PRECISION-TBI: a study protocol for a vanguard prospective cohort study to enhance understanding and management of moderate to severe traumatic brain injury in Australia.

INTRODUCTION: Traumatic brain injury (TBI) is a heterogeneous condition in terms of pathophysiology and clinical course. Outcomes from moderate to severe TBI (msTBI) remain poor despite concerted research efforts. The heterogeneity of clinical management represents a barrier to progress in this area. PRECISION-TBI is a prospective, observational, cohort study that will establish a clinical research network across major neurotrauma centres in Australia. This network will enable the ongoing collection of injury and clinical management data from patients with msTBI, to quantify variations in processes of care between sites. It will also pilot high-frequency data collection and analysis techniques, novel clinical interventions, and comparative effectiveness methodology. METHODS AND ANALYSIS: PRECISION-TBI will initially enrol 300 patients with msTBI with Glasgow Coma Scale (GCS) <13 requiring intensive care unit (ICU) admission for invasive neuromonitoring from 10 Australian neurotrauma centres. Demographic data and process of care data (eg, prehospital, emergency and surgical intervention variables) will be collected. Clinical data will include prehospital and emergency department vital signs, and ICU physiological variables in the form of high frequency neuromonitoring data. ICU treatment data will also be collected for specific aspects of msTBI care. Six-month extended Glasgow Outcome Scores (GOSE) will be collected as the key outcome. Statistical analysis will focus on measures of between and within-site variation. Reports documenting performance on selected key quality indicators will be provided to participating sites. ETHICS AND DISSEMINATION: Ethics approval has been obtained from The Alfred Human Research Ethics Committee (Alfred Health, Melbourne, Australia). All eligible participants will be included in the study under a waiver of consent (hospital data collection) and opt-out (6 months follow-up). Brochures explaining the rationale of the study will be provided to all participants and/or an appropriate medical treatment decision-maker, who can act on the patient's behalf if they lack capacity. Study findings will be disseminated by peer-review publications. TRIAL REGISTRATION NUMBER: NCT05855252.

High-quality full genome assembly of historic Xylella fastidiosa strains from ICMP collection using a hybrid sequencing approach.

High-quality complete genomes of five Xylella fastidiosa strains were assembled by combining Nanopore and Illumina sequencing data. Among these, International Collection of Micro-organisms from Plants (ICMP) 8731, ICMP 8742 and ICMP 8745 belong to subspecies fastidiosa while ICMP 8739 and ICMP 8740 were determined as subspecies multiplex. The strains were further classified into sequence types.

Comparative genomics and genomic diversity of Pseudomonas syringae clade 2b-a in Australia.

BACKGROUND: A zucchini disease outbreak with unusual symptoms associated with Pseudomonas syringae clade 2b was identified in Bundaberg, Australia during autumn 2016. To investigate the genetic diversity of the 11 Australian isolates obtained from the outbreak, the genomes were compared to the publicly available P. syringae strains in phylogroup 2. RESULTS: Average nucleotide identity refined the P. syringae clade 2b-a into four clusters (Cluster A, B, C1 and C2), an expansion from the previously identified A, B and C. Australian isolates were in Cluster A, C1 and C2. Genomic analyses highlighted several key factors that may contribute to the virulence of these isolates. Six orthologous groups, including three virulence factors, were associated with P. syringae phylogroup 2 cucurbit-infecting strains. A region of genome plasticity analysis identified a type VI secretion system pathway in clade 2b-a strains which could also contribute to virulence. Pathogenicity assays on isolates KL004-k1, KFR003-1 and 77-4C, as representative isolates of Cluster A, C1 and C2, respectively, determined that all three isolates can infect pumpkin, squash, watermelon and zucchini var. Eva with different levels of disease severity. Subsequently, type III effectors were investigated and four type III effectors (avrRpt2, hopZ5, hopC1 and hopH1) were associated with host range. The hopZ effector family was also predicted to be associated with disease severity. CONCLUSIONS: This study refined the taxonomy of the P. syringae clade 2b-a, supported the association between effector profile and pathogenicity in cucurbits established in a previous study and provides new insight into important genomic features of these strains. This study also provided a detailed and comprehensive resource for future genomic and functional studies of these strains.

