GENType: all-in-one preimplantation genetic testing by pedigree haplotyping and copy number profiling suitable for third-party reproduction.

STUDY QUESTION: Is it possible to develop a comprehensive pipeline for all-in-one preimplantation genetic testing (PGT), also suitable for parents-only haplotyping and, for the first time, third-party reproduction? SUMMARY ANSWER: Optimized reduced representation sequencing (RRS) by GENType, along with a novel analysis platform (Hopla), enables cheap, accurate and comprehensive PGT of blastocysts, even without the inclusion of additional family members or both biological parents for genome-wide embryo haplotyping. WHAT IS KNOWN ALREADY: Several haplotyping strategies have proven to be effective for comprehensive PGT. However, these methods often rely on microarray technology, whole-genome sequencing (WGS) or a combination of strategies, hindering sample throughput and cost-efficiency. Moreover, existing tools (including other RRS-based strategies) require both prospective biological parents for embryo haplotyping, impeding application in a third-party reproduction setting. STUDY DESIGN, SIZE, DURATION: This study included a total of 257 samples. Preliminary technical validation was performed on 81 samples handpicked from commercially available cell lines. Subsequently, a clinical validation was performed on a total of 72 trophectoderm biopsies from 24 blastocysts, tested for a monogenic disorder (PGT-M) (n = 15) and/or (sub)chromosomal aneuploidy (PGT-SR/PGT-A) (n = 9). Once validated, our pipeline was implemented in a diagnostic setting on 104 blastocysts for comprehensive PGT. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were whole-genome amplified (WGA) and processed by GENType. Quality metrics, genome-wide haplotypes, b-allele frequencies (BAFs) and copy number profiles were generated by Hopla. PGT-M results were deduced from relative haplotypes, while PGT-SR/PGT-A results were inferred from read-count analysis and BAF profiles. Parents-only haplotyping was assessed by excluding additional family members from analysis and using an independently diagnosed embryo as phasing reference. Suitability for third-party reproduction through single-parent haplotyping was evaluated by excluding one biological parent from analysis. Results were validated against reference PGT methods. MAIN RESULTS AND THE ROLE OF CHANCE: Genome-wide haplotypes of single cells were highly accurate (mean > 99%) compared to bulk DNA. Unbalanced chromosomal abnormalities (>5 Mb) were detected by GENType. For both PGT-M as well as PGT-SR/PGT-A, our technology demonstrated 100% concordance with reference PGT methods for diverse WGA methods. Equally, for parents-only haplotyping and single-parent haplotyping (of autosomal dominant disorders and X-linked disorders), PGT-M results were fully concordant. Furthermore, the origin of trisomies in PGT-M embryos was correctly deciphered by Hopla. LIMITATIONS, REASONS FOR CAUTION: Intrinsic to linkage-analysis strategies, de novo single-nucleotide variants remain elusive. Moreover, parents-only haplotyping is not a stand-alone approach and requires prior diagnosis of at least one reference embryo by an independent technology (i.e. direct mutation analysis) for haplotype phasing. Using a haplotyping approach, the presence of a homologous recombination site across the chromosome is biologically required to distinguish meiotic II errors from mitotic errors during trisomy origin investigation. WIDER IMPLICATIONS OF THE FINDINGS: We offer a generic, fully automatable and accurate pipeline for PGT-M, PGT-A and PGT-SR as well as trisomy origin investigation without the need for personalized assays, microarray technology or WGS. The unique ability to perform single-parent assisted haplotyping of embryos paves the way for cost-effective PGT in a third-party reproduction setting. STUDY FUNDING/COMPETING INTEREST(S): L.D.W. is supported by the Research Foundation Flanders (FWO; 1S74619N). L.R. and B.M. are funded by Ghent University and M.B., S.S., K.T., F.V.M. and A.D. are supported by Ghent University Hospital. Research in the N.C. lab was funded by Ghent University, VIB and Kom op Tegen Kanker. A.D.K and N.C. are co-inventors of patent WO2017162754A1. The other authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

The potential of glycomics as prognostic biomarkers in liver disease and liver transplantation.

The study of glycomics is a novel and fascinating approach for the development of biomarkers. It has become clear that in the field of liver disease specific glycomic patters are present in specific disease states, which has led to the development of diagnostic biomarkers. In this manuscript, we will describe two new applications of this technology for the development of prognostic biomarkers. The first biomarker is associated with the risk of hepatocellular carcinoma development in patients with compensated cirrhosis. The second biomarker is present in perfusate and is related to the risk of primary non function occurrence after liver transplantation. The technology used for these biomarkers could easily be implemented on routine capillary electrophoresis equipment.

