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Rationale: The role of NADPH oxidase activation in pneumonia is complex because reactive oxygen species contribute to both microbial killing and regulation of the acute pulmonary infiltrate. The relative importance of each role remains poorly defined in community-acquired pneumonia.Objectives: We evaluated the contribution of NADPH oxidase-derived reactive oxygen species to the pathogenesis of pneumococcal pneumonia, addressing both the contribution to microbial killing and regulation of the inflammatory response.Methods: Mice deficient in the gp91(phox) component of the phagocyte NADPH oxidase were studied after pneumococcal challenge.Measurements and Main Results: gp91(phox)(-/-) mice demonstrated no defect in microbial clearance as compared with wild-type C57BL/6 mice. A significant increase in bacterial clearance from the lungs of gp91(phox)(-/-) mice was associated with increased numbers of neutrophils in the lung, lower rates of neutrophil apoptosis, and enhanced activation. Marked alterations in pulmonary cytokine/chemokine expression were also noted in the lungs of gp91(phox)(-/-) mice, characterized by elevated levels of tumor necrosis factor-alpha, KC, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and IL-6. The greater numbers of neutrophils in gp91(phox)(-/-) mice were not associated with increased lung injury. Levels of neutrophil elastase in bronchoalveolar lavage were not decreased in gp91(phox)(-/-) mice.Conclusions: During pneumococcal pneumonia, NADPH oxidase-derived reactive oxygen species are redundant for host defense but limit neutrophil recruitment and survival. Decreased NADPH oxidase-dependent reactive oxygen species production is well tolerated and improves disease outcome during pneumococcal pneumonia by removing neutrophils from the tight constraints of reactive oxygen species-mediated regulation. [ABSTRACT FROM AUTHOR]

Embryogenesis is a vulnerable time. Mutations in developmental cells can result in the wide dissemination of cells predisposed to disease within mature organs. We characterised the evolutionary history of four synchronous renal tumours from a 14-year-old girl using whole genome sequencing alongside single cell and bulk transcriptomic sequencing. Phylogenetic reconstruction timed the origin of all tumours to a multipotent embryonic cell committed to the right kidney, around 4 weeks post-conception. Biochemical and structural analysis of their shared MTOR mutation, absent from normal tissues, demonstrates enhanced protein flexibility, enabling a FAT domain hinge to dramatically increase activity of mTORC1 and mTORC2. Developmental mutations, not usually detected in traditional genetic screening, have vital clinical importance in guiding prognosis, targeted treatment, and family screening decisions for paediatric tumours., (© 2024. The Author(s).)

Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau ( VHL ) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL , and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL -null ccRCC cell lines and impairs tumour establishment and growth in vivo . This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL -null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling., Competing Interests: Declaration of interests The authors declare no competing interests.

