Linking methanotroph phenotypes to genotypes using a simple spatially resolved model ecosystem.

Connecting genes to phenotypic traits in bacteria is often challenging because of a lack of environmental context in laboratory settings. Laboratory-based model ecosystems offer a means to better account for environmental conditions compared with standard planktonic cultures and can help link genotypes and phenotypes. Here, we present a simple, cost-effective, laboratory-based model ecosystem to study aerobic methane-oxidizing bacteria (methanotrophs) within the methane-oxygen counter gradient typically found in the natural environment of these organisms. Culturing the methanotroph Methylomonas sp. strain LW13 in this system resulted in the formation of a distinct horizontal band at the intersection of the counter gradient, which we discovered was not due to increased numbers of bacteria at this location but instead to an increased amount of polysaccharides. We also discovered that different methanotrophic taxa form polysaccharide bands with distinct locations and morphologies when grown in the methane-oxygen counter gradient. By comparing transcriptomic data from LW13 growing within and surrounding this band, we identified genes upregulated within the band and validated their involvement in growth and band formation within the model ecosystem using knockout strains. Notably, deletion of these genes did not negatively affect growth using standard planktonic culturing methods. This work highlights the use of a laboratory-based model ecosystem that more closely mimics the natural environment to uncover bacterial phenotypes missing from standard laboratory conditions, and to link these phenotypes with their genetic determinants.

Federated Learning Survival Model and Potential Radiotherapy Decision Support Impact Assessment for Non-small Cell Lung Cancer Using Real-World Data.

AIMS: The objective of this study was to develop a two-year overall survival model for inoperable stage I-III non-small cell lung cancer (NSCLC) patients using routine radiation oncology data over a federated (distributed) learning network and evaluate the potential of decision support for curative versus palliative radiotherapy. METHODS: A federated infrastructure of data extraction, de-identification, standardisation, image analysis, and modelling was installed for seven clinics to obtain clinical and imaging features and survival information for patients treated in 2011-2019. A logistic regression model was trained for the 2011-2016 curative patient cohort and validated for the 2017-2019 cohort. Features were selected with univariate and model-based analysis and optimised using bootstrapping. System performance was assessed by the receiver operating characteristic (ROC) and corresponding area under curve (AUC), C-index, calibration metrics and Kaplan-Meier survival curves, with risk groups defined by model probability quartiles. Decision support was evaluated using a case-control analysis using propensity matching between treatment groups. RESULTS: 1655 patient datasets were included. The overall model AUC was 0.68. Fifty-eight percent of patients treated with palliative radiotherapy had a low-to-moderate risk prediction according to the model, with survival times not significantly different (p = 0.87 and 0.061) from patients treated with curative radiotherapy classified as high-risk by the model. When survival was simulated by risk group and model-indicated treatment, there was an estimated 11% increase in survival rate at two years (p < 0.01). CONCLUSION: Federated learning over multiple institution data can be used to develop and validate decision support systems for lung cancer while quantifying the potential impact of their use in practice. This paves the way for personalised medicine, where decisions can be based more closely on individual patient details from routine care.

Dosimetric Impact of Delineation and Motion Uncertainties on the Heart and Substructures in Lung Cancer Radiotherapy.

