An anciently diverged family of RNA binding proteins maintain correct splicing of a class of ultra-long exons through cryptic splice site repression.

Previously, we showed that the germ cell-specific nuclear protein RBMXL2 represses cryptic splicing patterns during meiosis and is required for male fertility (Ehrmann et al., 2019). Here, we show that in somatic cells the similar yet ubiquitously expressed RBMX protein has similar functions. RBMX regulates a distinct class of exons that exceed the median human exon size. RBMX protein-RNA interactions are enriched within ultra-long exons, particularly within genes involved in genome stability, and repress the selection of cryptic splice sites that would compromise gene function. The RBMX gene is silenced during male meiosis due to sex chromosome inactivation. To test whether RBMXL2 might replace the function of RBMX during meiosis we induced expression of RBMXL2 and the more distantly related RBMY protein in somatic cells, finding each could rescue aberrant patterns of RNA processing caused by RBMX depletion. The C-terminal disordered domain of RBMXL2 is sufficient to rescue proper splicing control after RBMX depletion. Our data indicate that RBMX and RBMXL2 have parallel roles in somatic tissues and the germline that must have been conserved for at least 200 million years of mammalian evolution. We propose RBMX family proteins are particularly important for the splicing inclusion of some ultra-long exons with increased intrinsic susceptibility to cryptic splice site selection.

SLC4A11 mediates ammonia import and promotes cancer stemness in hepatocellular carcinoma.

End stage liver disease is marked by portal hypertension, systemic elevations in ammonia, and development of hepatocellular carcinoma (HCC). While these clinical consequences of cirrhosis are well described, it remains poorly understood whether hepatic insufficiency and the accompanying elevations in ammonia contribute to HCC carcinogenesis. Using preclinical models, we discovered that ammonia entered the cell through the transporter SLC4A11 and served as a nitrogen source for amino acid and nucleotide biosynthesis. Elevated ammonia promoted cancer stem cell properties in vitro and tumor initiation in vivo. Enhancing ammonia clearance reduced HCC stemness and tumor growth. In patients, elevations in serum ammonia were associated with an increased incidence of HCC. Taken together, this study forms the foundation for clinical investigations using ammonia lowering agents as potential therapies to mitigate HCC incidence and aggressiveness.

Sialylation Inhibition Can Partially Revert Acquired Resistance to Enzalutamide in Prostate Cancer Cells.

Prostate cancer is a lethal solid malignancy and a leading cause of cancer-related deaths in males worldwide. Treatments, including radical prostatectomy, radiotherapy, and hormone therapy, are available and have improved patient survival; however, recurrence remains a huge clinical challenge. Enzalutamide is a second-generation androgen receptor antagonist that is used to treat castrate-resistant prostate cancer. Among patients who initially respond to enzalutamide, virtually all acquire secondary resistance, and an improved understanding of the mechanisms involved is urgently needed. Aberrant glycosylation, and, in particular, alterations to sialylated glycans, have been reported as mediators of therapy resistance in cancer, but a link between tumour-associated glycans and resistance to therapy in prostate cancer has not yet been investigated. Here, using cell line models, we show that prostate cancer cells with acquired resistance to enzalutamide therapy have an upregulation of the sialyltransferase ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) and increased levels of α2,6-sialylated N-glycans. Furthermore, using the sialyltransferase inhibitor P-SiaFNEtoc, we discover that acquired resistance to enzalutamide can be partially reversed by combining enzalutamide therapy with sialic acid blockade. Our findings identify a potential role for ST6GAL1-mediated aberrant sialylation in acquired resistance to enzalutamide therapy for prostate cancer and suggest that sialic acid blockade in combination with enzalutamide may represent a novel therapeutic approach in patients with advanced disease. Our study also highlights the potential to bridge the fields of cancer biology and glycobiology to develop novel combination therapies for prostate cancer.

Primary cilia signaling in astrocytes mediates development and regional-specific functional specification.

Astrocyte diversity is greatly influenced by local environmental modulation. Here we report that the majority of astrocytes across the mouse brain possess a singular primary cilium localized to the cell soma. Comparative single-cell transcriptomics reveals that primary cilia mediate canonical SHH signaling to modulate astrocyte subtype-specific core features in synaptic regulation, intracellular transport, energy and metabolism. Independent of canonical SHH signaling, primary cilia are important regulators of astrocyte morphology and intracellular signaling balance. Dendritic spine analysis and transcriptomics reveal that perturbation of astrocytic cilia leads to disruption of neuronal development and global intercellular connectomes in the brain. Mice with primary ciliary-deficient astrocytes show behavioral deficits in sensorimotor function, sociability, learning and memory. Our results uncover a critical role for primary cilia in transmitting local cues that drive the region-specific diversification of astrocytes within the developing brain.

