Ongoing genome doubling promotes evolvability and immune dysregulation in ovarian cancer.

Whole-genome doubling (WGD) is a critical driver of tumor development and is linked to drug resistance and metastasis in solid malignancies. Here, we demonstrate that WGD is an ongoing mutational process in tumor evolution. Using single-cell whole-genome sequencing, we measured and modeled how WGD events are distributed across cellular populations within tumors and associated WGD dynamics with properties of genome diversification and phenotypic consequences of innate immunity. We studied WGD evolution in 65 high-grade serous ovarian cancer (HGSOC) tissue samples from 40 patients, yielding 29,481 tumor cell genomes. We found near-ubiquitous evidence of WGD as an ongoing mutational process promoting cell-cell diversity, high rates of chromosomal missegregation, and consequent micronucleation. Using a novel mutation-based WGD timing method, doubleTime , we delineated specific modes by which WGD can drive tumor evolution: (i) unitary evolutionary origin followed by significant diversification, (ii) independent WGD events on a pre-existing background of copy number diversity, and (iii) evolutionarily late clonal expansions of WGD populations. Additionally, through integrated single-cell RNA sequencing and high-resolution immunofluorescence microscopy, we found that inflammatory signaling and cGAS-STING pathway activation result from ongoing chromosomal instability and are restricted to tumors that remain predominantly diploid. This contrasted with predominantly WGD tumors, which exhibited significant quiescent and immunosuppressive phenotypic states. Together, these findings establish WGD as an evolutionarily 'active' mutational process that promotes evolvability and dysregulated immunity in late stage ovarian cancer.

A balance of clinical assessment and use of diagnostic imaging: A CT colonography comparative case report.

Computer tomography colonography (CTC) is a non-invasive procedure which has replaced barium enema. CTC uses helical images of a cleansed and gas-distended colon for the diagnosis and treatment of colonic neoplasms. This case study compares 2 patients: one with positive pathology (patient A) and another as comparator (patient B) with a similar pathology to discuss and debate possible treatment pathways. Patient (A) CTC showed 2 polyps: 6 mm and 10 mm, which the colorectal surgeons thought only needed follow-up. Our comparator (patient B) displayed a similar pathology which measured 9 mm. In this case (patient B), there was mutual agreement with the surgeons for polypectomy but without haematology involvement which was atypical of the usual pathway. The surgeons did not see the 9 mm polyp at polypectomy which could be due to observer error or radiology reporter error. Given that conventional colonoscopy is more sensitive in detecting polyps; a repeat of both tests could confirm the presence of polyp, however, the surgeons gave patient (B) a virtual appointment and requested a repeat CTC in 12 months. In colorectal medicine there can be variations in the treatment of patients with polyps. While a repeat of both tests could confirm the presence of polyp in patient (B), the surgeons' decisions regarding the patient's treatment reflected a balance of confidence in clinical assessment and use of diagnostic imaging which can reduce unnecessary requests and use of diagnostic tests.

The recognition of structured elements by a conserved groove distant from domains associated with catalysis is an essential determinant of RNase E.

RNase E is an endoribonuclease found in many bacteria, including important human pathogens. Within Escherichia coli, it has been shown to have a major role in both the maturation of all classes of RNA involved in translation and the initiation of mRNA degradation. Thus, knowledge of the major determinants of RNase E cleavage is central to our understanding and manipulation of bacterial gene expression. We show here that the binding of RNase E to structured RNA elements is crucial for the processing of tRNA, can activate catalysis and may be important in mRNA degradation. The recognition of structured elements by RNase E is mediated by a recently discovered groove that is distant from the domains associated with catalysis. The functioning of this groove is shown here to be essential for E. coli cell viability and may represent a key point of evolutionary divergence from the paralogous RNase G family, which we show lack amino acid residues conserved within the RNA-binding groove of members of the RNase E family. Overall, this work provides new insights into the recognition and cleavage of RNA by RNase E and provides further understanding of the basis of RNase E essentiality in E. coli.