AIMS: Delineation variations and organ motion produce difficult-to-quantify uncertainties in planned radiation doses to targets and organs at risk. Similar to manual contouring, most automatic segmentation tools generate single delineations per structure; however, this does not indicate the range of clinically acceptable delineations. This study develops a method to generate a range of automatic cardiac structure segmentations, incorporating motion and delineation uncertainty, and evaluates the dosimetric impact in lung cancer. MATERIALS AND METHODS: Eighteen cardiac structures were delineated using a locally developed auto-segmentation tool. It was applied to lung cancer planning CTs for 27 curative (planned dose ≥50 Gy) cases, and delineation variations were estimated by using ten mapping-atlases to provide separate substructure segmentations. Motion-related cardiac segmentation variations were estimated by auto-contouring structures on ten respiratory phases for 9/27 cases that had 4D-planning CTs. Dose volume histograms (DVHs) incorporating these variations were generated for comparison. RESULTS: Variations in mean doses (Dmean), defined as the range in values across ten feasible auto-segmentations, were calculated for each cardiac substructure. Over the study cohort the median variations for delineation uncertainty and motion were 2.20-11.09 Gy and 0.72-4.06 Gy, respectively. As relative values, variations in Dmean were between 18.7%-65.3% and 7.8%-32.5% for delineation uncertainty and motion, respectively. Doses vary depending on the individual planned dose distribution, not simply on segmentation differences, with larger dose variations to cardiac structures lying within areas of steep dose gradient. CONCLUSION: Radiotherapy dose uncertainties from delineation variations and respiratory-related heart motion were quantified using a cardiac substructure automatic segmentation tool. This predicts the 'dose range' where doses to structures are most likely to fall, rather than single DVH curves. This enables consideration of these uncertainties in cardiotoxicity research and for future plan optimisation. The tool was designed for cardiac structures, but similar methods are potentially applicable to other OARs.

Focused Ultrasound Blood-Brain Barrier Opening Arrests the Growth and Formation of Cerebral Cavernous Malformations.

BACKGROUND: Cerebral cavernous malformations (CCM) are vascular lesions within the central nervous system, consisting of dilated and hemorrhage-prone capillaries. CCMs can cause debilitating neurological symptoms, and surgical excision or stereotactic radiosurgery are the only current treatment options. Meanwhile, transient blood-brain barrier opening (BBBO) with focused ultrasound (FUS) and microbubbles is now understood to exert potentially beneficial bioeffects, such as stimulation of neurogenesis and clearance of amyloid-ß. Here, we tested whether FUS BBBO could be deployed therapeutically to control CCM formation and progression in a clinically-representative murine model. METHODS: CCMs were induced in mice by postnatal, endothelial-specific Krit1 ablation. FUS was applied for BBBO with fixed peak-negative pressures (PNPs; 0.2-0.6 MPa) or passive cavitation detection-modulated PNPs. Magnetic resonance imaging (MRI) was used to target FUS treatments, evaluate safety, and measure longitudinal changes in CCM growth after BBBO. RESULTS: FUS BBBO elicited gadolinium accumulation primarily at the perilesional boundaries of CCMs, rather than lesion cores. Passive cavitation detection and gadolinium contrast enhancement were comparable in CCM and wild-type mice, indicating that Krit1 ablation does not confer differential sensitivity to FUS BBBO. Acutely, CCMs exposed to FUS BBBO remained structurally stable, with no signs of hemorrhage. Longitudinal MRI revealed that FUS BBBO halted the growth of 94% of CCMs treated in the study. At 1 month, FUS BBBO-treated lesions lost, on average, 9% of their pre-sonication volume. In contrast, non-sonicated control lesions grew to 670% of their initial volume. Lesion control with FUS BBBO was accompanied by a marked reduction in the area and mesenchymal appearance of Krit mutant endothelium. Strikingly, in mice receiving multiple BBBO treatments with fixed PNPs, de novo CCM formation was significantly reduced by 81%. Mock treatment plans on MRIs of patients with surgically inaccessible lesions revealed their lesions are amenable to FUS BBBO with current clinical technology. CONCLUSIONS: Our results establish FUS BBBO as a novel, non-invasive modality that can safely arrest murine CCM growth and prevent their de novo formation. As an incisionless, MR image-guided therapy with the ability to target eloquent brain locations, FUS BBBO offers an unparalleled potential to revolutionize the therapeutic experience and enhance the accessibility of treatments for CCM patients.

Magnetic Resonance Imaging of Mouse Cerebral Cavernomas Reveal Differential Lesion Progression and Variable Permeability to Gadolinium.