Impact of fasting on the gastric volume of critically ill patients before extubation: a prospective observational study using gastric ultrasound.

Background: A period of fasting before tracheal extubation of ventilated patients in the ICU is common practice, aiming to reduce gastric volume and aspiration risk. As the volume of gastric content is unknown at the time of extubation, the efficacy of this practice is uncertain. Methods: A prospective, observational study using gastric ultrasound was undertaken. Images were obtained at four time points: (i) at baseline, with gastric feeds running; (ii) after suctioning of gastric contents through a gastric tube; (iii) after a 4 h period with no gastric feed running; and (iv) after both a 4 h fasting period and gastric tube suctioning. The primary outcome was the proportion of patients classed as low risk of aspiration with each intervention, using qualitative and quantitative gastric ultrasound. Results: Fifty-four patients in the ICU were enrolled. Forty-four (81%) subjects had images that were suitable for analysis. Suctioning of stomach content through a gastric tube and fasting were equivalent with 39/44 (88.6%) and 5/44 (11.4%) subjects classified as low risk and at risk of aspiration, respectively. A period of fasting followed by suction resulted in 41/44 (93.2%) patients being at low risk. Conclusions: Suctioning of stomach contents through the gastric tube and a 4 h fasting period appear equivalent at reducing gastric volume below a safe threshold. A small percentage did not reach the threshold despite all interventions.

Characterization of Pseudomonas syringae Isolated from Systemic Infection of Zucchini in Australia.

Zucchini plants with symptoms including twisted petioles, necrotic leaves, crown rot, and internal fruit rot were found in Bundaberg, Australia, at a commercial field for the first time during late autumn 2016, resulting in direct yield losses of 70 to 80%. Three Pseudomonas syringae strains isolated from symptomatic leaf (KL004-k1), crown (77-4C), and fruit (KFR003-1) were characterized and their pathogenicity evaluated on pumpkin, rockmelon, squash, and zucchini. Biochemical assays showed typical results for P. syringae. The three isolates differed, however, in that two produced fluorescent pigment (KFR003-1 and 77-4C) whereas the third, KL004-k1, was nonfluorescent. Multilocus sequence analysis classified the isolates to phylogroup 2b. The single-nucleotide polymorphism analysis of core genome from the Australian and closely related international isolates of P. syringae showed two separate clusters. The Australian isolates were clustered based on fluorescent phenotype. Pathogenicity tests demonstrated that all three isolates moved systemically within the inoculated plants and induced necrotic leaf symptoms in zucchini plants. Their identities were confirmed with specific PCR assays for P. syringae and phylogroup 2. Pathogenicity experiments also showed that the Eva variety of zucchini was more susceptible than the Rosa variety for all three isolates. Isolate KL004-k1 was more virulent than 77-4C on pumpkin, rockmelon, squash, and zucchini. This study expands the knowledge of P. syringae isolates that infect cucurbits and provides useful information for growers about the relative susceptibility of a range of cucurbit species.

Risk stratification in the investigation of pulmonary nodules in a high-risk cohort: positron emission tomography/computed tomography outperforms clinical risk prediction algorithms.

