Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.

BACKGROUND: The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency. METHOD: Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies. RESULT: We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration. CONCLUSION: Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.

Facial Function in Bell Palsy in a Cohort of Children Randomized to Prednisolone or Placebo 12 Months After Diagnosis.

BACKGROUND: Information on the medium-term recovery of children with Bell palsy or acute idiopathic lower motor neuron facial paralysis is limited. METHODS: We followed up children aged 6 months to <18 years with Bell palsy for 12 months after completion of a randomized trial on the use of prednisolone. We assessed facial function using the clinician-administered House-Brackmann scale and the modified parent-administered House-Brackmann scale. RESULTS: One hundred eighty-seven children were randomized to prednisolone (n = 93) or placebo (n = 94). At six months, the proportion of patients who had recovered facial function based on the clinician-administered House-Brackmann scale was 98% (n = 78 of 80) in the prednisolone group and 93% (n = 76 of 82) in the placebo group. The proportion of patients who had recovered facial function based on the modified parent-administered House-Brackmann scale was 94% (n = 75 of 80) vs 89% (n = 72 of 81) at six months (OR 1.88; 95% CI 0.60, 5.86) and 96% (n = 75 of 78) vs 92% (n = 73 of 79) at 12 months (OR 3.12; 95% CI 0.61, 15.98). CONCLUSIONS: Although the vast majority had complete recovery of facial function at six months, there were some children without full recovery of facial function at 12 months, regardless of prednisolone use.

Pain in children with Bell's palsy: secondary analysis of a randomised controlled trial.

OBJECTIVE: To describe the prevalence and severity of pain experienced by children with Bell's palsy over the first 6 months of illness and its association with the severity of facial paralysis. METHODS: This was a secondary analysis of data obtained in a phase III, triple-blinded, randomised, placebo-controlled trial of prednisolone for the treatment of Bell's palsy in children aged 6 months to <18 years conducted between 13 October 2015 and 23 August 2020 in Australia and New Zealand. Children were recruited within 72 hours of symptom onset and pain was assessed using a child-rated visual analogue scale (VAS), a child-rated Faces Pain Score-Revised (FPS-R) and/or a parent-rated VAS at baseline, and at 1, 3 and 6 months until recovered, and are reported combined across treatment groups. RESULTS: Data were available for 169 of the 187 children randomised from at least one study time point. Overall, 37% (62/169) of children reported any pain at least at one time point. The frequency of any pain reported using the child-rated VAS, child-rated FPS-R and parent-rated VAS was higher at the baseline assessment (30%, 23% and 27%, respectively) compared with 1-month (4%, 0% and 4%, respectively) and subsequent follow-up assessments. At all time points, the median pain score on all three scales was 0 (no pain). CONCLUSIONS: Pain in children with Bell's palsy was infrequent and primarily occurred early in the disease course and in more severe disease. The intensity of pain, if it occurs, is very low throughout the clinical course of disease. TRIAL REGISTRATION NUMBER: ACTRN12615000563561.

Hydraulic segmentation explains differences in loss of branch conductance caused by fire.

The hydraulic death hypothesis suggests that fires kill trees by damaging the plant's hydraulic continuum in addition to stem cambium. A corollary to this hypothesis is that plants that survive fires possess 'pyrohydraulic' traits that prevent heat-induced embolism formation in the xylem and aid post-fire survival. We examine whether hydraulic segmentation within stem xylem may act as such a trait. To do so, we measured the percentage loss of conductance (PLC) and vulnerability to embolism axially along segments of branches exposed to heat plumes in two differing species, fire-tolerant Eucalyptus cladocalyx F. Muell and fire-sensitive Kiggelaria africana L., testing model predictions that fire-tolerant species would exhibit higher degrees of hydraulic segmentation (greater PLC in the distal parts of the branch than the basal) than fire-intolerant species (similar PLC between segments). Following exposure to a heat plume, K. africana suffered between 73 and 84% loss of conductance in all branch segments, whereas E. cladocalyx had 73% loss of conductance in whole branches, including the distal tips, falling to 29% in the most basal part of the branch. There was no evidence for differences in resistance segmentation between the species, and there was limited evidence for differences in distal vulnerability to embolism across the branches. Hydraulic segmentation in E. cladocalyx may enable it to resprout effectively post-fire with a functional hydraulic system. The lack of hydraulic segmentation in K. africana reveals the need to understand possible trade-offs associated with hydraulic segmentation in long-lived woody species with respect to drought and fire.