BACKGROUND: Cerebral cavernous malformations, also known as cavernous angiomas, are blood vessel abnormalities comprised of clusters of grossly enlarged and hemorrhage-prone capillaries. The prevalence in the general population, including asymptomatic cases, is estimated to be 0.5%. Some patients develop severe symptoms, including seizures and focal neurological deficits, whereas others remain asymptomatic. The causes of this remarkable presentation heterogeneity within a primarily monogenic disease remain poorly understood. METHODS: We established a chronic mouse model of cerebral cavernous malformations, induced by postnatal ablation of Krit1 with Pdgfb-CreERT2, and examined lesion progression in these mice with T2-weighted 7T magnetic resonance imaging (MRI). We also established a modified protocol for dynamic contrast-enhanced MRI and produced quantitative maps of gadolinium tracer gadobenate dimeglumine. After terminal imaging, brain slices were stained with antibodies against microglia, astrocytes, and endothelial cells. RESULTS: These mice develop cerebral cavernous malformations lesions gradually over 4 to 5 months of age throughout the brain. Precise volumetric analysis of individual lesions revealed nonmonotonous behavior, with some lesions temporarily growing smaller. However, the cumulative lesional volume invariably increased over time and after about 2 months followed a power trend. Using dynamic contrast-enhanced MRI, we produced quantitative maps of gadolinium in the lesions, indicating a high degree of heterogeneity in lesional permeability. MRI properties of the lesions were correlated with cellular markers for endothelial cells, astrocytes, and microglia. Multivariate comparisons of MRI properties of the lesions with cellular markers for endothelial and glial cells revealed that increased cell density surrounding lesions correlates with stability, whereas denser vasculature within and surrounding the lesions may correlate with high permeability. CONCLUSIONS: Our results lay a foundation for better understanding individual lesion properties and provide a comprehensive preclinical platform for testing new drug and gene therapies for controlling cerebral cavernous malformations.

Validation of a Fully Automated Hybrid Deep Learning Cardiac Substructure Segmentation Tool for Contouring and Dose Evaluation in Lung Cancer Radiotherapy.

BACKGROUND AND PURPOSE: Accurate and consistent delineation of cardiac substructures is challenging. The aim of this work was to validate a novel segmentation tool for automatic delineation of cardiac structures and subsequent dose evaluation, with potential application in clinical settings and large-scale radiation-related cardiotoxicity studies. MATERIALS AND METHODS: A recently developed hybrid method for automatic segmentation of 18 cardiac structures, combining deep learning, multi-atlas mapping and geometric segmentation of small challenging substructures, was independently validated on 30 lung cancer cases. These included anatomical and imaging variations, such as tumour abutting heart, lung collapse and metal artefacts. Automatic segmentations were compared with manual contours of the 18 structures using quantitative metrics, including Dice similarity coefficient (DSC), mean distance to agreement (MDA) and dose comparisons. RESULTS: A comparison of manual and automatic contours across all cases showed a median DSC of 0.75-0.93 and a median MDA of 2.09-3.34 mm for whole heart and chambers. The median MDA for great vessels, coronary arteries, cardiac valves, sinoatrial and atrioventricular conduction nodes was 3.01-8.54 mm. For the 27 cases treated with curative intent (planned target volume dose ≥50 Gy), the median dose difference was -1.12 to 0.57 Gy (absolute difference of 1.13-3.25%) for the mean dose to heart and chambers; and -2.25 to 4.45 Gy (absolute difference of 0.94-6.79%) for the mean dose to substructures. CONCLUSION: The novel hybrid automatic segmentation tool reported high accuracy and consistency over a validation set with challenging anatomical and imaging variations. This has promising applications in substructure dose calculations of large-scale datasets and for future studies on long-term cardiac toxicity.

Applications of focused ultrasound-mediated blood-brain barrier opening.

The blood brain barrier (BBB) plays a critically important role in the regulation of central nervous system (CNS) homeostasis, but also represents a major limitation to treatments of brain pathologies. In recent years, focused ultrasound (FUS) in conjunction with gas-filled microbubble contrast agents has emerged as a powerful tool for transiently and non-invasively disrupting the BBB in a targeted and image-guided manner, allowing for localized delivery of drugs, genes, or other therapeutic agents. Beyond the delivery of known therapeutics, FUS-mediated BBB opening also demonstrates the potential for use in neuromodulation and the stimulation of a range of cell- and tissue-level physiological responses that may prove beneficial in disease contexts. Clinical trials investigating the safety and efficacy of FUS-mediated BBB opening are well underway, and offer promising non-surgical approaches to treatment of devastating pathologies. This article reviews a range of pre-clinical and clinical studies demonstrating the tremendous potential of FUS to fundamentally change the paradigm of treatment for CNS diseases.

