Joanna Crawford

Caspase-4 and caspase-11 (CASP4/11) sense bacterial lipopolysaccharide (LPS). Currently available inhibitors of CASP4/11 also block the activity of caspase-1 (CASP1), which restricts their usefulness in the study of CASP4/11 functions, as well as their clinical potential for the treatment of LPS-linked diseases through CASP4/11 inhibition. Here, we identify mitochondrial cardiolipin as a selective inhibitor of CASP4/11-dependent cell death and inflammatory cytokine secretion, without affecting CASP1 function. Cardiolipin targets the CARD domain of CASP4/11, impeding its interaction with LPS to restrain CASP4/11 activation, thereby suppressing LPS-induced systemic inflammation in vivo. By identifying cardiolipin as a selective inhibitor of CASP4/11, we provide an urgently needed tool for studying caspase-4/11 and noncanonical inflammasome functions in inflammatory pathways and LPS-induced pathogenesis.

Spatial transcriptomics (ST) offers enormous potential to decipher the biological and pathological heterogeneity in precious archival cancer tissues. Traditionally, these tissues have rarely been used and only examined at a low throughput, most commonly by histopathological staining. ST adds thousands of times as many molecular features to histopathological images, but critical technical issues and limitations require more assessment of how ST performs on fixed archival tissues. In this work, we addressed this in a cancer‐heterogeneity pipeline, starting with an exploration of the whole transcriptome by two sequencing‐based ST protocols capable of measuring coding and non‐coding RNAs. We optimised the two protocols to work with challenging formalin‐fixed paraffin‐embedded (FFPE) tissues, derived from skin. We then assessed alternative imaging methods, including multiplex RNAScope single‐molecule imaging and multiplex protein imaging (CODEX). We evaluated the methods’ performance for tissues stored from 4 to 14 years ago, covering a range of RNA qualities, allowing us to assess variation. In addition to technical performance metrics, we determined the ability of these methods to quantify tumour heterogeneity. We integrated gene expression profiles with pathological information, charting a new molecular landscape on the pathologically defined tissue regions. Together, this work provides important and comprehensive experimental technical perspectives to consider the applications of ST in deciphering the cancer heterogeneity in archived tissues. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Perioperative immune checkpoint inhibitor (ICI) trials for intermediate high-risk clear cell renal cell carcinoma (ccRCC) have failed to consistently demonstrate improved patient outcomes. These unsuccessful ICI trials suggest that the tumour infiltrating immunophenotypes, termed here as the immune cell types, states and their spatial location within the tumour microenvironment (TME), were unfavourable for ICI treatment. Defining the tumour infiltrating immune cells may assist with the identification of predictive immunophenotypes within the TME that are favourable for ICI treatment. To define the immunophenotypes within the ccRCC TME, fresh para-tumour (pTME, n = 2), low-grade (LG, n = 4, G1-G2) and high-grade (HG, n = 4, G3-G4) tissue samples from six patients with ccRCC presenting at a tertiary referral hospital underwent spatial transcriptomics sequencing (ST-seq). Within the generated ST-seq datasets, immune cell types and states, termed here as exhausted/pro-tumour state or non-exhausted/anti-tumour state, were identified using multiple publicly available single-cell RNA and T-cell receptor sequencing datasets as references. HG TMEs revealed abundant exhausted/pro-tumour immune cells with no consistent increase in expression of PD-1, PD-L1 and CTLA4 checkpoints and angiogenic genes. Additional HG TME immunophenotype characteristics included: pro-tumour tissue-resident monocytes with consistently increased expression of HAVCR2 and LAG3 checkpoints; an exhausted CD8⁺ T cells sub-population with stem-like progenitor gene expression; and pro-tumour tumour-associated macrophages and monocytes within the recurrent TME with the expression of TREM2. Whilst limited by a modest sample size, this study represents the largest ST-seq dataset on human ccRCC. Our study reveals that high-risk ccRCC TMEs are infiltrated by exhausted/pro-tumour immunophenotypes lacking specific checkpoint gene expression confirming that HG ccRCC TME are immunogenic but not ICI favourable.

Identifying tissue-specific molecular signatures of active regulatory elements is critical to understanding gene regulatory mechanisms. In this study, transcription start sites (TSS) and enhancers were identified using Cap analysis of gene expression (CAGE) across endometrial stromal cell (ESC) samples obtained from women with (n = 4) and without endometriosis (n = 4). ESC TSSs and enhancers were compared to those reported in other tissue and cell types in FANTOM5 and were integrated with RNA-seq and ATAC-seq data from the same samples for regulatory activity and network analyses. CAGE tag count differences between women with and without endometriosis were statistically tested and tags within close proximity to genetic variants associated with endometriosis risk were identified. Over 90% of tag clusters mapping to promoters were observed in cells and tissues in FANTOM5. However, some potential cell-type-specific promoters and enhancers were also observed. Regions of open chromatin identified using ATAC-seq provided further evidence of the active transcriptional regions identified by CAGE. Despite the small sample number, there was evidence of differences associated with endometriosis at 210 consensus clusters, including IGFBP5, CALD1 and OXTR. ESC TSSs were also located within loci associated with endometriosis risk from genome-wide association studies. This study provides novel evidence of transcriptional differences in endometrial stromal cells associated with endometriosis and provides a valuable cell-type specific resource of active TSSs and enhancers in endometrial stromal cells.