Agreement Between House-Brackmann and Sunnybrook Facial Nerve Grading Systems in Bell's Palsy in Children: Secondary Analysis of a Randomized, Placebo-Controlled Multicenter Trial.

BACKGROUND: There is limited evidence on the use of facial nerve function grading scales in acute facial nerve paralysis in children. OBJECTIVE: To investigate the agreement between and the usability of the House-Brackmann and Sunnybrook scales in children with idiopathic facial paralysis (Bell's palsy) and to compare their ease of administration. METHODS: Data from a randomized controlled trial in children aged 6 months to <18 years with Bell's palsy was used. Children were recruited within 72 hours of symptom onset and assessed using the House-Brackmann and the Sunnybrook scales at baseline and at 1, 3, and 6 months until recovered. Agreement between the scales was assessed using the intraclass correlation coefficient (ICC) at each time point and using a Bland-Altman plot. Ease of administration was assessed using an 11-point Likert scale. RESULTS: Comparative data were available for 169 of the 187 children randomized. The ICC between the 2 scales across all time points was 0.92 (95% confidence interval [CI] 0.91-0.93), at baseline 0.37 (95% 0.25, 0.51), at 1 month 0.91 (95% CI 0.89-0.94), at 3 months 0.85 (95% CI 0.80-0.89), and at 6 months 0.96 (95% CI 0.95-0.97). The median score for the ease of administration for the House-Brackmann and Sunnybrook scales was 3 (interquartile range [IQR]: 1-5) and 7 (IQR: 4-8) respectively (P < .001, Wilcoxon signed-rank test). CONCLUSIONS: There was excellent agreement between House-Brackmann and Sunnybrook scales, with poorer agreement at baseline. Clinicians found the House-Brackmann scale easier to administer. These findings suggest that both scales can be applied in children.

Consistent responses to moisture stress despite diverse growth forms within mountain fynbos communities.

Understanding climate change impacts on the Cape Floristic Region requires improved knowledge of plant physiological responses to the environment. Studies examining physiological responses of mountain fynbos have consisted of campaign-based measurements, capturing snapshots of plant water relations and photosynthesis. We examine conclusions drawn from prior studies by tracking in situ physiological responses of three species, representing dominant growth forms (proteoid, ericoid, restioid), over 2 years using miniature continuous sap flow technology, long-term observations of leaf/culm water potential and gas exchange, and xylem vulnerability to embolism. We observed considerable inter-specific variation in the timing and extent of seasonal declines in productivity. Shallow-rooted Erica monsoniana exhibited steep within-season declines in sap flow and water potentials, and pronounced inter-annual variability in total daily sap flux (Js). Protea repens showed steady reductions in Js across both years, despite deeper roots and less negative water potentials. Cannomois congesta-a shallow-rooted restioid-was least negatively impacted. Following rehydrating rain at the end of summer, gas exchange recovery was lower in the drier year compared with the normal year, but did not differ between species. Loss of function in the drier year was partially accounted for by loss of xylem transport capacity in Erica and Cannomois, but not Protea. Hitherto unseen water use patterns, including inter-annual variability of gas exchange associated with contrasting water uptake properties, reveal that species use different mechanisms to cope with summer dry periods. Revealing physiological responses of key growth forms enhances predictions of plant function within mountain fynbos under future conditions.

Efficacy of Prednisolone for Bell Palsy in Children: A Randomized, Double-Blind, Placebo-Controlled, Multicenter Trial.