BACKGROUND: Clinical prediction models and 18-fluorine-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) are used for the assessment of solitary pulmonary nodules (SPN); however, a biopsy is still required before treatment, which carries risk. AIM: To determine the combined predictive benefit of one such model combined with modern PET/CT data to improve decision-making about biopsy prior to treatment and possibly reduce costs. METHODS: Patients with a SPN undergoing 18F-FDG-PET/CT from January 2011 to December 2012 were retrospectively identified; 143 patients met inclusion criteria. PET/CT studies were rated (5-point visual scale), and CT characteristics were determined. Tissue was obtained by endobronchial ultrasonography with guide sheath (EBUS-GS), CT-guided biopsy and/or surgery. EBUS-transbronchial needle aspiration (TBNA) was used instead of nodule biopsy if there were PET-positive sub-centimetre lymph nodes. RESULTS: The prediction model yielded an area under the receiver operating characteristic curve (AUC-ROC) of 64% (95% confidence interval (CI) 0.55-0.75). PET/CT increased this to 75% (95% CI 0.65-0.84). The 11% improvement is statistically significant. PET/CT score was the best single predictor for malignancy. A PET score of 1-2 had a specificity of 100% (CI 0.73-1.0), whereas a score of 4-5 had a sensitivity of only 76% (CI 0.68-0.84). No significant difference in clinical prediction scores between groups was noted. PET/CT showed the greatest benefit in true negatives and in detecting small mediastinal lymph nodes to allow EBUS-TBNA with a higher diagnostic rate. Cost analysis did not support a policy of resection-without-tissue diagnosis. CONCLUSION: PET/CT improves the clinical prediction of SPN, but its greatest use is in proving benignity. High PET scores had high false positive rates and did not add to clinical prediction. PET should be incorporated early in decision-making to allow for more effective biopsy strategies.

Surfactant protein-C chromatin-bound green fluorescence protein reporter mice reveal heterogeneity of surfactant protein C-expressing lung cells.

The regeneration of alveolar epithelial cells is a critical aspect of alveolar reorganization after lung injury. Although alveolar Type II (AT2) cells have been described as progenitor cells for alveolar epithelia, more remains to be understood about how their progenitor cell properties are regulated. A nuclear, chromatin-bound green fluorescence protein reporter (H2B-GFP) was driven from the murine surfactant protein-C (SPC) promoter to generate SPC H2B-GFP transgenic mice. The SPC H2B-GFP allele allowed the FACS-based enrichment and gene expression profiling of AT2 cells. Approximately 97% of AT2 cells were GFP-labeled on Postnatal Day 1, and the percentage of GFP-labeled AT2 cells decreased to approximately 63% at Postnatal Week 8. Isolated young adult SPC H2B-GFP(+) cells displayed proliferation, differentiation, and self-renewal capacity in the presence of lung fibroblasts in a Matrigel-based three-dimensional culture system. Heterogeneity within the GFP(+) population was revealed, because cells with distinct alveolar and bronchiolar gene expression arose in three-dimensional cultures. CD74, a surface marker highly enriched on GFP(+) cells, was identified as a positive selection marker, providing 3-fold enrichment for AT2 cells. In vivo, GFP expression was induced within other epithelial cell types during maturation of the distal lung. The utility of the SPC H2B-GFP murine model for the identification of AT2 cells was greatest in early postnatal lungs and more limited with age, when some discordance between SPC and GFP expression was observed. In adult mice, this allele may allow for the enrichment and future characterization of other SPC-expressing alveolar and bronchiolar cells, including putative stem/progenitor cell populations.

Enhanced shape-selective recognition of anion guests through complexation-induced organization of porphyrin hosts.

We present a fortuitous discovery of enhanced shape-selective recognition of anion guests that stems from a complexation-induced conformational change in porphyrin hosts upon anion binding. Porphyrin hosts reported here exist in a conformation that is not favorable to guest binding. Anions that bind strongly are those that can induce a conformational change in the host to allow guest binding. Furthermore, guests that mimic the shape of the newly formed pocket bind the strongest.

Lung stem cell self-renewal relies on BMI1-dependent control of expression at imprinted loci.