Maternal gut bacteria drive intestinal inflammation in offspring with neurodevelopmental disorders by altering the chromatin landscape of CD4+ T cells.

Children with autism spectrum disorders often display dysregulated immune responses and related gastrointestinal symptoms. However, the underlying mechanisms leading to the development of both phenotypes have not been elucidated. Here, we show that mouse offspring exhibiting autism-like phenotypes due to prenatal exposure to maternal inflammation were more susceptible to developing intestinal inflammation following challenges later in life. In contrast to its prenatal role in neurodevelopmental phenotypes, interleukin-17A (IL-17A) generated immune-primed phenotypes in offspring through changes in the maternal gut microbiota that led to postnatal alterations in the chromatin landscape of naive CD4+ T cells. The transfer of stool samples from pregnant mice with enhanced IL-17A responses into germ-free dams produced immune-primed phenotypes in offspring. Our study provides mechanistic insights into why children exposed to heightened inflammation in the womb might have an increased risk of developing inflammatory diseases in addition to neurodevelopmental disorders.

Patterns of palliative radiotherapy fractionation for brain metastases patients in New South Wales, Australia.

BACKGROUND AND PURPOSE: There is a paucity of studies examining variation in the use of palliative radiation therapy (RT) fractionation for brain metastases. The aim of this study is to assess variation in palliative RT fractionation given for brain metastases in New South Wales (NSW), Australia, and identify factors associated with variation. MATERIALS AND METHODS: This is a population-based cohort of patients who received whole brain RT (WBRT) for brain metastases (2009-2014), as captured in the NSW Central Cancer Registry. A logistic regression model was used to identify factors associated with fractionation type. RESULTS: Of the 2,698 patients that received WBRT, 1,389 courses (51%) were < 6 fractions, 1,050 courses (39%) were 6-10 fractions, and 259 courses (10%) were > 10 fractions. Older patients were more likely to be treated with shorter courses (P < 0.0001). Patients with primary lung cancers were more likely to receive shorter courses compared with other primary cancers (P < 0.0001). Patients without surgical excision were more likely to receive < 6 fractions compared to those who underwent surgical excision. Shorter courses were more likely to be delivered to patients with the most disadvantaged socioeconomic status (SES) compared with patients with the least disadvantaged SES (P < 0.0001). There were significant fluctuations in the proportion of courses using lower number of fractions over time from 2009 to 2014, but no apparent trend (P = 0.02). There was wide variation in the proportion of shorter courses across residence local health districts, ranging from 24% to 69% for < 6 fractions, 21% to 72% for 6-10 fractions, and 4% to 20% for > 10 fractions (P < 0.0001). CONCLUSION: This study has identified significant unwarranted variations in fractionation for WBRT in NSW. Accelerating the uptake of shorter fractionation regimens, if warranted through evidence, should be prioritised to enhance evidence-based care.

Patterns of use of palliative radiotherapy fractionation for bone metastases and 30-day mortality.

BACKGROUND AND PURPOSE: Adoption of single-fraction radiation therapy (SFRT) has not been universal in the palliative treatment of bone metastases, despite evidence supporting its safety and efficacy. The aim of this study was to assess SFRT use for bone metastases in New South Wales (NSW), Australia, and the rate of 30-day mortality (30DM). MATERIALS AND METHODS: This is a population-based cohort of patients who received palliative radiation therapy (RT) for bone metastases (2009-2014), as captured in the NSW Central Cancer Registry. A logistic regression model was used to identify factors associated with fractionation type. The proportion of patients dying within 30-days from treatment start date was calculated. RESULTS: Of the 14,602 courses of palliative RT delivered for bone metastases, 30% were SFRT. SFRT was more likely to be delivered to older patients: ≥80 years (34%) versus < 60 years (28%). Patients with lower socioeconomic status (SES) (35%) were more likely to receive SFRT compared with higher SES (25%). SFRT delivered to patients from outer regional area of residence (34%) were higher compared to those from the major city (29%). The proportion of SFRT delivered to patients with comorbidities ≥2 (34%) was higher than patients with no comorbidity (29%). SFRT was associated with higher 30DM of 21% compared with 11% for multi-fraction RT (MFRT). CONCLUSION: SFRT is underused for the treatment of bone metastases in NSW. This is an impetus to develop tools making SFRT obligatory in this setting unless there is good justification not to.

