Biology subjects

Callewaert, N.

Publications and source records attributed to Callewaert, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Addition of DMSO to T cell cultures skews differentiation towards a memory phenotype.

Dimethyl sulfoxide (DMSO) is a polar aprotic organic solvent that is widely used in biological applications. It is routinely applied as a cryoprotectant for long-term cell freezing as well as to dissolve peptides or drugs for immune cell functional assays. We report on a remarkable impact of low concentrations of DMSO on in vitro expansion of the total CD3+ pool isolated from human PBMCs as well as on the purified CD8+ T cell fraction thereof in the presence of different cytokine combinations typically used for therapeutic T cell expansion. Characterizing survival, proliferation, activation, exhaustion and differentiation, we demonstrate that DMSO at low concentrations substantially skews the differentiation of T cells towards a memory phenotype in a dose-dependent way. This is a desirable outcome for the field of adoptive T cell therapies for cancer, where it has been established that T cells with a memory phenotype exert superior anti-cancer immune responses.

immunology↗

bioRxiv2026

A Glycosylation-Dependent Checkpoint Restrains Intestinal Intra-Epithelial Lymphocyte Activation

Intraepithelial lymphocytes (IELs) are abundant in the intestinal epithelium, where they maintain barrier integrity and provide immune defense. Because of their potent cytotoxic and effector potential, IEL activity must be tightly controlled to prevent tissue damage. However, the mechanisms that calibrate IEL responsiveness remain unclear. Here, we identify glucosaminyl (N-acetyl) transferase 2 (Gcnt2) as a key restrainer of both natural and induced gut IEL. Among T cells, Gcnt2 is uniquely enriched in the intestine and partly dependent on retinoic acid signaling. GCNT2-mediated branched glycosylation marks IELs with signatures of tissue adaptation and reduced TCR responsiveness. Genetic ablation of Gcnt2 enhanced IEL degranulation, cytokine production, and cytotoxicity upon stimulation, improving bacterial clearance and limiting infection-induced disease, while aggravating the pathological consequences of strong T cell activation. Mechanistically, GCNT2-mediated glycosylation of CD45 reduced its phosphatase activity, thereby dampening TCR signaling and effector responses. Together, these findings reveal GCNT2 as a glycosylation-dependent checkpoint that fine-tunes IEL effector functions, uncovering a novel mechanism by which the intestinal immune system balances responsiveness and tolerance. SummaryGCNT2-mediated I-branching glycosylation of CD45 restrains IEL activation.

immunology↗

bioRxiv2025

ENGINEERING AND PRECLINICAL EVALUATION OF DRIED FOOD-FORMULATED YEAST-SECRETED ANTIBODIES AS PERI-EXPOSURE PROPHYLAXIS AGAINST CLOSTRIDIOIDES DIFFICILE DISEASE.

Clostridioides difficile gastrointestinal infection (CDI) causes significant disease burden, often following gut dysbiosis induced by antibiotic treatment in at-risk individuals. A non-invasive, easy to use targeted prophylaxis in such cases of acute elevated CDI risk, especially in those with a history of recurrent CDI, would prevent much suffering. In this study, we envisioned a simple prophylactic intervention for CDI enabled by the oral administration of yeast-secreted dried food-formulated C. difficile toxin A (TcdA)-and B (TcdB)-neutralizing VHH-based antibodies. We engineered two antibody versions: a previously reported tetravalent VHH (VHH4) comprising dual specificities for TcdA and TcdB, and its murine IgA Fc fusion counterpart (VHH4-mIgA Fc). We explored Generally Recognized as Safe (GRAS) Komagataella phaffii yeast as a low-cost, highly scalable production platform. In a first experiment, we produced the antibodies at small scale in shake flask yeast cultivations and formulated the secreted antibody-containing growth medium into standard-composition dried mouse feed pellets. The antibody food pellet-administration intervention was tested in a C. difficile spore-challenged mouse model in which CDI leads to 50% mortality of untreated animals. We started the antibody-feed prophylaxis dosed at 10 nmol/g pellet one day prior to C. difficile challenge and continued until the infection was cleared with ad libitum feeding. We found that the VHH4 feed almost completely prevented mortality and reduced disease-related weight loss. Despite its higher in vitro potency against TcdA and equipotent potency against TcdB, the VHH4-mIgA Fc fusion did not confer additional in vivo benefit, and further work focused on the simpler VHH4 format. Scalable high-cell-density bioreactor production of VHH4 yielded equally effective prophylactic formulations at the same dose of 10 nmol/g pellet, achieving complete survival and reduced disease burden. Our findings suggest that, with further antibody- and process development, oral prophylaxis with scalable, cost-effective, yeast-produced, dried food-formulated anti-toxin VHHs could offer a simple non-invasive, non-antibiotic prophylaxis option for the high-risk (r)CDI patient population.