Human tissue-engineered skeletal muscle: a novel 3D in vitro model for drug disposition and toxicity after intramuscular injection.

The development of laboratory-grown tissues, referred to as organoids, bio-artificial tissue or tissue-engineered constructs, is clearly expanding. We describe for the first time how engineered human muscles can be applied as a pre- or non-clinical model for intramuscular drug injection to further decrease and complement the use of in vivo animal studies. The human bio-artificial muscle (BAM) is formed in a seven day tissue engineering procedure during which human myoblasts fuse and differentiate to aligned myofibers in an extracellular matrix. The dimensions of the BAM constructs allow for injection and follow-up during several days after injection. A stereotactic setup allows controllable injection at multiple sites in the BAM. We injected several compounds; a dye, a hydrolysable compound, a reducible substrate and a wasp venom toxin. Afterwards, direct reflux, release and metabolism were assessed in the BAM constructs in comparison to 2D cell culture and isolated human muscle strips. Spectrophotometry and luminescence allowed to measure the release of the injected compounds and their metabolites over time. A release profile over 40 hours was observed in the BAM model in contrast to 2D cell culture, showing the capacity of the BAM model to function as a drug depot. We also determined compound toxicity on the BAMs by measuring creatine kinase release in the medium, which increased with increasing toxic insult. Taken together, we show that the BAM is an injectable human 3D cell culture model that can be used to measure release and metabolism of injected compounds in vitro.

Elucidation of the molecular mechanisms of two nanobodies that inhibit thrombin-activatable fibrinolysis inhibitor activation and activated thrombin-activatable fibrinolysis inhibitor activity.

UNLABELLED: Essentials Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for cardiovascular disorders. TAFI inhibitory nanobodies represent a promising step in developing profibrinolytic therapeutics. We have solved three crystal structures of TAFI in complex with inhibitory nanobodies. Nanobodies inhibit TAFI through distinct mechanisms and represent novel profibrinolytic leads. SUMMARY: Background Thrombin-activatable fibrinolysis inhibitor (TAFI) is converted to activated TAFI (TAFIa) by thrombin, plasmin, or the thrombin-thrombomodulin complex (T/TM). TAFIa is antifibrinolytic, and high levels of TAFIa are associated with an increased risk for cardiovascular disorders. TAFI-inhibitory nanobodies represent a promising approach for developing profibrinolytic therapeutics. Objective To elucidate the molecular mechanisms of inhibition of TAFI activation and TAFIa activity by nanobodies with the use of X-ray crystallography and biochemical characterization. Methods and results We selected two nanobodies for cocrystallization with TAFI. VHH-a204 interferes with all TAFI activation modes, whereas VHH-i83 interferes with T/TM-mediated activation and also inhibits TAFIa activity. The 3.05-Å-resolution crystal structure of TAFI-VHH-a204 reveals that the VHH-a204 epitope is localized to the catalytic moiety (CM) in close proximity to the TAFI activation site at Arg92, indicating that VHH-a204 inhibits TAFI activation by steric hindrance. The 2.85-Å-resolution crystal structure of TAFI-VHH-i83 reveals that the VHH-i83 epitope is located close to the presumptive thrombomodulin-binding site in the activation peptide (AP). The structure and supporting biochemical assays suggest that VHH-i83 inhibits TAFIa by bridging the AP to the CM following TAFI activation. In addition, the 3.00-Å-resolution crystal structure of the triple TAFI-VHH-a204-VHH-i83 complex demonstrates that the two nanobodies can simultaneously bind to TAFI. Conclusions This study provides detailed insights into the molecular mechanisms of TAFI inhibition, and reveals a novel mode of TAFIa inhibition. VHH-a204 and VHH-i83 merit further evaluation as potential profibrinolytic therapeutics.

Influenza-binding sialylated polymer coated gold nanoparticles prepared via RAFT polymerization and reductive amination.

We report on a straightforward strategy to fabricate bioactive glycosylated gold nanoparticles via a combination of RAFT polymerization, carbohydrate ligation through reductive amination and thiol-gold self-assembly. This approach is used for the design of gold nanoparticles decorated with the complex sialylated glycan Neu5Ac-α-2-6-Gal, and we demonstrate multivalent and specific recognition between the nanoparticles, lectins and hemagglutinin on the surface of the influenza virus.

Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models.

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.

N-glycosylation engineering of biopharmaceutical expression systems.