Siderophore-Linked Ruthenium Catalysts for Targeted Allyl Ester Prodrug Activation within Bacterial Cells.

Due to rising resistance, new antibacterial strategies are needed, including methods for targeted antibiotic release. As targeting vectors, chelating molecules called siderophores that are released by bacteria to acquire iron have been investigated for conjugation to antibacterials, leading to the clinically approved drug cefiderocol. The use of small-molecule catalysts for prodrug activation within cells has shown promise in recent years, and here we investigate siderophore-linked ruthenium catalysts for the activation of antibacterial prodrugs within cells. Moxifloxacin-based prodrugs were synthesised, and their catalyst-mediated activation was demonstrated under anaerobic, biologically relevant conditions. In the absence of catalyst, decreased antibacterial activities were observed compared to moxifloxacin versus Escherichia coli K12 (BW25113). A series of siderophore-linked ruthenium catalysts were investigated for prodrug activation, all of which displayed a combinative antibacterial effect with the prodrug, whereas a representative example displayed little toxicity against mammalian cell lines. By employing complementary bacterial growth assays, conjugates containing siderophore units based on catechol and azotochelin were found to be most promising for intracellular prodrug activation.

Activity-directed expansion of a series of antibacterial agents.

The feasibility of using activity-directed synthesis to drive antibacterial discovery was investigated. An array of 220 Pd-catalysed microscale reactions was executed, and the crude product mixtures were evaluated for activity against Staphylococcus aureus. Scale-up of the hit reactions, purification and evaluation, enabled expansion of a class of antibacterial quinazolinones. The novel antibacterials had MICs from 0.016 µg mL-1 (i.e. 38 nM) to 2-4 µg mL-1 against S. aureus ATCC29213.

Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli.

Escherichia coli endoribonuclease E has a major influence on gene expression. It is essential for the maturation of ribosomal and transfer RNA as well as the rapid degradation of messenger RNA. The latter ensures that translation closely follows programming at the level of transcription. Recently, one of the hallmarks of RNase E, i.e. its ability to bind via a 5'-monophosphorylated end, was shown to be unnecessary for the initial cleavage of some polycistronic tRNA precursors. Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and a 5'-sensor mutant of RNase E in vitro that, contrary to current models, 5'-monophosphate-independent, 'direct entry' cleavage is a major pathway for degrading and processing RNA. Moreover, we present further evidence that direct entry is facilitated by RNase E binding simultaneously to multiple unpaired regions. These simple requirements may maximize the rate of degradation and processing by permitting multiple sites to be surveyed directly without being constrained by 5'-end tethering. Cleavage was detected at a multitude of sites previously undescribed for RNase E, including ones that regulate the activity and specificity of ribosomes. A potentially broad role for RNase G, an RNase E paralogue, in the trimming of 5'-monophosphorylated ends was also revealed.

Adjacent single-stranded regions mediate processing of tRNA precursors by RNase E direct entry.

The RNase E family is renowned for being central to the processing and decay of all types of RNA in many species of bacteria, as well as providing the first examples of endonucleases that can recognize 5'-monophosphorylated ends thereby increasing the efficiency of cleavage. However, there is increasing evidence that some transcripts can be cleaved efficiently by Escherichia coli RNase E via direct entry, i.e. in the absence of the recognition of a 5'-monophosphorylated end. Here, we provide biochemical evidence that direct entry is central to the processing of transfer RNA (tRNA) in E. coli, one of the core functions of RNase E, and show that it is mediated by specific unpaired regions that are adjacent, but not contiguous to segments cleaved by RNase E. In addition, we find that direct entry at a site on the 5' side of a tRNA precursor triggers a series of 5'-monophosphate-dependent cleavages. Consistent with a major role for direct entry in tRNA processing, we provide additional evidence that a 5'-monophosphate is not required to activate the catalysis step in cleavage. Other examples of tRNA precursors processed via direct entry are also provided. Thus, it appears increasingly that direct entry by RNase E has a major role in bacterial RNA metabolism.