Author Correction: Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20098-z.

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.

Lateralized Expression of Cortical Perineuronal Nets during Maternal Experience is Dependent on MECP2.

Cortical neuronal circuits along the sensorimotor pathways are shaped by experience during critical periods of heightened plasticity in early postnatal development. After closure of critical periods, measured histologically by the formation and maintenance of extracellular matrix structures called perineuronal nets (PNNs), the adult mouse brain exhibits restricted plasticity and maturity. Mature PNNs are typically considered to be stable structures that restrict synaptic plasticity on cortical parvalbumin+ (PV+) GABAergic neurons. Changes in environment (i.e., novel behavioral training) or social contexts (i.e., motherhood) are known to elicit synaptic plasticity in relevant neural circuitry. However, little is known about concomitant changes in the PNNs surrounding the cortical PV+ GABAergic neurons. Here, we show novel changes in PNN density in the primary somatosensory cortex (SS1) of adult female mice after maternal experience [called surrogate (Sur)], using systematic microscopy analysis of a whole brain region. On average, PNNs were increased in the right barrel field and decreased in the left forelimb regions. Individual mice had left hemisphere dominance in PNN density. Using adult female mice deficient in methyl-CpG-binding protein 2 (MECP2), an epigenetic regulator involved in regulating experience-dependent plasticity, we found that MECP2 is critical for this precise and dynamic expression of PNN. Adult naive Mecp2-heterozygous (Het) females had increased PNN density in specific subregions in both hemispheres before maternal experience, compared with wild-type (WT) littermate controls. The laterality in PNN expression seen in naive Het (NH) was lost after maternal experience in Sur Het (SH) mice, suggesting possible intact mechanisms for plasticity. Together, our results identify subregion and hemisphere-specific alterations in PNN expression in adult females, suggesting extracellular matrix plasticity as a possible neurobiological mechanism for adult behaviors in rodents.

Sonoselective transfection of cerebral vasculature without blood-brain barrier disruption.

Treatment of many pathologies of the brain could be improved markedly by the development of noninvasive therapeutic approaches that elicit robust, endothelial cell-selective gene expression in specific brain regions that are targeted under MR image guidance. While focused ultrasound (FUS) in conjunction with gas-filled microbubbles (MBs) has emerged as a noninvasive modality for MR image-guided gene delivery to the brain, it has been used exclusively to transiently disrupt the blood-brain barrier (BBB), which may induce a sterile inflammation response. Here, we introduce an MR image-guided FUS method that elicits endothelial-selective transfection of the cerebral vasculature (i.e., "sonoselective" transfection), without opening the BBB. We first determined that activating circulating, cationic plasmid-bearing MBs with pulsed low-pressure (0.1 MPa) 1.1-MHz FUS facilitates sonoselective gene delivery to the endothelium without MRI-detectable disruption of the BBB. The degree of endothelial selectivity varied inversely with the FUS pressure, with higher pressures (i.e., 0.3-MPa and 0.4-MPa FUS) consistently inducing BBB opening and extravascular transfection. Bulk RNA sequencing analyses revealed that the sonoselective low-pressure regimen does not up-regulate inflammatory or immune responses. Single-cell RNA sequencing indicated that the transcriptome of sonoselectively transfected brain endothelium was unaffected by the treatment. The approach developed here permits targeted gene delivery to blood vessels and could be used to promote angiogenesis, release endothelial cell-secreted factors to stimulate nerve regrowth, or recruit neural stem cells.