BACKGROUND AND OBJECTIVE: Corticosteroids are used to treat the early stages of idiopathic facial paralysis (Bell's palsy) in children, but their effectiveness is uncertain. We set out to determine if prednisolone improves the proportion of children with Bell's palsy with complete recovery at one month. METHODS: We conducted a double-blind, placebo-controlled, randomised trial of prednisolone in children presenting to emergency departments with Bell's palsy. Patients aged 6 months to less than 18 years, recruited within 72 hours after symptom onset, were randomly assigned to receive 10 days of treatment with oral prednisolone (approximately 1 mg/kg) or placebo. The primary outcome was complete recovery of facial function at 1 month rated on the House-Brackmann scale. Secondary outcomes included facial function, adverse events and pain up to 6 months. Target recruitment was n=540 (270 per group). RESULTS: Between 13 October 2015 to 23 August 2020, 187 children were randomised (94 to prednisolone and 93 to placebo) and included in the intention-to-treat analysis. At 1 month, the proportions of patients who had recovered facial function were 49% (n=43/87) in the prednisolone group compared with 57% (n=50/87) in the placebo group (risk difference -8.1%, 95% CI -22.8 to 6.7; adjusted odds ratio [aOR] 0.7, 95% CI 0.4 to 1.3). At 3 months these proportion were 90% (n=71/79) for the prednisolone group versus 85% (n=72/85) for the placebo group (risk difference 5.2%, 95%, CI -5.0 to 15.3; aOR 1.2, 95% CI 0.4 to 3.0) and at 6 months 99% (n=77/78) and 93% (n=76/82) respectively (risk difference 6.0%, 95% CI -0.1 to 12.2; aOR 3.0 95% CI 0.5 to 17.7) There were no serious adverse events and little evidence for group differences in secondary outcomes. DISCUSSION: In children with Bell's palsy the vast majority recover without treatment. The study, although underpowered, does not provide evidence that early treatment with prednisolone improves complete recovery. REGISTRATION: Registered with the Australian New Zealand Clinical Trials Registry ACTRN12615000563561, registered 1 June 2015, ://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=368505&isReview=true CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for children with Bell's palsy, prednisolone does not significantly change recovery of complete facial function at one month. However, the study lacked the precision to exclude an important harm or benefit from prednisolone.

A real-world comparison of in-person vs. virtual contraceptive care trainings for clinicians and support staff.

OBJECTIVES: To conduct an exploratory analysis comparing in-person vs. virtual training programs about contraceptive care among clinicians and staff at 14 healthcare agencies in Washington state. METHODS: Survey data from in-person trainings were collected between July 2019 and March 2020 and from virtual trainings between June 2020 and January 2021. PRIMARY OUTCOMES: changes in contraceptive knowledge, understanding of contraceptive counseling and care, and participant engagement and experience with the training. RESULTS: Post-survey response rates for in-person trainings were 82% for clinicians and 72% for support staff while post-survey response rates for virtual trainings were 48% for clinicians and 43% for staff. Average knowledge scores for in-person clinician trainings increased from 63% prior to training to 80% post-training (p < 0.05), knowledge scores for virtual clinician trainings increased from 72% to 86% (p < 0.05), and the pre-to-post change in scores between training modalities was similar (p > 0.05 for the score difference). Average knowledge scores among in-person support staff trainings increased from 63% to 84% (p < 0.05), scores among virtual support staff trainings increased from 68% to 87% (p < 0.05) and, again, the pre-to-post change in scores between training modalities was similar (p > 0.05 for the score difference). Only minimal differences in survey scores between modalities were observed on most measures of participant engagement and experience with the trainings (p > 0.05 for most score differences). CONCLUSIONS: These exploratory results suggest that in-person and virtual contraceptive care trainings yielded comparable results among both clinicians and support staff. IMPLICATIONS: Results from this post-hoc analysis of survey data suggest a general equivalency of effectiveness between in-person trainings and virtual trainings, although in-person trainings may be more satisfying or engaging for participants. Further work and research is needed to inform strategies for making virtual trainings more engaging and satisfying for participants.

Biome boundary maintained by intense belowground resource competition in world's thinnest-rooted plant community.

Recent findings point to plant root traits as potentially important for shaping the boundaries of biomes and for maintaining the plant communities within. We examined two hypotheses: 1) Thin-rooted plant strategies might be favored in biomes with low soil resources; and 2) these strategies may act, along with fire, to maintain the sharp boundary between the Fynbos and Afrotemperate Forest biomes in South Africa. These biomes differ in biodiversity, plant traits, and physiognomy, yet exist as alternative stable states on the same geological substrate and in the same climate conditions. We conducted a 4-y field experiment to examine the ability of Forest species to invade the Fynbos as a function of growth-limiting nutrients and belowground plant-plant competition. Our results support both hypotheses: First, we found marked biome differences in root traits, with Fynbos species exhibiting the thinnest roots reported from any biome worldwide. Second, our field manipulation demonstrated that intense belowground competition inhibits the ability of Forest species to invade Fynbos. Nitrogen was unexpectedly the resource that determined competitive outcome, despite the long-standing expectation that Fynbos is severely phosphorus constrained. These findings identify a trait-by-resource feedback mechanism, in which most species possess adaptive traits that modify soil resources in favor of their own survival while deterring invading species. Our findings challenge the long-held notion that biome boundaries depend primarily on external abiotic constraints and, instead, identify an internal biotic mechanism-a selective feedback among traits, plant-plant competition, and ecosystem conditions-that, along with contrasting fire regime, can act to maintain biome boundaries.