Evaluating the evidence for genotype-informed Bayesian dosing of tacrolimus in children undergoing solid organ transplantation: A systematic literature review.

Tacrolimus, a calcineurin inhibitor, is a highly effective immunosuppressant used in solid organ transplantation (SOT). However, it is characterized by a narrow therapeutic range and high inter-patient variability in pharmacokinetics. Standard weight-based dosing followed by empiric dose titration is suboptimal in controlling drug concentrations, increasing risk of rejection or toxicity, particularly in the initial months post transplantation. This review explores the potential of combined pre-transplant genotyping and pharmacokinetic (PK) modelling to improve tacrolimus dosing in paediatric SOT recipients. A systematic search of Medline, Embase and Cochrane databases identified studies published between March 2013 and March 2023 that investigated genotype- and PK model-informed tacrolimus dosing in children post-SOT. The Newcastle-Ottawa Scale assessed study quality. Seven studies encompassing paediatric kidney, heart, liver and lung transplants reported using genotype and model-informed dosing. A combination of clinical and genetic factors significantly impacts tacrolimus clearance and thus initial dose recommendation. Body size, transplant organ and co-medications were consistently important, while either time post-transplant or haematocrit emerged in some studies. Several models were identified, however, with limitations evident in some and with absence of evidence for their effectiveness in optimizing initial and subsequent dosing. This review highlights the development of PK models in paediatric SOT that integrate genotype and clinical covariates to personalize early tacrolimus dosing. While promising, prospective studies are needed to validate and confirm their effectiveness in improving time to therapeutic concentrations and reducing under- or overexposure. This approach has the potential to optimize tacrolimus therapy in paediatric SOT, thereby improving outcomes.

Phase 2 Trial Assessing Toxicity of Personalized Response-Based Radiation Treatment in Patients With Locally Advanced Non-Small Cell Lung Cancer.

PURPOSE: Local failure rates after treatment for locally advanced non-small cell lung cancer (NSCLC) remain high. Efforts to improve local control with a uniform dose escalation or dose escalation to midtreatment positron emission tomography (PET)-avid residual disease have been limited by heightened toxicity. This trial aimed to refine response-based adaptive radiation therapy (RT) and minimize toxicity by incorporating fluorodeoxyglucose-PET (FDG-PET) and ventilation-perfusion single-photon emission computed tomography (SPECT) imaging midtreatment. METHODS AND MATERIALS: A total of 47 patients with stage IIA to III unresectable NSCLC were prospectively enrolled in this single-institution trial (NCT02492867). Patients received concurrent chemoradiation therapy with personalized response-based adaptive RT over 30 fractions incorporating ventilation-perfusion single-photon emission computed tomography and FDG-PET. The first 21 fractions (46.2 Gy at 2.2 Gy/fraction) were delivered to the tumor while minimizing the dose to the SPECT-defined functional lung. The plan was then adapted for the final 9 fractions (2.2-3.8 Gy/fraction) up to a total of 80.4 Gy, based on the midtreatment FDG-PET tumor response to escalate the dose to the residual tumor while minimizing the dose to the SPECT-defined functional lung. Nonprogressing patients received consolidative carboplatin, paclitaxel, or durvalumab. The primary endpoint of the study was ≥ grade 2 lung and esophageal toxicities. Secondary endpoints included time to local progression, tumor response, and overall survival. RESULTS: At 1 year posttreatment, the rates of grade 2 and grade 3 pneumonitis were 21.3% and 2.1%, respectively, with no difference in pneumonitis rates among patients who received and did not receive adjuvant durvalumab (P = .74). Although there were no grade 3 esophageal-related toxicities, 66.0% of patients experienced grade 2 esophagitis. The 1- and 2-year local control rates were 94.5% (95% CI, 87.4%-100%) and 87.5% (95% CI, 76.7%-100%), respectively. Overall survival was 82.8% (95% CI, 72.6%-94.4%) at 1 year and 62.3% (95% CI, 49.6%-78.3%) at 2 years. CONCLUSIONS: Response-based adaptive dose-escalation accounting for tumor change and normal tissue function during treatment provided excellent local control, comparable toxicity to standard chemoradiation therapy, and did not increase toxicity with adjuvant immunotherapy.

Outbreak of Ralstonia pickettii associated with contamination of saline products distributed internationally, the United Kingdom, 2024.