New spatial molecular technologies are poised to transform our understanding and treatment of urological cancers. By mapping the spatial molecular architecture of tumours, these platforms uncover the complex heterogeneity within and around individual malignancies, offering novel insights into disease development, progression, diagnosis, and treatment. They enable tracking of clonal phylogenetics in situ and immune-cell interactions in the tumour microenvironment. A whole transcriptome/genome/proteome-level spatial analysis is hypothesis generating, particularly in the areas of risk stratification and precision medicine. Current challenges include reagent costs, harmonisation of protocols, and computational demands. Nonetheless, the evolving landscape of the technology and evolving machine learning applications have the potential to overcome these barriers, pushing towards a future of personalised cancer therapy, leveraging detailed spatial cellular and molecular data. PATIENT SUMMARY: Tumours are complex and contain many different components. Although we have been able to observe some of these differences visually under the microscope, until recently, we have not been able to observe the genetic changes that underpin cancer development. Scientists are now able to explore molecular/genetic differences using approaches such as "spatial transcriptomics" and "spatial proteomics", which allow them to see genetic and cellular variation across a region of normal and cancerous tissue without destroying the tissue architecture. Currently, these technologies are limited by high associated costs, and a need for powerful and complex computational analysis workflows. Future advancements and results through these new technologies may assist patients and their doctors as they make decisions about treating their cancer., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Clinical imaging tools to probe aggressiveness of renal masses are lacking, and T2-weighted imaging as an integral part of magnetic resonance imaging protocol only provides qualitative information. We developed high-resolution and accelerated T2 mapping methods based on echo merging and using k-t undersampling and reduced flip angles (TEMPURA) and tested their potential to quantify differences between renal tumour subtypes and grades., Methods: Twenty-four patients with treatment-naïve renal tumours were imaged: seven renal oncocytomas (RO); one eosinophilic/oncocytic renal cell carcinoma; two chromophobe RCCs (chRCC); three papillary RCCs (pRCC); and twelve clear cell RCCs (ccRCC). Median, kurtosis, and skewness of T2 were quantified in tumours and in the normal-adjacent kidney cortex and were compared across renal tumour subtypes and between ccRCC grades., Results: High-resolution TEMPURA depicted the tumour structure at improved resolution compared to conventional T2-weighted imaging. The lowest median T2 values were present in pRCC (high-resolution, 51 ms; accelerated, 45 ms), which was significantly lower than RO (high-resolution; accelerated, p = 0.012) and ccRCC (high-resolution, p = 0.019; accelerated, p = 0.008). ROs showed the lowest kurtosis (high-resolution, 3.4; accelerated, 4.0), suggestive of low intratumoural heterogeneity. Lower T2 values were observed in higher compared to lower grade ccRCCs (grades 2, 3 and 4 on high-resolution, 209 ms, 151 ms, and 106 ms; on accelerated, 172 ms, 160 ms, and 102 ms, respectively), with accelerated TEMPURA showing statistical significance in comparison (p = 0.037)., Conclusions: Both high-resolution and accelerated TEMPURA showed marked potential to quantify differences across renal tumour subtypes and between ccRCC grades., Trial Registration: ClinicalTrials.gov, NCT03741426 . Registered on 13 November 2018., Relevance Statement: The newly developed T 2 mapping methods have improved resolution, shorter acquisition times, and promising quantifiable readouts to characterise incidental renal masses., (© 2024. The Author(s).)

A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin's pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced.

Characterization of somatic mutations at single-cell resolution is essential to study cancer evolution, clonal mosaicism and cell plasticity. Here, we describe SComatic, an algorithm designed for the detection of somatic mutations in single-cell transcriptomic and ATAC-seq (assay for transposase-accessible chromatin sequence) data sets directly without requiring matched bulk or single-cell DNA sequencing data. SComatic distinguishes somatic mutations from polymorphisms, RNA-editing events and artefacts using filters and statistical tests parameterized on non-neoplastic samples. Using >2.6 million single cells from 688 single-cell RNA-seq (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) data sets spanning cancer and non-neoplastic samples, we show that SComatic detects mutations in single cells accurately, even in differentiated cells from polyclonal tissues that are not amenable to mutation detection using existing methods. Validated against matched genome sequencing and scRNA-seq data, SComatic achieves F1 scores between 0.6 and 0.7 across diverse data sets, in comparison to 0.2-0.4 for the second-best performing method. In summary, SComatic permits de novo mutational signature analysis, and the study of clonal heterogeneity and mutational burdens at single-cell resolution., (© 2023. The Author(s).)

Urinary tract infections (UTI) account for a substantial financial burden globally. Over 75% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which have demonstrated an extraordinarily rapid growth rate in vivo. This rapid growth rate appears paradoxical given that urine and the human urinary tract are relatively nutrient-restricted. Thus, we lack a fundamental understanding of how uropathogens propel growth in the host to fuel pathogenesis. Here, we used large in silico, in vivo, and in vitro screens to better understand the role of UPEC transport mechanisms and their contributions to uropathogenesis. In silico analysis of annotated transport systems indicated that the ATP-binding cassette (ABC) family of transporters was most conserved among uropathogenic bacterial species, suggesting their importance. Consistent with in silico predictions, we determined that the ABC family contributed significantly to fitness and virulence in the urinary tract: these were overrepresented as fitness factors in vivo (37.2%), liquid media (52.3%), and organ agar (66.2%). We characterized 12 transport systems that were most frequently defective in screening experiments by generating in-frame deletions. These mutant constructs were tested in urovirulence phenotypic assays and produced differences in motility and growth rate. However, deletion of multiple transport systems was required to achieve substantial fitness defects in the cochallenge murine model. This is likely due to genetic compensation among transport systems, highlighting the centrality of ABC transporters in these organisms. Therefore, these nutrient uptake systems play a concerted, critical role in pathogenesis and are broadly applicable candidate targets for therapeutic intervention., Competing Interests: Competing interests statement:The authors declare no competing interest.