Parents' and carers' impression of "quality" within a Paediatric Emergency Department.

BACKGROUND: Quality improvement systems are needed to overcome the 'Quality Gap' - difference between evidence-based guidelines and the care delivered. While there are a large array of potential quality assurance measures exists in the Paediatric Emergency Department, parent's/carer's perception of these is unknown. This study aimed to identify what 'quality of care' means to parents/carers of Paediatric Emergency Department (PED) patients, further determine which aspects of these are most important to them. Also, to identify which of the existing PED quality measures are most important to parents/carers, and their preferred method of providing feedback. METHODS: A Modified Rand-Delphi study was performed with parents/carers as the expert group and consensus was obtained from them via three web-based surveys. All parents/carers of children attending a tertiary paediatric hospital during six-week in winter were eligible- no exclusions. Quality measures scoring at least 7 on a 9-point Likert scale during the final survey were considered "very important", while those scoring at least an 8 were considered "extremely important". RESULTS: One hundred four parents/carers responded from a total of 1095 participants. Parents/carers generated 527 free text entries, to the initial survey on what 'quality of care' means. These were mapped to 48 quality measure which they ranked on subsequent surveys. Eighteen quality measures were considered very important by at least 90% of respondents. Of these, six were considered extremely important by at least 70% of respondents: 'Thorough medical assessment' (84%); 'A triage system' (84%); 'Experienced and knowledgeable staff that are skilled in paediatrics' (77%); 'Resources and equipment available to provide care' (72%); and 'Clear follow up plans and reviews that are communicated and scheduled' (72%). Parents/carers considered existing quality measures as important with 'timely treatment of a critical condition' as the most important. Most participants preferred to provide anonymous feedback (N = 69, 66%), online (N = 77, 72%) after discharge (N = 82, 70%). CONCLUSION: We have elicited what 'quality of care' means to parents/carers, and which aspects are most important to them. Parents/carers consider commonly used PED quality measure as very important. However, they are less important than outcomes generated by themselves. Further parents/carers in this study preferred to provide feedback that was anonymous and electronically distributed after they leave the ED.

Does a trade-off between growth plasticity and resource conservatism mediate post-fire shrubland responses to rainfall seasonality?

Growth plasticity may allow fire-prone species to maximize their recovery rates during temporary, sporadic periods of rainfall availability in the post-fire environment. However, moisture-driven growth plasticity could be maladaptive in nutrient-limited environments that require tighter control of growth and resource use. We investigated whether a trade-off between plasticity and conservatism mediates growth responses to altered rainfall seasonality in neighbouring shrubland communities that occupy different soils. We monitored post-fire vegetation regrowth in two structurally similar, Mediterranean-type shrublands for 3 years. We investigated the effects of experimentally altered rainfall seasonality on post-fire species' growth rates. We found that moisture-driven growth plasticity was higher among species occupying the fertile soils of the renosterveld site relative to those occupying the nutrient-poor soils of the fynbos site. This resulted in higher overall responsiveness of post-fire recovery patterns in renosterveld to experimental shifts in rainfall seasonality. In post-fire shrubland communities, the trade-off between moisture-dependent growth plasticity and resource conservatism could be mediated by soil nutrient availability. Therefore, edaphic differences between structurally similar shrublands could lead to differences in their sensitivity to post-fire rainfall seasonality.

Risk of Leukemia in Children With Peripheral Facial Palsy.