We describe an outbreak of Ralstonia pickettii in the United Kingdom, with isolates genetically indistinguishable from a 2023 Australian outbreak linked to internationally distributed saline solutions. Confirmed cases (n = 3) had bacteraemia, clinically relevant infection, indwelling venous lines and frequent healthcare contact. Multi-stakeholder intervention was required including product recall and risk communications. We recommend a low threshold for investigating clusters of Ralstonia species and similar opportunistic pathogens, considering contaminated product sources. Effective mitigation requires multi-agency partnership and international collaboration.

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry.

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically limited by the number of fluorescent parameters that can be imaged at a single time. Most immune cell subsets have been defined using flow cytometry by using complex combinations of protein markers, often requiring four or more parameters to conclusively identify, which is beyond the capabilities of most conventional microscopes. As flow cytometry dissociates tissues and loses spatial information, there is a need for techniques that can retain spatial information while interrogating the roles of complex cell types. These issues are addressed here by creating a method for expanding the number of fluorescent parameters that can be imaged by collecting the signals of spectrally overlapping fluorophores and using spectral unmixing to separate the signals of each individual fluorophore. These images are then processed using an analysis pipeline to take high-parameter histology images and extract single cells from these images so that the unique fluorescent properties of each cell can be analyzed at a single-cell level. Using flow cytometry-like gating strategies, cells can then be profiled into subsets and mapped back onto the histology sections to not only quantify their abundance, but also establish how they interact with the tissue environment. Overall, the simplicity and potential of using histoflow cytometry to study complex immune populations in histology sections is demonstrated.

Pharmacogenomic studies of fertility outcomes in pediatric cancer survivors - A systematic review.

For the same age, sex, and dosage, there can be significant variation in fertility outcomes in childhood cancer survivors. Genetics may explain this variation. This study aims to: (i) review the genetic contributions to infertility, (ii) search for pharmacogenomic studies looking at interactions of cancer treatment, genetic predisposition and fertility-related outcomes. Systematic searches in MEDLINE Ovid, Embase Classic+Embase, and PubMed were conducted using the following selection criteria: (i) pediatric, adolescent, and young adult cancer survivors, below 25 years old at the time of diagnosis, (ii) fertility outcome measures after cancer therapy, (iii) genetic considerations. Studies were excluded if they were (i) conducted in animal models, (ii) were not published in English, (iii) editorial letters, (iv) theses. Articles were screened in Covidence by at least two independent reviewers, followed by data extraction and a risk of bias assessment using the Quality in Prognostic Studies tool. Eight articles were reviewed with a total of 29 genes. Outcome measures included sperm concentration, azoospermia, AMH levels, assessment of premature menopause, ever being pregnant or siring a pregnancy. Three studies included replication cohorts, which attempted replication of SNP findings for NPY2R, BRSK1, FANCI, CYP2C19, CYP3A4, and CYP2B6. Six studies were rated with a high risk of bias. Differing methods may explain a lack of replication, and small cohorts may have contributed to few significant findings. Larger, prospective longitudinal studies with an unbiased genome-wide focus will be important to replicate significant results, which can be applied clinically.

Impact of lung cancer screening on stage migration and mortality among the national Veterans Health Administration population with lung cancer.

BACKGROUND: Despite randomized trials demonstrating a mortality benefit to low-dose computed tomography screening to detect lung cancer, uptake of lung cancer screening (LCS) has been slow, and the benefits of screening remain unclear in clinical practice. METHODS: This study aimed to assess the impact of screening among patients in the Veterans Health Administration (VA) health care system diagnosed with lung cancer between 2011 and 2018. Lung cancer stage at diagnosis, lung cancer-specific survival, and overall survival between patients with cancer who did and did not receive screening before diagnosis were evaluated. We used Cox regression modeling and inverse propensity weighting analyses with lead time bias adjustment to correlate LCS exposure with patient outcomes. RESULTS: Of 57,919 individuals diagnosed with lung cancer in the VA system between 2011 and 2018, 2167 (3.9%) underwent screening before diagnosis. Patients with screening had higher rates of stage I diagnoses (52% vs. 27%; p ≤ .0001) compared to those who had no screening. Screened patients had improved 5-year overall survival rates (50.2% vs. 27.9%) and 5-year lung cancer-specific survival (59.0% vs. 29.7%) compared to unscreened patients. Among screening-eligible patients who underwent National Comprehensive Cancer Network guideline-concordant treatment, screening resulted in substantial reductions in all-cause mortality (adjusted hazard ratio [aHR], 0.79; 95% confidence interval [CI], 0.67-0.92; p = .003) and lung-specific mortality (aHR, 0.61; 95% CI, 0.50-0.74; p < .001). CONCLUSIONS: While LCS uptake remains limited, screening was associated with earlier stage diagnoses and improved survival. This large national study corroborates the value of LCS in clinical practice; efforts to widely adopt this vital intervention are needed.

