictyostelium, rescued a knockout of phyA but not gnt1. Though recombinant Phgt was also unable to glycosylate Dictyostelium HO-Skp1, it could hydrolyze UDP-GlcNAc and modify a synthetic hydroxypeptide from Dictyostelium Skp1. Pythium encodes two highly similar Skp1 isoforms, but only Skp1A was efficiently hydroxylated and glycosylated in vitro. While kinetic analysis revealed no evidence for processive processing of Skp1, the physical linkage of the two activities implies dedication to Skp1 in vivo. These findings indicate a widespread occurrence of the Skp1 modification pathway across protist phylogeny, suggest that both Gnt1 and PhyA are specific for Skp1 and indicate that the second Skp1 provides a bypass mechanism for O2-regulation in Pythium and other protists that conserve this gene., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As the protozoan parasite Toxoplasma gondii disseminates through its host, it responds to environmental changes by altering its gene expression, metabolism, and other processes. Oxygen is one variable environmental factor, and properly adapting to changes in oxygen levels is critical to prevent the accumulation of reactive oxygen species and other cytotoxic factors. Thus, oxygen-sensing proteins are important, and among these, 2-oxoglutarate-dependent prolyl hydroxylases are highly conserved throughout evolution. Toxoplasma expresses two such enzymes, TgPHYa, which regulates the SCF-ubiquitin ligase complex, and TgPHYb. To characterize TgPHYb, we created a Toxoplasma strain that conditionally expresses TgPHYb and report that TgPHYb is required for optimal parasite growth under normal growth conditions. However, exposing TgPHYb-depleted parasites to extracellular stress leads to severe decreases in parasite invasion, which is likely due to decreased abundance of parasite adhesins. Adhesin protein abundance is reduced in TgPHYb-depleted parasites as a result of inactivation of the protein synthesis elongation factor eEF2 that is accompanied by decreased rates of translational elongation. In contrast to most other oxygen-sensing proteins that mediate cellular responses to low O 2 , TgPHYb is specifically required for parasite growth and protein synthesis at high, but not low, O 2 tensions as well as resistance to reactive oxygen species. In vivo , reduced TgPHYb expression leads to lower parasite burdens in oxygen-rich tissues. Taken together, these data identify TgPHYb as a sensor of high O 2 levels, in contrast to TgPHYa, which supports the parasite at low O 2 IMPORTANCE Because oxygen plays a key role in the growth of many organisms, cells must know how much oxygen is available. O 2 -sensing proteins are therefore critical cellular factors, and prolyl hydroxylases are the best-studied type of O 2 -sensing proteins. In general, prolyl hydroxylases trigger cellular responses to decreased oxygen availability. But, how does a cell react to high levels of oxygen? Using the protozoan parasite Toxoplasma gondii , we discovered a prolyl hydroxylase that allows the parasite to grow at elevated oxygen levels and does so by regulating protein synthesis. Loss of this enzyme also reduces parasite burden in oxygen-rich tissues, indicating that sensing both high and low levels of oxygen impacts the growth and physiology of Toxoplasma ., (Copyright © 2019 Florimond et al.)

Skp1 is a subunit of the SCF ( S kp1/ C ullin 1/ F -box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from Toxoplasma gondii is hydroxylated by an O 2 -dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba Dictyostelium , where it regulates SCF assembly and O 2 -dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates the addition of the final two sugars in Dictyostelium , generating Galα1, 3Galα1,3Fucα1,2Galβ1,3GlcNAcα1-. Here, we found that Toxoplasma utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for the addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [ 3 H]glucose from UDP-[ 3 H]glucose to the trisaccharide form of Skp1 in a glt1 -dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1 and labeled only Skp1 in glt1 Δ extracts, suggesting specificity for Skp1. glt1 -knock-out parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

All-trans-retinoic acid may be an important molecular signal in the postnatal control of eye size. The goal of this study was to identify retinoic acid-binding proteins secreted by the choroid and sclera during visually guided ocular growth. Following photoaffinity labeling with all-trans-[11,12-(3)H]retinoic acid, the most abundant labeled protein detected in the conditioned medium of choroid or sclera had an apparent Mr of 27,000 Da. Following purification and mass spectrometry, the Mr 27,000 band was identified as apolipoprotein A-I. Affinity capture of the radioactive Mr 27,000 band by anti-chick apolipoprotein A-I antibodies confirmed its identity as apolipoprotein A-I. Photoaffinity labeling and fluorescence quenching experiments demonstrated that binding of retinoic acid to apolipoprotein A-I is 1) concentration-dependent, 2) selective for all-trans-retinoic acid, and 3) requires the presence of apolipoprotein A-I-associated lipids for retinoid binding. Expression of apolipoprotein A-I mRNA and protein synthesis were markedly up-regulated in choroids of chick eyes during the recovery from induced myopia, and apolipoprotein A-I mRNA was significantly increased in choroids following retinoic acid treatment. Together, these data suggest that apolipoprotein A-I may participate in a regulatory feedback mechanism with retinoic acid to control the action of retinoic acid on ocular targets during postnatal ocular growth., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Multiple species of cellular slime mold (CSM) amoebae share overlapping subterranean environments near the soil surface. Despite similar life-styles, individual species form independent starvation-induced fruiting bodies whose spores can renew the life cycle. N-glycans associated with the cell surface glycocalyx have been predicted to contribute to interspecific avoidance, resistance to pathogens, and prey preference. N-glycans from five CSM species that diverged 300-600 million years ago and whose genomes have been sequenced were fractionated into neutral and acidic pools and profiled by MALDI-TOF-MS. Glycan structure models were refined using linkage specific antibodies, exoglycosidase digestions, MALDI-MS/MS, and chromatographic studies. Amoebae of the type species Dictyostelium discoideum express modestly trimmed high mannose N-glycans variably modified with core α3-linked Fuc and peripherally decorated with 0-2 residues each of β-GlcNAc, Fuc, methylphosphate and/or sulfate, as reported previously. Comparative analyses of D. purpureum, D. fasciculatum, Polysphondylium pallidum, and Actyostelium subglobosum revealed that each displays a distinctive spectrum of high-mannose species with quantitative variations in the extent of these modifications, and qualitative differences including retention of Glc, mannose methylation, and absence of a peripheral GlcNAc, fucosylation, or sulfation. Starvation-induced development modifies the pattern in all species but, except for universally observed increased mannose-trimming, the N-glycans do not converge to a common profile. Correlations with glycogene repertoires will enable future reverse genetic studies to eliminate N-glycomic differences to test their functions in interspecific relations and pathogen evasion.

O(2) sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1-F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1-cullin-1-F-box protein-Rbx1 subcomplex of E3(SCF)Ub ligases. E3(SCF)Ub ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O(2) availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA-Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We propose that O(2)-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3(SCF)Ub ligases that regulate O(2)-dependent developmental progression., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)