molecular biology↗

bioRxiv2024

Yeast-based production platform for potent and stable heavy chain-only antibodies

Monoclonal antibodies are the leading drug of the biopharmaceutical market because of their high specificity and tolerability, but the current CHO-based manufacturing platform remains expensive and time-consuming leading to limited accessibility, especially in the case of diseases with high incidence and pandemics. Therefore, there is an urgent need for an alternative production system. In this study, we present a rapid and cost-effective microbial platform for heavy chain-only antibodies (VHH-Fc) in the methylotrophic yeast Komagataella phaffii (aka Pichia pastoris). We demonstrate the potential of this platform using a simplified single-gene VHH-Fc fusion construct instead of the conventional monoclonal antibody format, as this is more easily expressed in Pichia pastoris. We demonstrate that the Pichia-produced VHH-Fc fusion construct is stable and that a Pichia-produced VHH-Fc directed against the SARS-CoV-2 spike has potent SARS-CoV-2 neutralizing activity in vitro and in vivo. We expect that this platform will pave the way towards faster and cheaper development and production of broadly neutralizing single-chain antibodies in yeast.

microbiology↗

bioRxiv2023

Ultrapotent SARS coronavirus-neutralizing single-domain antibodies that bind a conserved membrane proximal epitope of the spike

Currently circulating SARS-CoV-2 variants have gained complete or significant resistance to all SARS-CoV-2-neutralizing antibodies that have been used in the clinic. Such antibodies can prevent severe disease in SARS-CoV-2 exposed patients for whom vaccines may not provide optimal protection. Here, we describe single-domain antibodies (VHHs), also known as nanobodies, that can broadly neutralize SARS-CoV-2 with unusually high potency. Structural analysis revealed their binding to a unique, highly conserved, membrane proximal, quaternary epitope in the S2 subunit of the spike. Furthermore, a VHH-human IgG1 Fc fusion, efficiently expressed in Chinese hamster ovary cells as a stable antibody construct, protected hamsters against SARS-CoV-2 replication in a therapeutic setting when administered systemically at low dose. This VHH-based antibody represents a new candidate anti-COVID-19 biologic that targets the Achilles heel of the viral spike.

microbiology↗

bioRxiv2023

N-glycosylation engineering in chimeric antigen receptor T cells enhances anti-tumor activity

Chimeric antigen receptor (CAR) T cell therapy has had limited success in solid tumors, requiring novel enhancement strategies. Modifying the glycocalyx of CAR T cells is unexplored; we report on genome-editing of the MGAT5 gene to abolish human CAR T N-glycan poly-LacNAc modifications. This boosted tumor control in carcinoma and lymphoma models, for donors of whom the non-engineered CAR T cells largely failed in tumor control. More blood-circulating MGAT5 KO CD70 nanoCAR T cells were found, exhibiting potent tumor cell-killing activity ex vivo, while non-glycoengineered CAR T cells faltered. MGAT5 KO CD70 nanoCAR T cells also mediated durable anti-tumor immunity, improving control of secondary carcinoma challenge months later. Single-cell transcriptomics revealed increased mitotic activity and type I interferon signaling, indicating sustained intratumoral activation. The glyco-engineered cells had unaltered antigen sensitivity and dependence on T cell growth factors, preserving key safety features. MGAT5 KO is readily compatible with clinical manufacturing, representing a promising approach to enhance CAR T cell therapy.

immunology↗

bioRxiv2022

OPENPichia: building a free-to-operate Komagataella phaffii protein expression toolkit