Minimising Adverse Drug Reactions and Verifying Economic Legitimacy-Pharmacogenomics Implementation in Children (MARVEL- PIC): protocol for a national randomised controlled trial of pharmacogenomics implementation.

INTRODUCTION: DNA-informed prescribing (termed pharmacogenomics, PGx) is the epitome of personalised medicine. Despite international guidelines existing, its implementation in paediatric oncology remains sparse. METHODS AND ANALYSIS: Minimising Adverse Drug Reactions and Verifying Economic Legitimacy-Pharmacogenomics Implementation in Children is a national prospective, multicentre, randomised controlled trial assessing the impact of pre-emptive PGx testing for actionable PGx variants on adverse drug reaction (ADR) incidence in patients with a new cancer diagnosis or proceeding to haematopoetic stem cell transplant. All ADRs will be prospectively collected by surveys completed by parents/patients using the National Cancer Institute Pediatric Patient Reported [Ped-PRO]-Common Terminology Criteria for Adverse Events (CTCAE) (weeks 1, 6 and 12). Pharmacist will assess for causality and severity in semistructured interviews using the CTCAE and Liverpool Causality Assessment Tool. The primary outcome is a reduction in ADRs among patients with actionable PGx variants, where an ADR will be considered as any CTCAE grade 2 and above for non-haematological toxicities and any CTCAE grade 3 and above for haematological toxicities Cost-effectiveness of pre-emptive PGx (secondary outcome) will be compared with standard of care using hospital inpatient and outpatient data along with the validated Childhood Health Utility 9D Instrument. Power and statistics considerations: A sample size of 440 patients (220 per arm) will provide 80% power to detect a 24% relative risk reduction in the primary endpoint of ADRs (two-sided α=5%, 80% vs 61%), allowing for 10% drop-out. ETHICS AND DISSEMINATION: The ethics approval of the trial has been obtained from the Royal Children's Hospital Ethics Committee (HREC/89083/RCHM-2022). The ethics committee of each participating centres nationally has undertaken an assessment of the protocol and governance submission. TRIAL REGISTRATION NUMBER: NCT05667766.

Spatial organisation of fungi in soil biocrusts of the Kalahari is related to bacterial community structure and may indicate ecological functions of fungi in drylands.

Biological soil crusts, or biocrusts, are microbial communities found in soil surfaces in drylands and in other locations where vascular plant cover is incomplete. They are functionally significant for numerous ecosystem services, most notably in the C fixation and storage due to the ubiquity of photosynthetic microbes. Whereas carbon fixation and storage have been well studied in biocrusts, the composition, function and characteristics of other organisms in the biocrust such as heterotrophic bacteria and especially fungi are considerably less studied and this limits our ability to gain a holistic understanding of biocrust ecology and function. In this research we characterised the fungal community in biocrusts developed on Kalahari Sand soils from a site in southwest Botswana, and combined these data with previously published bacterial community data from the same site. By identifying organisational patterns in the community structure of fungi and bacteria, we found fungi that were either significantly associated with biocrust or the soil beneath biocrusts, leading to the conclusion that they likely perform functions related to the spatial organisation observed. Furthermore, we showed that within biocrusts bacterial and fungal community structures are correlated with each other i.e., a change in the bacterial community is reflected by a corresponding change in the fungal community. Importantly, this correlation but that this correlation does not occur in nearby soils. We propose that different fungi engage in short-range and long-range interactions with dryland soil surface bacteria. We have identified fungi which are candidates for further studies into their potential roles in biocrust ecology at short ranges (e.g., processing of complex compounds for waste management and resource provisioning) and longer ranges (e.g., translocation of resources such as water and the fungal loop model). This research shows that fungi are likely to have a greater contribution to biocrust function and dryland ecology than has generally been recognised.

Sialic acid blockade inhibits the metastatic spread of prostate cancer to bone.