Most children with peripheral facial palsy will not have a cause identified. Although leukemia can cause facial nerve palsy, the magnitude of the risk is unknown and recommendations for investigations are variable. We are currently conducting a randomized, placebo-controlled trial of prednisolone for the treatment of Bell's palsy in children within the Paediatric Research in Emergency Departments International Collaborative emergency research network. In the course of the assessment for eligibility of the trial, from 644 acute-onset facial palsy presentations we identified 5 children with previously undiagnosed leukemia. We estimate the rate of leukemia in children with acute-onset facial palsy who present to emergency departments to be 0.6% (95% confidence interval 0.2% to 1.6%). In accordance with these cases, we suggest consideration of a screening CBC count for acute-onset peripheral facial palsy presentations in children before initiation of corticosteroid treatment.

Seasonal variation in tap water δ2H and δ18O isotopes reveals two tap water worlds.

Stable isotope ratios of hydrogen and oxygen (δ2H and δ18O) in tap water provide important insights into the way that people interact with and manage the hydrological cycle. Understanding how these interactions vary through space and time allows for the management of these resources to be improved, and for isotope data to be useful in other disciplines. The seasonal variation of δ2H and δ18O in tap water within South Africa was assessed to identify municipalities that are supplied by seasonally invariant sources that have long residence periods, such as groundwater, and those supplied by sources that vary seasonally in a manner consistent with evapoconcentration, such as surface water-the proposed two tap water "worlds". Doing so allows for the cost-effective spatial interpolation of δ2H and δ18O values that likely reflect that of groundwater, removing the residual error introduced by other sources that are dependent on discrete, isolated factors that cannot be spatially generalised. Applying the proposed disaggregation may also allow for the efficient identification of municipalities that are dependent on highly variable or depleted surface water resources, which are more likely to be vulnerable to climate and demographic changes.

Emergency department utilisation by vulnerable paediatric populations during the COVID-19 pandemic.

OBJECTIVE: To determine if changes to community-based services have effected paediatric ED attendances for mental health issues and neonates during the COVID-19 pandemic. METHODS: Analysis of total presentations, presentations with a mental health diagnoses and presentation of neonates during the early stages of the pandemic compared with the previous year for four Victorian hospitals. RESULTS: There was a 47.2% decrease in total presentations compared with 2019, with a 35% increase in mental health diagnoses and a 2% increase in neonatal presentations. CONCLUSION: Vulnerable paediatric patients are seeking care elsewhere during the pandemic because of the closure of community services.

Parent perspectives on children and young people's mental health services in Victoria - What's wrong and how to fix it: A multi-site qualitative study.

AIM: The number of children and young people presenting to emergency departments (EDs) with anxiety and depression is increasing. We aimed to determine parent perspectives on: (i) barriers to accessing non-ED mental health services; and (ii) improving access in the paediatric mental health service system. METHODS: Qualitative study with parents of children and young people aged 0-19 years who attended one of four EDs across Victoria between October 2017 and September 2018 and received a primary diagnosis of anxiety or depression. EXCLUSION CRITERIA: child or young person without a parent/guardian, or presented with self-harm or suicide attempt. Eligible participants completed semi-structured phone interviews. Interviews were recorded and transcripts were coded and analysed using content analysis. RESULTS: A total of 72 parents completed interviews. The average child age was 14 years (standard deviation 2.5) and two thirds identified as female (64%). A total of 57% of children and young people presented with a primary diagnosis of anxiety. Parents reported barriers in accessing care including: service shortages and inaccessibility, underresourced schools, lack of clinician mental health expertise, lack of child-clinician rapport, inconsistent care, financial constraints, lack of mental health awareness among parents, and stigma. Parents want expanded and improved access to services, more respite and support services, supportive schools, and improved mental health education for parents. CONCLUSIONS: Parents of children and young people attending the ED for anxiety and depression are generally dissatisfied with services for child mental health. Solutions that enable parents to better care for their child in the community are needed to improve care.

Understanding parent-reported factors that influence children and young people's anxiety and depression presentations to emergency departments: A multi-site study.