Recent Advances in the Use of Focused Ultrasound for Magnetic Resonance Image-Guided Therapeutic Nanoparticle Delivery to the Central Nervous System.

Targeting systemically-administered drugs and genes to specific regions of the central nervous system (CNS) remains a challenge. With applications extending into numerous disorders and cancers, there is an obvious need for approaches that facilitate the delivery of therapeutics across the impervious blood-brain barrier (BBB). Focused ultrasound (FUS) is an emerging treatment method that leverages acoustic energy to oscillate simultaneously administered contrast agent microbubbles. This FUS-mediated technique temporarily disrupts the BBB, allowing ordinarily impenetrable agents to diffuse and/or convect into the CNS. Under magnetic resonance image guidance, FUS and microbubbles enable regional targeting-limiting the large, and potentially toxic, dosage that is often characteristic of systemically-administered therapies. Subsequent to delivery across the BBB, therapeutics face yet another challenge: penetrating the electrostatically-charged, mesh-like brain parenchyma. Non-bioadhesive, encapsulated nanoparticles can help overcome this additional barrier to promote widespread treatment in selected target areas. Furthermore, nanoparticles offer significant advantages over conventional systemically-administered therapeutics. Surface modifications of nanoparticles can be engineered to enhance targeted cellular uptake, and nanoparticle formulations can be tailored to control many pharmacokinetic properties such as rate of drug liberation, distribution, and excretion. For instance, nanoparticles loaded with gene plasmids foster relatively stable transfection, thus obviating the need for multiple, successive treatments. As the formulations and applications of these nanoparticles can vary greatly, this review article provides an overview of FUS coupled with polymeric or lipid-based nanoparticles currently utilized for drug delivery, diagnosis, and assessment of function in the CNS.

The population benefit of evidence-based radiotherapy: 5-Year local control and overall survival benefits.

BACKGROUND: To describe the population benefit of radiotherapy in a high-income setting if evidence-based guidelines were routinely followed. METHODS: Australian decision tree models were utilized. Radiotherapy alone (RT) benefit was defined as the absolute proportional benefit of radiotherapy compared with no treatment for radical indications, and of radiotherapy over surgery alone for adjuvant indications. Chemoradiotherapy (CRT) benefit was the absolute incremental benefit of concurrent chemoradiotherapy over RT. Five-year local control (LC) and overall survival (OS) benefits were measured. Citation databases were systematically queried for benefit data. Meta-analysis and sensitivity analysis were performed. FINDINGS: 48% of all cancer patients have indications for radiotherapy, 34% curative and 14% palliative. RT provides 5-year LC benefit in 10.4% of all cancer patients (95% Confidence Interval 9.3, 11.8) and 5-year OS benefit in 2.4% (2.1, 2.7). CRT provides 5-year LC benefit in an additional 0.6% of all cancer patients (0.5, 0.6), and 5-year OS benefit for an additional 0.3% (0.2, 0.4). RT benefit was greatest for head and neck (LC 32%, OS 16%), and cervix (LC 33%, OS 18%). CRT LC benefit was greatest for rectum (6%) and OS for cervix (3%) and brain (3%). Sensitivity analysis confirmed a robust model. INTERPRETATION: Radiotherapy provides significant 5-year LC and OS benefits as part of evidence-based cancer care. CRT provides modest additional benefits.

Can the Aviation Industry be Useful in Teaching Oncology about Safety?

Healthcare practitioners have long considered aviation as a domain from which much can be learned about safety. Over the past 30 years, attempts have been made to apply aviation safety-related concepts to healthcare. Although some applications have been successful, a few decades later, many healthcare safety experts have learned that the appeal of the aviation-healthcare analogy is an illusion. Both domains are so basically dissimilar that simple adoption of aviation concepts will not be successful. However, what has succeeded is healthcare's adaptation of specific aviation safety concepts. Three concepts, investment in safety, human factors and safety management systems, are described and examples are given of adapted applications to healthcare/clinical oncology. Finally, there is a need to ensure that these concepts are applied systematically throughout healthcare rather than sporadically and without a centralised mandate, to help ensure success and improved patient and provider safety.