BACKGROUND: Bone metastasis is a common consequence of advanced prostate cancer. Bisphosphonates can be used to manage symptoms, but there are currently no curative treatments available. Altered tumour cell glycosylation is a hallmark of cancer and is an important driver of a malignant phenotype. In prostate cancer, the sialyltransferase ST6GAL1 is upregulated, and studies show ST6GAL1-mediated aberrant sialylation of N-glycans promotes prostate tumour growth and disease progression. METHODS: Here, we monitor ST6GAL1 in tumour and serum samples from men with aggressive prostate cancer and using in vitro and in vivo models we investigate the role of ST6GAL1 in prostate cancer bone metastasis. FINDINGS: ST6GAL1 is upregulated in patients with prostate cancer with tumours that have spread to the bone and can promote prostate cancer bone metastasis in vivo. The mechanisms involved are multi-faceted and involve modification of the pre-metastatic niche towards bone resorption to promote the vicious cycle, promoting the development of M2 like macrophages, and the regulation of immunosuppressive sialoglycans. Furthermore, using syngeneic mouse models, we show that inhibiting sialylation can block the spread of prostate tumours to bone. INTERPRETATION: Our study identifies an important role for ST6GAL1 and α2-6 sialylated N-glycans in prostate cancer bone metastasis, provides proof-of-concept data to show that inhibiting sialylation can suppress the spread of prostate tumours to bone, and highlights sialic acid blockade as an exciting new strategy to develop new therapies for patients with advanced prostate cancer. FUNDING: Prostate Cancer Research and the Mark Foundation For Cancer Research, the Medical Research Council and Prostate Cancer UK.

Reappraisal of sauropod dinosaur diversity in the Upper Cretaceous Winton Formation of Queensland, Australia, through 3D digitisation and description of new specimens.

Skeletal remains of sauropod dinosaurs have been known from Australia for over 100 years. Unfortunately, the classification of the majority of these specimens to species level has historically been impeded by their incompleteness. This has begun to change in the last 15 years, primarily through the discovery and description of several partial skeletons from the Cenomanian-lower Turonian (lower Upper Cretaceous) Winton Formation in central Queensland, with four species erected to date: Australotitan cooperensis, Diamantinasaurus matildae, Savannasaurus elliottorum, and Wintonotitan wattsi. The first three of these appear to form a clade (Diamantinasauria) of early diverging titanosaurs (or close relatives of titanosaurs), whereas Wintonotitan wattsi is typically recovered as a distantly related non-titanosaurian somphospondylan. Through the use of 3D scanning, we digitised numerous specimens of Winton Formation sauropods, facilitating enhanced comparison between type and referred specimens, and heretofore undescribed specimens. We present new anatomical information on the holotype specimen of Diamantinasaurus matildae, and describe new remains pertaining to twelve sauropod individuals. Firsthand observations and digital analysis enabled previously proposed autapomorphic features of all four named Winton Formation sauropod species to be identified in the newly described specimens, with some specimens exhibiting putative autapomorphies of more than one species, prompting a reassessment of their taxonomic validity. Supported by a specimen-level phylogenetic analysis, we suggest that Australotitan cooperensis is probably a junior synonym of Diamantinasaurus matildae, but conservatively regard it herein as an indeterminate diamantinasaurian, meaning that the Winton Formation sauropod fauna now comprises three (rather than four) valid diamantinasaurian species: Diamantinasaurus matildae, Savannasaurus elliottorum, and Wintonotitan wattsi, with the latter robustly supported as a member of the clade for the first time. We refer some of the newly described specimens to these three species and provide revised diagnoses, with some previously proposed autapomorphies now regarded as diamantinasaurian synapomorphies. Our newly presented anatomical data and critical reappraisal of the Winton Formation sauropods facilitates a more comprehensive understanding of the mid-Cretaceous sauropod palaeobiota of central Queensland.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Affinity Purification-Mass Spectrometry and Single Fiber Physiology/Proteomics Reveals Mechanistic Insights of C18ORF25.

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in the skeletal muscle, following acute exercise in humans. Phosphorylation was shown to improve the ex vivo skeletal muscle contractile function in mice, but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein/protein interactions. Several nucleocytoplasmic and contractile-associated proteins were identified, which revealed a subset of GTPases that associate with C18ORF25 in an AMPK- and S67 phosphorylation-dependent manner. We confirmed that C18ORF25 is localized to the nucleus and the contractile apparatus in the skeletal muscle. Mice lacking C18Orf25 display defects in calcium handling specifically in fast-twitch muscle fibers. To investigate these mechanisms, we developed an integrated single fiber physiology and single fiber proteomic platform. The approach enabled a detailed assessment of various steps in the excitation-contraction pathway including SR calcium handling and force generation, followed by paired single fiber proteomic analysis. This enabled us to identify >700 protein/phenotype associations and 36 fiber-type specific differences, following loss of C18Orf25. Taken together, our data provide unique insights into the function of C18ORF25 and its role in skeletal muscle physiology.