OBJECTIVE: Victorian ED data show increased presentations for anxiety and depression in children. We aimed to determine parent-reported factors contributing to these presentations. METHODS: Qualitative study with parents of children and young people aged 0-17 years who attended one of four EDs across Victoria between October 2017 and September 2018 and received a primary diagnosis of anxiety or depression (excluding self-harm or suicide attempt). Eligible parents completed semi-structured phone interviews, which were audio-recorded and transcribed. Transcripts were coded and qualitatively analysed using thematic analysis. RESULTS: Seventy parents completed interviews. The average age of children and young people was 14 years (standard deviation 2.4) and 63% (n = 44) identified as female. Thirty (43%) children received a primary diagnosis of depression, compared to 40 (57%) children who received a primary diagnosis of anxiety. The majority of respondents were mothers (n = 59; 84%). Key themes as to why families presented to EDs included: listening to trusted professionals, desperation, a feeling of no alternative, respecting their child's need to feel safe and to rule out a potentially serious medical condition. CONCLUSIONS: Parents bring their children to the ED for many reasons. Policy makers, managers and clinicians should work with parents to develop alternative approaches that provide families with community-based support, particularly for younger children and after hours, in order to provide an appropriate source of care for children and young people with anxiety and depression.

Maintenance of active chromatin states by HMGN2 is required for stem cell identity in a pluripotent stem cell model.

BACKGROUND: Members of the HMGN protein family modulate chromatin structure and influence epigenetic modifications. HMGN1 and HMGN2 are highly expressed during early development and in the neural stem/progenitor cells of the developing and adult brain. Here, we investigate whether HMGN proteins contribute to the chromatin plasticity and epigenetic regulation that is essential for maintaining pluripotency in stem cells. RESULTS: We show that loss of Hmgn1 or Hmgn2 in pluripotent embryonal carcinoma cells leads to increased levels of spontaneous neuronal differentiation. This is accompanied by the loss of pluripotency markers Nanog and Ssea1, and increased expression of the pro-neural transcription factors Neurog1 and Ascl1. Neural stem cells derived from these Hmgn-knockout lines also show increased spontaneous neuronal differentiation and Neurog1 expression. The loss of HMGN2 leads to a global reduction in H3K9 acetylation, and disrupts the profile of H3K4me3, H3K9ac, H3K27ac and H3K122ac at the Nanog and Oct4 loci. At endodermal/mesodermal genes, Hmgn2-knockout cells show a switch from a bivalent to a repressive chromatin configuration. However, at neuronal lineage genes whose expression is increased, no epigenetic changes are observed and their bivalent states are retained following the loss of HMGN2. CONCLUSIONS: We conclude that HMGN1 and HMGN2 maintain the identity of pluripotent embryonal carcinoma cells by optimising the pluripotency transcription factor network and protecting the cells from precocious differentiation. Our evidence suggests that HMGN2 regulates active and bivalent genes by promoting an epigenetic landscape of active histone modifications at promoters and enhancers.

Ramification has little impact on shoot hydraulic efficiency in the sexually dimorphic genus Leucadendron (Proteaceae).

Despite the diversity of branching architectures in plants, the impact of this morphological variation on hydraulic efficiency has been poorly studied. Branch junctions are commonly thought to be points of high hydraulic resistance, but adjustments in leaf area or xylem conduit abundance or dimensions could compensate for the additional hydraulic resistance of nodal junctions at the level of the entire shoot. Here we used the sexually dimorphic genus Leucadendron (Proteaceae) to test whether variation in branch ramification impacts shoot hydraulic efficiency. We found that branch ramification was related to leaf traits via Corner's rules such that more highly ramified shoots had smaller leaves, but that branch ramification had little consistent impact on shoot hydraulic efficiency, whether measured on a leaf area or stem cross-sectional area basis. These results suggest that the presumed increase in resistance associated with branching nodes can be compensated by other adjustments at the shoot level (e.g. leaf area adjustments, increased ramification to add additional branches in parallel rather than in series) that maintain hydraulic efficiency at the level of the entire shoot. Despite large morphological differences between males and females in the genus Leucadendron, which are due to differences in pollination and reproduction between the sexes, the physiological differences between males and females are minimal.

Lidocaine and phenylephrine versus saline placebo nasal spray for the pain and distress of nasogastric tube insertion in young children and infants: a randomised, double-blind, controlled trial.

