Biology subjects

Callewaert, N.

Publications and source records attributed to Callewaert, N..

23 records · Page 2Linked to original sources

bioRxiv2019

A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing

Methylation profiling of circulating cell-free DNA (cfDNA) is of great interest as a liquid biopsy assay for the detection and monitoring of cancer and other pathologies. Here we describe circulating cell-free DNA reduced representation bisulfite sequencing (cf-RRBS), enabling the use of highly effective RRBS on fragmented plasma cfDNA. This method enriches the CpG-rich RRBS target regions by enzymatic degradation of all off-target DNA rather than by targeted capture, in contrast to previous methods. Critical steps are fully enzymatic in a single-tube, making it rapid, cost-effective, robust, and easily implemented on a liquid-handling station for high-throughput sample preparation. We benchmark cf-RRBS results to those obtained by previous more complex methods and exemplify its use for accurate non-invasive subtyping of lung cancer, a frequent onco-pathology task. cf-RRBS enables any molecular pathology lab to tap into the cfDNA methylome, only making use of off-the-shelf reagents and open-source data analysis tools. One Sentence SummaryNovel methodology enables facile and cost-effective methylation profiling of fragmented plasma DNA, allowing for routine liquid biopsy clinical diagnostics, exemplified here for differential diagnosis of lung cancer subtypes.

genomics↗

bioRxiv2019

Reference genome for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine

Mycobacterium bovis bacillus Calmette-Guerin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). This study reports on an integrated genome analysis workflow for BCG, resulting in the completely assembled genome sequence of BCG Danish 1331 (07/270), one of the WHO reference strains for BCG vaccines. We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of this vaccine strain.

microbiology↗

bioRxiv2018

SapM mutation to improve the BCG vaccine: genomic, transcriptomic and preclinical safety characterization

The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine shows variable efficacy in protection against adult tuberculosis (TB). Earlier, we have described a BCG mutant vaccine with a transposon insertion in the gene coding for the secreted acid phosphatase SapM, which led to enhanced long-term survival of vaccinated mice challenged with TB infection. To facilitate development of this mutation as part of a future improved live attenuated TB vaccine, we have now characterized the genome and transcriptome of this sapM::Tn mutant versus parental BCG Pasteur. Furthermore, we show that the sapM::Tn mutant had an equal low pathogenicity as WT BCG upon intravenous administration to immunocompromised SCID mice, passing this important safety test. Subsequently, we investigated the clearance of this improved vaccine strain following vaccination and found a more effective innate immune control over the sapM::Tn vaccine bacteria as compared to WT BCG. This leads to a fast contraction of IFN{gamma} producing Th1 and Tc1 cells after sapM::Tn BCG vaccination. These findings corroborate that a live attenuated vaccine that affords improved long-term survival upon TB infection can be obtained by a mutation that further attenuates BCG. These findings suggest that an analysis of the effectiveness of innate immune control of the vaccine bacteria could be instructive also for other live attenuated TB vaccines that are currently under development, and encourage further studies of SapM mutation as a strategy in developing a more protective live attenuated TB vaccine.

microbiology↗

bioRxiv2018

Clinical assay for direct assessment of IgG galactosylation in serum using endoglycosidase S

Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohns and lupus, but also other pathologies like alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and are hallmarked by a reduced level of serum IgG galactosylation. IgG (under)galactosylation is a promising biomarker to assess disease severity, and monitor and adjust therapy. However, this biomarker has not been implemented in routine clinical chemistry due to a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput.\n\nWe addressed this issue by using endo-{beta}-N-acetylglucosaminidase from Streptococcus pyogenes (endoS) to specifically release Fc N-glycans in whole serum. The entire assay can be completed in a few hours and only entails adding endoS and labeling the glycans with APTS. Glycans are then readily analyzed through capillary electrophoresis. We demonstrate in two independent patient cohorts that IgG undergalactosylation levels obtained with this assay correlate very well with scores calculated from PNGaseF-released glycans of purified antibodies. Our new assay allows to directly and specifically measure the degree of IgG galactosylation in serum through a fast and completely liquid phase protocol, without the requirement for antibody purification. This should help advancing this biomarker towards clinical implementation.

biochemistry↗

bioRxiv2018

Beyond the ribosome: proteome-wide secretability studies using SECRiFY

While transcriptome- and proteome-wide technologies to assess processes in protein biogenesis are now widely available, we still lack global approaches to assay post-ribosomal biogenesis events, in particular those occurring in the eukaryotic secretory system. We here developed a method, SECRiFY, to simultaneously assess the secretability of >105 protein fragments by two yeast species, S. cerevisiae and P. pastoris, using custom fragment libraries, surface display and a sequencing-based readout. Screening human proteome fragments with a median size of 50 - 100 amino acids, we generated datasets that enable datamining into protein features underlying secretability, revealing a striking role for intrinsic disorder and chain flexibility. SECRiFY is the first methodology that generates sufficient amounts of annotated data for advanced machine learning methods to deduce secretability predictors. The finding that secretability is indeed a learnable feature of protein sequences is of significant impact in the broad area of recombinant protein expression and de novo protein design.

molecular biology↗