N-glycosylation, the enzymatic coupling of oligosaccharides to specific asparagine residues of nascent polypeptide chains, is one of the most widespread post-translational modifications. Following transfer of an N-glycan precursor in the ER, this structure is further modified by a number of glycosidases and glyco-syltransferases in the ER and the Golgi complex. The processing reactions occurring in the ER are highly conserved between lower and higher eukaryotes. In contrast, the reactions that take place in the Golgi complex are species- and cell type-specific. Due to its non-template driven nature, glycoproteins typically occur as a mixture of glycoforms. Since N-glycans influence circulation half-life, tissue distribution, and biological activity each glycoform has its own pharmacokinetic, pharmacodynamic and efficacy profile. Moreover, modification of glycoproteins with non-human oligosaccharides can result in undesired immunogenicity. Therefore, engineering of the N-glycosylation pathway of most currently used heterologous protein expression systems (bacteria, mammalian cells, insect cells, yeasts and plants) is actively pursued by several academic and industrial laboratories. These research efforts are in the first place directed at humanizing the N-glycosylation pathway and eliminating immunogenic glycotopes. Moreover, one wants to establish new structure-function relationships of different glycoforms, which helps to decreasing the complexity of the N-glycan repertoire towards one defined N-glycan structure. In this review, we discuss the most important recent milestones in the glycoengineering field.

Reversible changes in serum immunoglobulin galactosylation during the immune response and treatment of inflammatory autoimmune arthritis.

OBJECTIVES: Improved DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) technology was used to monitor the changes in the galactosylation status of serum immunoglobulins during the immune response and therapy of autoimmune arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in susceptible DBA/1 mice and the undergalactosylation status (UGS) of serum immunoglobulins was determined using the improved DSA-FACE technology. Prophylactic intravenous tolerisation with type II collagen as well as semitherapeutic treatment with dexamethasone (DEX) were performed and UGS was analysed. Next, the serum immunoglobulin glycosylation profiles of patients with rheumatoid arthritis (RA) and spondyloarthropathy (SpA) were studied and changes in the UGS scores during anti-tumour necrosis factor (TNF)alpha therapy followed. RESULTS: In the longitudinal CIA study, the undergalactosylation state of immunoglobulins was found to be significantly correlated with the clinical arthritis scores. Upon collagen-specific tolerisation as well as glucocorticoid semitherapeutic treatment, improvement of the clinical arthritis scores correlated with decreased levels of UGS. It was also demonstrated that withdrawal of DEX was associated with an increased UGS score. Interestingly, reversibility in the UGS was also shown during treatment of patients with RA and SpA with anti-TNFalpha. CONCLUSIONS: These findings demonstrate that the UGS of serum immunoglobulins changes during the disease course of CIA and that this UGS is inhibited by antigen-specific and antigen-independent treatment procedures. The observation that Ig galactosylation is a reversible process is also documented during treatment of patients with RA and SpA with anti-TNFalpha.

Novel tools for the study of class I alpha-mannosidases: a chromogenic substrate and a substrate-analog inhibitor.

The use of chromogenic substrates for evaluation of class I alpha-mannosidase is described. 2('),4(')-Dinitrophenyl-alpha-D-mannopyranoside allows rapid and sensitive assays of enzymatic activities, e.g., of heterologously expressed alpha-1,2-mannosidase from Trichoderma reesei. Interaction constants of several ligands with alpha-mannosidases from class I and II could also be determined. Furthermore, novel types of inhibitors derived from D-lyxose are presented. Methyl-alpha-D-lyxopyranosyl-(1(')-->2)-alpha-D-mannopyranoside is a potent inhibitor of the alpha-1,2-mannosidase from T. reesei (K(i)=600 microM) and since it probably spans subsites -1/+1, this disaccharide could be valuable in crystallographic studies of class I alpha-mannosidases.

Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris.

Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-alpha-D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of alpha-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man(5)GlcNAc(2) was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.

Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment.

The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycoprotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a challenging task. We present here methodology allowing the medium-throughput analysis of N-glycans derived from low picomole amounts of glycoproteins using the standard DNA-sequencing equipment available in any life sciences laboratory. The high sensitivity of the overall analytical process (from glycoprotein to results) is obtained using state-of-the-art deglycosylation procedures combined with a highly efficient and reproducible novel postderivatization cleanup step involving Sephadex G10 packed 96-well filterplates. All sample preparation steps (enzymatic deglycosylation with PNGase F, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderivatization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresis and MALDI-TOF-MS platforms already in use in some glycobiology labs and anticipates the higher throughput that will be offered by the capillary-array-based DNA sequencers currently penetrating the market. The described technology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.

Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase.

A cDNA encoding 1,2-alpha-D-mannosidase mds 1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56,266 Da and shows high similarity to the amino acid sequences of 1,2-alpha-D-mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris. Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae alpha-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analyzed. The enzyme was characterized as a class-I mannosidase.