Hot droughts, droughts attributed to below-average precipitation and exceptional warmth, are increasingly common in the twenty-first century, yet little is known about their effect on coniferous tree growth because of their historical rarity. In much of the American West, including California, radial tree growth is principally driven by precipitation, and narrow ring widths are typically associated with either drier or drought conditions. However, for species growing at high elevations (e.g., Larix lyalli, Pinus albicaulis), growth can be closely aligned with above-average temperatures with maximum growth coinciding with meteorological drought, suggesting that the growth effects of drought span from adverse to beneficial depending on location. Here, we compare radial growth responses of three high-elevation old-growth pines (Pinus jeffreyi, P. lambertiana, and P. contorta) growing in the San Jacinto Mountains, California, during a twenty-first-century hot drought (2000-2020) largely caused by exceptional warmth and a twentieth-century drought (1959-1966) principally driven by precipitation deficits. Mean radial growth during the hot drought was 12% above average while 18% below average during the mid-century drought illustrating that the consequences of environmental stress exhibit spatiotemporal variability. We conclude that the effects of hot droughts on tree growth in high-elevation forests may produce responses different than what is commonly associated with extended dry periods for much of western North America's forested lands at lower elevational ranges and likely applies to other mountainous regions (e.g., Mediterranean Europe) defined by summer-dry conditions. Thus, the climatological/biological interactions discovered in Southern California may offer clues to the unique nature of high-elevation forested ecosystems globally., (© 2024. The Author(s) under exclusive licence to International Society of Biometeorology.)

How individual T cells compete for and respond to IL-2 at the molecular level, and, as a consequence, how this shapes population dynamics and the selection of high-affinity clones is still poorly understood. Here we describe how the RNA binding protein ZFP36L1, acts as a sensor of TCR affinity to promote clonal expansion of high-affinity CD8 T cells. As part of an incoherent feed-forward loop, ZFP36L1 has a nonredundant role in suppressing multiple negative regulators of cytokine signaling and mediating a selection mechanism based on competition for IL-2. We suggest that ZFP36L1 acts as a sensor of antigen affinity and establishes the dominance of high-affinity T cells by installing a hierarchical response to IL-2., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Cognitive dysfunction following radiotherapy (RT) is one of the most common complications associated with RT delivered to the brain, but the precise mechanisms behind this dysfunction are not well understood, and to date, there are no preventative measures or effective treatments. To improve patient outcomes, a better understanding of the effects of radiation on the brain's functional systems is required. Functional magnetic resonance imaging (fMRI) has shown promise in this regard, however, compared to neural activity, hemodynamic measures of brain function are slow and indirect. Understanding how RT acutely and chronically affects functional brain organization requires more direct examination of temporally evolving neural dynamics as they relate to cerebral hemodynamics for bridging with human studies. In order to adequately study the underlying mechanisms of RT-induced cognitive dysfunction, the development of clinically mimetic RT protocols in animal models is needed. To address these challenges, we developed a fractionated whole-brain RT protocol (3Gy/day for 10 days) and applied longitudinal wide field optical imaging (WFOI) of neural and hemodynamic brain activity at 1, 2, and 3 months post RT. At each time point, mice were subject to repeated behavioral testing across a variety of sensorimotor and cognitive domains. Disruptions in cortical neuronal and hemodynamic activity observed 1 month post RT were significantly worsened by 3 months. While broad changes were observed in functional brain organization post RT, brain regions most impacted by RT occurred within those overlapping with the mouse default mode network and other association areas similar to prior reports in human subjects. Further, significant cognitive deficits were observed following tests of novel object investigation and responses to auditory and contextual cues after fear conditioning. Our results fill a much-needed gap in understanding the effects of whole-brain RT on systems level brain organization and how RT affects neuronal versus hemodynamic signaling in the cortex. Having established a clinically-relevant injury model, future studies can examine therapeutic interventions designed to reduce neuroinflammation-based injury following RT. Given the overlap of sequelae that occur following RT with and without chemotherapy, these tools can also be easily incorporated to examine chemotherapy-related cognitive impairment., (© 2023. The Author(s).)