In the standard toolkit for recombinant protein expression, the yeast known in biotechnology as Pichia pastoris (formally: Komagataella phaffii) takes up the position between E. coli and HEK293 or CHO mammalian cells, and is used by thousands of laboratories both in academia and industry. The organism is eukaryotic yet microbial, and grows to extremely high cell densities while secreting proteins into its fully defined growth medium, using very well established strong inducible or constitutive promoters. Many products made in Pichia are in the clinic and in industrial markets. Pichia is also a favoured host for the rapidly emerging area of precision fermentation for the manufacturing of food proteins. However, the earliest steps in the development of the industrial strain (NRRL Y-11430/CBS 7435) that is used throughout the world were performed prior to 1985 in industry (Phillips Petroleum Company) and are not in the public domain. Moreover, despite the long expiry of associated patents, the patent deposit NRRL Y-11430/CBS 7435 that is the parent to all commonly used industrial strains, is not or no longer made freely available through the resp. culture collections. This situation is far from ideal for what is a major chassis for synthetic biology, as it generates concern that novel applications of the system are still encumbered by licensing requirements of the very basic strains. In the spirit of open science and freedom to operate for what is a key component of biotechnology, we set out to resolve this by using genome sequencing of type strains, reverse engineering where necessary, and comparative protein expression and strain characterisation studies. We find that the industrial strains derive from the K. phaffii type strain lineage deposited as 54-11.239 in the UC Davis Phaff Yeast Strain collection by Herman Phaff in 1954. This type strain has valid equivalent deposits that are replicated/derived from it in other yeast strain collections, incl. in ARS-NRRL NRRL YB-4290 (deposit also made by Herman Phaff) and NRRL Y-7556, CBS 2612 and NCYC 2543. We furthermore discovered that NRRL Y-11430 and its derivatives carry an ORF-truncating mutation in the HOC1 cell wall synthesis gene, and that reverse engineering of a similar mutation in the NCYC 2543 type strain imparts the high transformability that is characteristic of the industrial strains. Uniquely, the NCYC 2543 type strain, which we propose to call OPENPichia henceforth, is freely available from the NCYC culture collection, incl. resale and commercial production licenses at nominal annual licensing fees1. Furthermore, our not-for-profit research institute VIB has also acquired a resale/distribution license from NCYC, which we presently use to openly provide to end-users our genome-sequenced OPENPichia subclone strain and its derivatives, i.e., currently the highly transformable hoc1tr and the his4 auxotrophic mutants. To complement the OPENPichia platform, a fully synthetic modular gene expression vector building toolkit was developed, which is also openly distributed, for any purpose. We invite other researchers to contribute to our open science resource-building effort to establish a new unencumbered standard chassis for Pichia synthetic biology.

molecular biology↗

bioRxiv2022

PhosphoLingo: protein language models for phosphorylation site prediction

Protein post-translational modifications (PTMs) play an important role in numerous biological processes by significantly affecting protein structure and dynamics. Effective computational methods that provide a sequence-based prediction of PTM sites are desirable to guide functional experiments. Whereas these methods typically train neural networks on one-hot encoded amino acid sequences, protein language models carry higher-level pattern information that may improve sequence based prediction performance and hence constitute the current edge of the field. In this study, we first evaluate the training of convolutional neural networks on top of various protein language models for sequence based PTM prediction. Our results show substantial prediction accuracy improvements for various PTMs with current procedures of dataset compilation and model performance evaluation. We then used model interpretation methods to study what these advanced models actually base their learning on. Importantly for the entire field of PTM site predictors trained on proteomics-derived data, our model interpretation and transferability experiments reveal that the current approach to compile training datasets based on proteomics data leads to an artefactual protease-specific training bias that is exploited by the prediction models. This results in an overly optimistic estimation of prediction accuracy, an important caveat in the application of advanced machine learning approaches to PTM prediction based on proteomics data. We suggest a partial solution to reduce this data bias by implementing negative sample filtering, only allowing candidate PTM sites in matched peptides that are present in the experimental metadata. Availability and implementationThe prediction tool, with training and evaluation code, trained models, datasets, and predictions for various PTMs are available at https://github.com/jasperzuallaert/PhosphoLingo. Contactsven.degroeve@vib-ugent.be and nico.callewaert@vib-ugent.be Supplementary informationSupplementary materials are available at bioRxiv.

