A prognostic neural epigenetic signature in high-grade glioma.

Neural-tumor interactions drive glioma growth as evidenced in preclinical models, but clinical validation is limited. We present an epigenetically defined neural signature of glioblastoma that independently predicts patients' survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals a high abundance of malignant stemcell-like cells in high-neural glioblastoma, primarily of the neural lineage. These cells are further classified as neural-progenitor-cell-like, astrocyte-like and oligodendrocyte-progenitor-like, alongside oligodendrocytes and excitatory neurons. In line with these findings, high-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients, a high-neural signature is associated with decreased overall and progression-free survival. High-neural tumors also exhibit increased functional connectivity in magnetencephalography and resting-state magnet resonance imaging and can be detected via DNA analytes and brain-derived neurotrophic factor in patients' plasma. The prognostic importance of the neural signature was further validated in patients diagnosed with diffuse midline glioma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant. High-neural gliomas likely require a maximized surgical resection approach for improved outcomes.

Optimizing mycobacteria molecular diagnostics: No decontamination! Human DNA depletion? Greener storage at 4 °C!

Introduction: Tuberculosis (TB) is an infectious disease caused by the group of bacterial pathogens Mycobacterium tuberculosis complex (MTBC) and is one of the leading causes of death worldwide. Timely diagnosis and treatment of drug-resistant TB is a key pillar of WHO's strategy to combat global TB. The time required to carry out drug susceptibility testing (DST) for MTBC via the classic culture method is in the range of weeks and such delays have a detrimental effect on treatment outcomes. Given that molecular testing is in the range of hours to 1 or 2 days its value in treating drug resistant TB cannot be overstated. When developing such tests, one wants to optimize each step so that tests are successful even when confronted with samples that have a low MTBC load or contain large amounts of host DNA. This could improve the performance of the popular rapid molecular tests, especially for samples with mycobacterial loads close to the limits of detection. Where optimizations could have a more significant impact is for tests based on targeted next generation sequencing (tNGS) which typically require higher quantities of DNA. This would be significant as tNGS can provide more comprehensive drug resistance profiles than the relatively limited resistance information provided by rapid tests. In this work we endeavor to optimize pre-treatment and extraction steps for molecular testing. Methods: We begin by choosing the best DNA extraction device by comparing the amount of DNA extracted by five commonly used devices from identical samples. Following this, the effect that decontamination and human DNA depletion have on extraction efficiency is explored. Results: The best results were achieved (i.e., the lowest Ct values) when neither decontamination nor human DNA depletion were used. As expected, in all tested scenarios the addition of decontamination to our workflow substantially reduced the yield of DNA extracted. This illustrates that the standard TB laboratory practice of applying decontamination, although being vital for culture-based testing, can negatively impact the performance of molecular testing. As a complement to the above experiments, we also considered the best Mycobacterium tuberculosis DNA storage method to optimize molecular testing carried out in the near- to medium-term. Comparing Ct values following three-month storage at 4 °C and at -20 °C and showed little difference between the two. Discussion: In summary, for molecular diagnostics aimed at mycobacteria this work highlights the importance of choosing the right DNA extraction device, indicates that decontamination causes significant loss of mycobacterial DNA, and shows that samples preserved for further molecular testing can be stored at 4 °C, just as well at -20 °C. Under our experimental settings, human DNA depletion gave no significant improvement in Ct values for the detection of MTBC.

Multi-species host range of staphylococcal phages isolated from wastewater.

The host range of bacteriophages defines their impact on bacterial communities and genome diversity. Here, we characterize 94 novel staphylococcal phages from wastewater and establish their host range on a diversified panel of 117 staphylococci from 29 species. Using this high-resolution phage-bacteria interaction matrix, we unveil a multi-species host range as a dominant trait of the isolated staphylococcal phages. Phage genome sequencing shows this pattern to prevail irrespective of taxonomy. Network analysis between phage-infected bacteria reveals that hosts from multiple species, ecosystems, and drug-resistance phenotypes share numerous phages. Lastly, we show that phages throughout this network can package foreign genetic material enclosing an antibiotic resistance marker at various frequencies. Our findings indicate a weak host specialism of the tested phages, and therefore their potential to promote horizontal gene transfer in this environment.

Uncovering a hidden diversity: optimized protocols for the extraction of dsDNA bacteriophages from soil.

BACKGROUND: Bacteriophages (phages) are the most numerous biological entities on Earth and play a crucial role in shaping microbial communities. Investigating the bacteriophage community from soil will shed light not only on the yet largely unknown phage diversity, but may also result in novel insights towards their functioning in the global biogeochemical nutrient cycle and their significance in earthbound ecosystems. Unfortunately, information about soil viromes is rather scarce compared to aquatic environments, due to the heterogeneous soil matrix, which rises major technical difficulties in the extraction process. Resolving these technical challenges and establishing a standardized extraction protocol is, therefore, a fundamental prerequisite for replicable results and comparative virome studies. RESULTS: We here report the optimization of protocols for the extraction of phage DNA from agricultural soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. As an optimization strategy, soil samples were spiked with Listeria phage A511 (Myovirus), Staphylococcus phage 2638AΔLCR (Siphovirus) and Escherichia phage T7 (Podovirus) (each 106 PFU/g soil). The efficacy of phage (i) elution, (ii) filtration, (iii) concentration and (iv) DNA extraction methods was tested. Successful extraction routes were selected based on spiked phage recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes were then extracted with the optimized methods and shotgun sequenced. Our approach yielded sufficient amounts of inhibitor-free viral DNA for shotgun sequencing devoid of amplification prior library preparation, and low 16S rRNA gene contamination levels (≤ 0.2‰). Compared to previously published protocols, the number of bacterial read contamination was decreased by 65%. In addition, 379 novel putative complete soil phage genomes (≤ 235 kb) were obtained from over 13,000 manually identified viral contigs, promising the discovery of a large, previously inaccessible viral diversity. CONCLUSION: We have shown a considerably enhanced extraction of the soil phage community by protocol optimization that has proven robust in both culture-dependent as well as through viromic analyses. Our huge data set of manually curated soil viral contigs substantially increases the amount of currently available soil virome data, and provides insights into the yet largely undescribed soil viral sequence space.