Genomic and transcriptomic changes complement each other in the pathogenesis of sporadic Burkitt lymphoma

Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing

The genomic and transcriptional landscape of primary central nervous system lymphoma

Primary lymphomas of the central nervous system (PCNSL) are mainly diffuse large B-cell lymphomas (DLBCLs) confined to the central nervous system (CNS). Molecular drivers of PCNSL have not been fully elucidated. Here, we profile and compare the whole-genome and transcriptome landscape of 51 CNS lymphomas (CNSL) to 39 follicular lymphoma and 36 DLBCL cases outside the CNS. We find recurrent mutations in JAK-STAT, NFkB, and B-cell receptor signaling pathways, including hallmark mutations in MYD88 L265P (67%) and CD79B (63%), and CDKN2A deletions (83%). PCNSLs exhibit significantly more focal deletions of HLA-D (6p21) locus as a potential mechanism of immune evasion. Mutational signatures correlating with DNA replication and mitosis are significantly enriched in PCNSL. TERT gene expression is significantly higher in PCNSL compared to activated B-cell (ABC)-DLBCL. Transcriptome analysis clearly distinguishes PCNSL and systemic DLBCL into distinct molecular subtypes. Epstein-Barr virus (EBV)+ CNSL cases lack recurrent mutational hotspots apart from IG and HLA-DRB loci. We show that PCNSL can be clearly distinguished from DLBCL, having distinct expression profiles, IG expression and translocation patterns, as well as specific combinations of genetic alterations

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Flotillin scaffold activity contributes to type VII secretion system assembly in <i>Staphylococcus aureus</i>

<div><p>Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen <i>Staphylococcus aureus</i>, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. <i>Staphylococcus aureus</i> cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity <i>in vitro</i> and <i>in vivo</i>. Our results suggest that flotillin assists in the assembly of <i>S</i>. <i>aureus</i> membrane components that participate in infection and influences the infective potential of this pathogen.</p></div

Effect of FloA on reconstituted T7SS in <i>E</i>. <i>coli</i>.

<p>(A) BN-PAGE and immunoblot analysis of solubilized <i>E</i>. <i>coli</i> membranes expressing structural T7SS proteins EsaA, EssA, EssB and EssC alone or with FloA. EssB was detected using polyclonal antibodies. (B) Size exclusion chromatography on a Superose 6 column with solubilized membrane fractions expressing structural T7SS proteins EsaA, EssA, EssB and EssC alone (- FloA) or with FloA (+FloA). The fractions corresponding to the elution volumes at 8–21 ml were separated in SDS-PAGE and detected by immunoblotting with polyclonal anti-FloA, -EsaA, -EssB or -EssC antibodies.</p

FloA is important for intermolecular interactions of T7SS membrane proteins.

<p>(A) Subcellular localization of GFP-EssB. Bright field and green fluorescence of a complemented GFP-EssB controlled by its own promoter (P_esxA) in a wild type and a Δ<i>floA</i> background in stationary growth phase. Bar, 1.5 μm (right). Corresponding immunoblot analysis of P_esxA GFP-EssB strains using polyclonal anti-GFP antibodies. An unlabeled wild type strain served as a negative control (left). (B) BN-PAGE analysis of DSP-crosslinked membrane fractions of <i>S</i>. <i>aureus</i> expressing complemented FLAG-EssB on a wild type or a Δ<i>floA</i> background using a monoclonal anti-FLAG antibody (left). The right panel shows a pixel intensity analysis of this BN-PAGE. The top arrow indicates higher molecular weight oligomers of EssB and the bottom arrow, low molecular weight oligomers. (C) Pulldown analysis of FLAG-tagged EssB using FLAG-capture beads. The blots show the eluted fractions of wild type, Δ<i>floA</i> mutant and a ΔT7SS mutant expressing FLAG-EssB; an unlabeled wild type served as negative control. EsaA and EssC were detected using polyclonal antibodies, EssA-MARS was detected using a polyclonal antibody to the mCherry protein. Immunoblot of CoIP elution fractions (Co-immunoprecipitation) (left) and of the input membrane fractions (right). Besides full-length EsaA, several fragments were detected, but only one co-eluted with FLAG-tagged EssB in the pull-down experiment. (D) Bacterial three-hybrid assay to study EssB interaction with EssA, alone or with flotillin on a third plasmid (pSEVA). Quantification of T25-EssB and EssA-T18 interactions were assayed with β-galactosidase activity assay using empty plasmid (pSEVA641), plasmid bearing flotillin (pSEVA641<i>-floA</i>) or no pSEVA plasmid (-).</p

FloA mutant reduces secretion efficiency of T7SS substrates EsxA, EsxB and EsxC.