bioinformatics↗

bioRxiv2022

GlycoVHH: Introducing N-glycans on the camelid VHH antibody scaffold - Optimal sites and use for macrophage delivery

As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for therapeutic use in various disease indications, and as versatile reagents in research and diagnostics. To further increase the versatility of VHHs, we explored the VHH scaffold in a structure-guided approach to select regions where the introduction of an N-glycosylation N-X-T sequon and its associated glycan should not interfere with protein folding or epitope recognition. We expressed variants of such glycoengineered VHHs in the Pichia pastoris GlycoSwitchM5 strain, allowing us to pinpoint preferred sites at which Man5GlcNAc2-glycans can be introduced at high site occupancy without affecting antigen binding. A VHH carrying predominantly a Man5GlcNAc2 N-glycan at one of these preferred sites showed highly efficient, glycan-dependent uptake by Mf4/4 macrophages in vitro and by alveolar lung macrophages in vivo, illustrating one potential application of glyco-engineered VHHs: a glycan-based targeting approach for lung macrophage endolysosomal system delivery. The set of optimal artificial VHH N-glycosylation sites identified in this study can serve as a blueprint for targeted glyco-engineering of other VHHs, enabling site-specific functionalization through the rapidly expanding toolbox of synthetic glycobiology.

molecular biology↗

bioRxiv2021

Anti-SARS-CoV-2 human antibodies retaining neutralizing activity against SARS-CoV-2 B.1.1.529 (omicron)

SARS-CoV-2 B.1.1.529, designated omicron, was recently identified as a new variant of concern by WHO and is rapidly replacing SARS-CoV-2 delta as the most dominant variant in many countries. Unfortunately, because of the high number of mutations present in the spike of SARS-CoV-2 omicron, most monoclonal antibodies (mAbs) currently approved for treatment of COVID-19 lose their in vitro neutralizing activity against this variant. We recently described a panel of human anti-SARS-CoV-2 mAbs that potently neutralize SARS-CoV-2 Wuhan, D614G and variants alpha, beta, gamma and delta. In this work, we evaluated our mAb panel for potential in vitro activity against SARS-CoV-2 delta and omicron. Three mAbs from our panel retain neutralizing activity against both delta and omicron, with mAb 3B8 still resulting in complete neutralization at a concentration as low as 0.02 g/ml for both variants. Overall, our data indicate that mAb 3B8 may have the potential to become a game-changer in the fight against the continuously evolving SARS-CoV-2.

immunology↗

bioRxiv2021

Potent neutralizing anti-SARS-CoV-2 human antibodies cure infection with SARS-CoV-2 variants in hamster model

Treatment with neutralizing monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to COVID-19 management. Unfortunately, SARS-CoV-2 variants can escape several of these recently approved mAbs, highlighting the need for additional discovery and development. In a convalescent COVID-19 patient, we identified six mAbs, classified in four epitope groups, that potently neutralized SARS-CoV-2 Wuhan, alpha, beta, gamma and delta infection in vitro. In hamsters, mAbs 3E6 and 3B8 potently cured infection with SARS-CoV-2 Wuhan, beta and delta when administered post-viral infection at 5 mg/kg. Even at 0.2 mg/kg, 3B8 still reduced viral titers. Intramuscular delivery of DNA-encoded 3B8 resulted in in vivo mAb production of median serum levels up to 90 g/ml, and protected hamsters against delta infection. Overall, our data mark 3B8 as a promising candidate against COVID-19, and highlight advances in both the identification and gene-based delivery of potent human mAbs.

immunology↗

bioRxiv2021

Ring Finger Protein 213 Assembles into a Sensor for ISGylated Proteins with Antimicrobial Activity