BACKGROUND: Nasogastric tube insertion is a common but distressing procedure in young children. We aimed to compare the efficacy of topical local anaesthetic and vasoconstrictor nasal spray with placebo for distress related to nasogastric tube insertion. METHODS: We did a prospective, randomised, controlled, double-blind, superiority trial in a single tertiary paediatric emergency department in Australia. Eligible participants were children aged 6 months to 5 years who were planned to have a nasogastric tube inserted as part of their emergency department treatment. Patients were assigned using computer-generated block randomisation to receive lidocaine and phenylephrine nasal spray (10 mg lidocaine and 1 mg phenylephrine for children weighing 6-12 kg; 20 mg lidocaine and 2 mg phenylephrine for children weighing >12 kg), or 0·9% sodium chloride placebo nasal spray, before nasogastric insertion. Guardians, observers, and proceduralists were all masked to the intervention. The primary outcome was procedure-related distress, measured using the Face, Legs, Activity, Cry, and Consolability (FLACC) scale during the final attempt at nasogastric tube insertion. All patients were included in the primary analysis (intention-to-treat). FLACC scores were compared using the Wilcoxon rank-sum test, and categorical outcomes were compared using χ2 or Fisher's exact tests as appropriate. This study is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12614000092695. FINDINGS: Between July 30, 2014, and Aug 17, 2017, 107 children in a convenience sample were randomly assigned to receive lidocaine and phenylephrine nasal spray (n=54) or placebo (n=53). Seven children did not receive study medication (six no longer required nasogastric tube insertion and one withdrew consent). 50 children received each intervention; all were included in the analysis. There was no difference in median FLACC score at time of nasogastric tube insertion (9 [IQR 7-10] for lidocaine and phenylephrine vs 9 [IQR 8-10] for placebo; median difference between groups -1, 95% CI -2·7 to 0·7, p=0·21). Adverse effects of the spray or nasogastric tube insertion (most commonly vomiting and gagging) occurred in 14 (28%) of those who received lidocaine and phenylephrine and 21 (42%) of those who received placebo. INTERPRETATION: Lidocaine and phenylephrine nasal spray does not reduce procedure-related distress associated with nasogastric tube insertion in young children compared with placebo. Further research addressing distress associated with nasogastric tube insertion in young children is needed. FUNDING: Monash Health Senior Medical Staff Association, Australasian College for Emergency Medicine, Emergency Medicine Research Foundation.

Impact of scribes on emergency medicine doctors' productivity and patient throughput: multicentre randomised trial.

OBJECTIVES: To evaluate the changes in productivity when scribes were used by emergency physicians in emergency departments in Australia and assess the effect of scribes on throughput. DESIGN: Randomised, multicentre clinical trial. SETTING: Five emergency departments in Victoria used Australian trained scribes during their respective trial periods. Sites were broadly representative of Australian emergency departments: public (urban, tertiary, regional referral, paediatric) and private, not for profit. PARTICIPANTS: 88 physicians who were permanent, salaried employees working more than one shift a week and were either emergency consultants or senior registrars in their final year of training; 12 scribes trained at one site and rotated to each study site. INTERVENTIONS: Physicians worked their routine shifts and were randomly allocated a scribe for the duration of their shift. Each site required a minimum of 100 scribed and non-scribed shifts, from November 2015 to January 2018. MAIN OUTCOME MEASURES: Physicians' productivity (total patients, primary patients); patient throughput (door-to-doctor time, length of stay); physicians' productivity in emergency department regions. Self reported harms of scribes were analysed, and a cost-benefit analysis was done. RESULTS: Data were collected from 589 scribed shifts (5098 patients) and 3296 non-scribed shifts (23 838 patients). Scribes increased physicians' productivity from 1.13 (95% confidence interval 1.11 to 1.17) to 1.31 (1.25 to 1.38) patients per hour per doctor, representing a 15.9% gain. Primary consultations increased from 0.83 (0.81 to 0.85) to 1.04 (0.98 to 1.11) patients per hour per doctor, representing a 25.6% gain. No change was seen in door-to-doctor time. Median length of stay reduced from 192 (interquartile range 108-311) minutes to 173 (96-208) minutes, representing a 19 minute reduction (P<0.001). The greatest gains were achieved by placing scribes with senior doctors at triage, the least by using them in sub-acute/fast track regions. No significant harm involving scribes was reported. The cost-benefit analysis based on productivity and throughput gains showed a favourable financial position with use of scribes. CONCLUSIONS: Scribes improved emergency physicians' productivity, particularly during primary consultations, and decreased patients' length of stay. Further work should evaluate the role of the scribe in countries with health systems similar to Australia's. TRIAL REGISTRATION: ACTRN12615000607572 (pilot site); ACTRN12616000618459.