<p>Cells were grown to early stationary growth phase. Filtered supernatants (SN) were supplemented with recombinant YtnP as a control of equal loading, then precipitated and immunoblotted and proteins detected with anti-FLAG, -YtnP and -EsxC antibodies. Cell extracts (CE) were probed with anti-FLAG and -EsxC antibodies. Detection of GroEL served as loading control. For FLAG-labeled EsxA and EsxB, an unlabeled wild type strain served as negative control and ΔT7SS strain was used as a secretion-negative strain. For EsxC secretion, a ΔT7SS mutant was used as a negative control and a Δ<i>essB</i> mutant strain as a secretion negative strain.</p

Anti-FMM molecules interfere with T7SS complex formation and can inhibit secretion of T7SS substrates <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Effect of anti-FMM molecules on T7SS complex formation. BN-PAGE analysis of untreated vs. DSP-crosslinked membrane fractions of <i>S</i>. <i>aureus</i> cells grown to stationary phase in the presence of 20 μM SIM, 150 μM 5-DSA or 50 μM ZA. Pixel intensity analysis of DSP-crosslinked samples are shown (right). (B) Effect of anti-FMM molecules on secretion of T7SS substrates. Cells were grown to the end of exponential growth phase in the presence of 20 μM SIM, 150 μM 5-DSA or 50 μM ZA. Precipitated supernatant (SN) and corresponding cell extracts (CE) were separated in SDS-PAGE and tested in immunoblot with anti-FLAG or -EsxC antibodies. Recombinant YtnP was added to supernatants as loading control and probed with anti-YtnP antibodies; for cell extracts anti-GroEL was used as a loading control. (C) Indirect ELISA to study the ZA effect on antibody response to EsxA-D. Zaragozic acid was administered to BALB/c mice by intraperitoneal injection, followed by challenge with a sublethal dose of staphylococci. The procedure was repeated on days 14 and 28 to boost the antibody response. Graphs show IgM antibody titers to EsxA-D in untreated <i>vs</i>. treated mice. Statistical analysis was performed using an unpaired t-test (*P<0.05; **P<0.01).</p

Flotillin interacts with the T7SS protein EssB.

<p>(A) Immunoblot analysis of pulldown assay to show the <i>in vivo</i> EssB and FloA interaction in <i>S</i>. <i>aureus</i>. Lane 1 shows FLAG-EssB FloA-His elution fraction from a Ni-NTA column. Negative controls (lanes 2, 3) are single-labeled strains and lane 4 is an unlabeled strain; lane 5 is the FLAG-EssB FloA-His double-labeled strain membrane fraction as positive control. (B) STED (stimulated emission depletion) microscopy images of a <i>ΔspA</i> strain double-immunolabeled with anti-FloA (Alexa488) and anti-EssB (Alexa546) antibodies. Top panel shows overlay of red and green fluorescent signals, false colored with red and green, respectively. Three individual cells are highlighted and further analyzed in panel C. Bottom panel shows colocalization analysis performed with the ImageJ JACoP plugin. Each pixel containing signal in both red and green fluorescent channel is now represented with a white pixel. Bar, 1 μm. (C) Three representative cells showing EssB and FloA colocalization. Top rows show overlay of false colored red and green fluorescent signals on the left and single red (EssB; Alexa546) and green (FloA, Alexa488) fluorescent channels in the center. Image on the right shows overlay of the white signal of colocalization analysis with the false-colored red and green-fluorescent channels. Bar, 0.2 μm. Each bottom panel shows pixel intensity analysis clockwise around the outline of the cell starting at the top.</p