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we used a viral-like particle trapping technology to identify ISG15-binding proteins and discovered Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly-characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We found that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We showed that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1 (HSV-1), human respiratory syncytial virus (RSV) and coxsackievirus B3 (CVB3), and we observed a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide novel molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

cell biology↗

bioRxiv2021

GlyConnect: a glycan-based conjugation extension of the GlycoDelete technology

Recently, our lab developed GlycoDelete, a technology suite that allows a radical simplification of eukaryotic N-glycosylation. The technology allows to produce glycoproteins that carry single GlcNAc, LacNAc, or LacNAc-Sia type glycans on their N-linked glycosylation sequons. GlycoDelete-type N-glycans are uniquely suited for glycan-based conjugation purposes, as these provide a short, homogeneous and hydrophilic link to the protein backbone. Targeting GlycoDelete-glycans allows for highly site-specific conjugation at sites in the protein which are normally occupied by bulky glycans, thus ensuring minimal interference with protein structure and function. The current manuscript describes the evaluation and optimization of both chemical and chemo-enzymatic conjugation of molecules onto the GlycoDelete-type glycans of a limited set of benchmark proteins.

bioengineering↗

bioRxiv2021

Exploration of synergistic action of cell wall-degrading enzymes against Mycobacterium tuberculosis

SynopsisO_ST_ABSBackgroundC_ST_ABSThe major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope - a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer - which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb. Objectives(i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes. MethodsWe inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal -amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant. ResultsOur biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, -amylase and polysorbate 80. ConclusionsSynergistically acting cell wall degrading enzymes are potently inhibiting Mtb - which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

microbiology↗

bioRxiv2020

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-domain Camelid Antibodies

The pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV-1) and COVID-19 coronavirus (SARS-CoV-2) have all emerged into the human population with devastating consequences. These viruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics to combat these highly pathogenic coronaviruses. Here, we describe the isolation and characterization of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs are capable of potently neutralizing MERS-CoV or SARS-CoV-1 S pseudotyped viruses. The crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs block receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S, and demonstrate that this cross-reactive VHH is capable of neutralizing SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

microbiology↗

bioRxiv2020

Off-target glycans encountered along the synthetic biology route towards humanized N-glycans in Pichia pastoris

BackgroundThe glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. ResultsWhen expressing mouse interleukin-22 (mIL-22) in a Pichia pastoris (syn. Komagataella phaffi) GlycoSwitchM5 strain which had been optimized to produce Man5GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5GlcNAc2 and an unexpected, partially -mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis and NMR, revealed that this novel glycan was Man5GlcNAc2 modified with a Glc-1,2-Man{beta}-1,2-Man{beta}-1,3-Glc-1,3-R tetra-saccharide. Also the biosynthetic intermediates of this off-target modification were detected. Expression of a Golgi-targeted GlcNAc Transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5GlcNAc2 structure. We have also observed the off-target glycan on other glycoproteins produced in the GlycoSwitchM5 strain. This illustrates the intricacies of Golgi glycosylation pathways and cautions that the use of glyco-engineered expression host cells should always be accompanied by detailed glycan analysis of the particular therapeutic proteins being produced. ConclusionsOur findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in Pichia pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.

synthetic biology↗

bioRxiv2019

Use of a counterselectable transposon to create markerless knockouts from a 18,432-clone ordered M. bovis BCG mutant resource

Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach; despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian Pooling-Coordinate Sequencing allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which is undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described, which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the M. tb complex (18,432 clones, mutating 83% of the non-essential M. tb homologues), from which clean knockouts can be generated.\n\nImportanceWhile speeding up research for many fields of biology (e.g. yeast, plant, and C. elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner, and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of M. bovis BCG Danish targets 83% of all non-essential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g. drug resistance research, vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the M. tb complex. The stretch to a full collection of mutants in all non-essential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.

microbiology↗

bioRxiv2019

Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA: a proof-of-principle study

In the clinical management of pediatric solid tumors, histological examination of tumor tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumor is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner. Translational relevanceObtaining a correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. Furthermore, the diagnostic odyssey can result in anxiety and discomfort for these children. By applying a novel technique, reduced representation bisulfite sequencing on cell-free DNA (cf-RRBS), we show the feasibility of obtaining the histopathological diagnosis with a minimally invasive test on either plasma or cerebrospinal fluid. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Given that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in difficult cases.

genomics↗