A variety of methods have been developed to demultiplex pooled samples in a single cell RNA sequencing (scRNA-seq) experiment which either require hashtag barcodes or sample genotypes prior to pooling. We introduce scSplit which utilizes genetic differences inferred from scRNA-seq data alone to demultiplex pooled samples. scSplit also enables mapping clusters to original samples. Using simulated, merged, and pooled multi-individual datasets, we show that scSplit prediction is highly concordant with demuxlet predictions and is highly consistent with the known truth in cell-hashing dataset. scSplit is ideally suited to samples without external genotype information and is available at: https://github.com/jon-xu/scSplit

Mechanically activated (MA) ion channels convert physical forces into electrical signals. Despite the importance of this function, the involvement of mechanosensitive ion channels in human disease is poorly understood. Here we report heterozygous missense mutations in the gene encoding the MA ion channel TMEM63A that result in an infantile disorder resembling a hypomyelinating leukodystrophy. Four unrelated individuals presented with congenital nystagmus, motor delay, and deficient myelination on serial scans in infancy, prompting the diagnosis of Pelizaeus-Merzbacher (like) disease. Genomic sequencing revealed all four individuals carry heterozygous missense variants in the pore-forming domain of TMEM63A. These variants were confirmed to have arisen de novo in three of the four individuals. While the physiological role of TMEM63A is incompletely understood, it is highly expressed in oligodendrocytes and it has recently been shown to be a mechanically activated (MA) ion channel. Using patch clamp electrophysiology, we demonstrated that each of the modelled variants results in strongly attenuated stretch-activated currents when expressed in naïve cells. Unexpectedly, the clinical evolution of all four individuals has been surprisingly favorable, with substantial improvements in neurological signs and developmental progression. In the three individuals with follow-up scans after four years of age, the myelin deficit had almost completely resolved. Our results suggest a previously unappreciated role for mechanosensitive ion channels in myelin development.

Objective To describe the leukodystrophy caused by pathogenic variants in LARS2 and KARS, encoding mitochondrial leucyl transfer RNA (tRNA) synthase and mitochondrial and cytoplasmic lysyl tRNA synthase, respectively. Methods We composed a group of 5 patients with leukodystrophy, in whom whole-genome or whole-exome sequencing revealed pathogenic variants in LARS2 or KARS. Clinical information, brain MRIs, and postmortem brain autopsy data were collected. We assessed aminoacylation activities of purified mutant recombinant mitochondrial leucyl tRNA synthase and performed aminoacylation assays on patients9 lymphoblasts and fibroblasts. Results Patients had a combination of early-onset deafness and later-onset neurologic deterioration caused by progressive brain white matter abnormalities on MRI. Female patients with LARS2 pathogenic variants had premature ovarian failure. In 2 patients, MRI showed additional signs of early-onset vascular abnormalities. In 2 other patients with LARS2 and KARS pathogenic variants, magnetic resonance spectroscopy revealed elevated white matter lactate, suggesting mitochondrial disease. Pathology in one patient with LARS2 pathogenic variants displayed evidence of primary disease of oligodendrocytes and astrocytes with lack of myelin and deficient astrogliosis. Aminoacylation activities of purified recombinant mutant leucyl tRNA synthase showed a 3-fold loss of catalytic efficiency. Aminoacylation assays on patients9 lymphoblasts and fibroblasts showed about 50% reduction of enzyme activity. Conclusion This study adds LARS2 and KARS pathogenic variants as gene defects that may underlie deafness, ovarian failure, and leukodystrophy with mitochondrial signature. We discuss the specific MRI characteristics shared by leukodystrophies caused by mitochondrial tRNA synthase defects. We propose to add aminoacylation assays as biochemical diagnostic tools for leukodystrophies.

A variety of experimental and computational methods have been developed to demultiplex samples from pooled individuals in a single-cell RNA sequencing (scRNA-Seq) experiment which either require adding information (such as hashtag barcodes) or measuring information (such as genotypes) prior to pooling. We introduce scSplit which utilises genetic differences inferred from scRNA-Seq data alone to demultiplex pooled samples. scSplit also extracts a minimal set of high confidence presence/absence genotypes in each cluster which can be used to map clusters to original samples. Using a range of simulated, merged individual-sample as well as pooled multi-individual scRNA-Seq datasets, we show that scSplit is highly accurate and concordant with demuxlet predictions. Furthermore, scSplit predictions are highly consistent with the known truth in cell-hashing dataset. We also show that multiplexed-scRNA-Seq can be used to reduce batch effects caused by technical biases. scSplit is ideally suited to samples for which external genome-wide genotype data cannot be obtained (for example non-model organisms), or for which it is impossible to obtain unmixed samples directly, such as mixtures of genetically distinct tumour cells, or mixed infections. scSplit is available at: https://github.com/jon-xu/scSplit