BMI1 is required for the self-renewal of stem cells in many tissues including the lung epithelial stem cells, Bronchioalveolar Stem Cells (BASCs). Imprinted genes, which exhibit expression from only the maternally or paternally inherited allele, are known to regulate developmental processes, but what their role is in adult cells remains a fundamental question. Many imprinted genes were derepressed in Bmi1 knockout mice, and knockdown of Cdkn1c (p57) and other imprinted genes partially rescued the self-renewal defect of Bmi1 mutant lung cells. Expression of p57 and other imprinted genes was required for lung cell self-renewal in culture and correlated with repair of lung epithelial cell injury in vivo. Our data suggest that BMI1-dependent regulation of expressed alleles at imprinted loci, distinct from imprinting per se, is required for control of lung stem cells. We anticipate that the regulation and function of imprinted genes is crucial for self-renewal in diverse adult tissue-specific stem cells.

Characterization of the cell of origin for small cell lung cancer.

Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.

Primary tumor genotype is an important determinant in identification of lung cancer propagating cells.

Successful cancer therapy requires the elimination or incapacitation of all tumor cells capable of regenerating a tumor. Therapeutic advances therefore necessitate the characterization of the cells that are able to propagate a tumor in vivo. We show an important link between tumor genotype and isolation of tumor-propagating cells (TPCs). Three mouse models of the most common form of human lung cancer each had TPCs with a unique cell-surface phenotype. The cell-surface marker Sca1 did not enrich for TPCs in tumors initiated with oncogenic Kras, and only Sca1-negative cells propagated EGFR mutant tumors. In contrast, Sca1-positive cells were enriched for tumor-propagating activity in Kras tumors with p53 deficiency. Primary tumors that differ in genotype at just one locus can therefore have tumor-propagating cell populations with distinct markers. Our studies show that the genotype of tumor samples must be considered in studies to identify, characterize, and target tumor-propagating cells.

Eye donation in Sydney metropolitan palliative care units.

Organ donation is generally accepted within the medical profession as a beneficial practice with demand continuing to exceed supply. For patients who are dying from cancer opportunities for organ donation are generally limited to eye donation. Between July 1, 2006 and 30 June 2007 over 2000 deaths occurred in nine palliative care units (PCUs) in metropolitan Sydney. Of these deaths only 50 patients became eye donors. Donors came from only four of the nine inpatient PCUs. Of these four, two provided nearly 90% of the eye donations. Only two PCUs in the Sydney metropolitan area provide significant numbers of eye donations. There are likely to be a number of factors contributing to the low rate of eye donation from PCUs and these are discussed in detail.

Intrinsic sensory deprivation induced by neonatal capsaicin treatment induces changes in rat brain and behaviour of possible relevance to schizophrenia.

Schizophrenia is considered to be a neurodevelopmental disorder with origins in the prenatal or neonatal period. Brains from subjects with schizophrenia have enlarged ventricles, reduced cortical thickness (CT) and increased neuronal density in the prefrontal cortex compared with those from normal subjects. Subjects with schizophrenia have reduced pain sensitivity and niacin skin flare responses, suggesting that capsaicin-sensitive primary afferent neurons might be abnormal in schizophrenia. This study tested the hypothesis that intrinsic somatosensory deprivation, induced by neonatal capsaicin treatment, causes changes in the brains of rats similar to those found in schizophrenia. Wistar rats were treated with capsaicin, 50 mg kg(-1) subcutaneously, or vehicle (control) at 24-36 h of life. At 5-7 weeks behavioural observations were made, and brains removed, fixed and sectioned. The mean body weight of capsaicin-treated rats was not significantly different from control, but the mean brain weight of male, but not female, rats, was significantly lower than control. Capsaicin-treated rats were hyperactive compared with controls. The hyperactivity was abolished by haloperidol. Coronal brain sections of capsaicin-treated rats had smaller cross-sectional areas, reduced CT, larger ventricles and aqueduct, smaller hippocampal area and reduced corpus callosum thickness, than brain sections from control rats. Neuronal density was increased in several cortical areas and the caudate putamen, but not in the visual cortex. It is concluded that neonatal capsaicin treatment of rats produces brain changes that are similar to those found in brains of subjects with schizophrenia.