PADLOC: a web server for the identification of antiviral defence systems in microbial genomes.

Most bacteria and archaea possess multiple antiviral defence systems that protect against infection by phages, archaeal viruses and mobile genetic elements. Our understanding of the diversity of defence systems has increased greatly in the last few years, and many more systems likely await discovery. To identify defence-related genes, we recently developed the Prokaryotic Antiviral Defence LOCator (PADLOC) bioinformatics tool. To increase the accessibility of PADLOC, we describe here the PADLOC web server (freely available at https://padloc.otago.ac.nz), allowing users to analyse whole genomes, metagenomic contigs, plasmids, phages and archaeal viruses. The web server includes a more than 5-fold increase in defence system types detected (since the first release) and expanded functionality enabling detection of CRISPR arrays and retron ncRNAs. Here, we provide user information such as input options, description of the multiple outputs, limitations and considerations for interpretation of the results, and guidance for subsequent analyses. The PADLOC web server also houses a precomputed database of the defence systems in > 230,000 RefSeq genomes. These data reveal two taxa, Campylobacterota and Spriochaetota, with unusual defence system diversity and abundance. Overall, the PADLOC web server provides a convenient and accessible resource for the detection of antiviral defence systems.

3D bioprinting of tissue-specific osteoblasts and endothelial cells to model the human jawbone.

Jawbone differs from other bones in many aspects, including its developmental origin and the occurrence of jawbone-specific diseases like MRONJ (medication-related osteonecrosis of the jaw). Although there is a strong need, adequate in vitro models of this unique environment are sparse to date. While previous approaches are reliant e.g. on scaffolds or spheroid culture, 3D bioprinting enables free-form fabrication of complex living tissue structures. In the present work, production of human jawbone models was realised via projection-based stereolithography. Constructs were bioprinted containing primary jawbone-derived osteoblasts and vasculature-like channel structures optionally harbouring primary endothelial cells. After 28 days of cultivation in growth medium or osteogenic medium, expression of cell type-specific markers was confirmed on both the RNA and protein level, while prints maintained their overall structure. Survival of endothelial cells in the printed channels, co-cultured with osteoblasts in medium without supplementation of endothelial growth factors, was demonstrated. Constructs showed not only mineralisation, being one of the characteristics of osteoblasts, but also hinted at differentiation to an osteocyte phenotype. These results indicate the successful biofabrication of an in vitro model of the human jawbone, which presents key features of this special bone entity and hence appears promising for application in jawbone-specific research.

The Burramys Project: a conservationist's reach should exceed history's grasp, or what is the fossil record for?

The fossil record provides important information about changes in species diversity, distribution, habitat and abundance through time. As we understand more about these changes, it becomes possible to envisage a wider range of options for translocations in a world where sustainability of habitats is under increasing threat. The Critically Endangered alpine/subalpine mountain pygmy-possum, Burramys parvus (Marsupialia, Burramyidae), is threatened by global heating. Using conventional strategies, there would be no viable pathway for stopping this iconic marsupial from becoming extinct. The fossil record, however, has inspired an innovative strategy for saving this species. This lineage has been represented over 25 Myr by a series of species always inhabiting lowland, wet forest palaeocommunities. These fossil deposits have been found in what is now the Tirari Desert, South Australia (24 Ma), savannah woodlands of the Riversleigh World Heritage Area, Queensland (approx. 24-15 Ma) and savannah grasslands of Hamilton, Victoria (approx. 4 Ma). This palaeoecological record has led to the proposal overviewed here to construct a lowland breeding facility with the goal of monitoring the outcome of introducing this possum back into the pre-Quaternary core habitat for the lineage. If this project succeeds, similar approaches could be considered for other climate-change-threatened Australian species such as the southern corroboree frog (Pseudophryne corroboree) and the western swamp tortoise (Pseudemydura umbrina). This article is part of a discussion meeting issue 'The past is a foreign country: how much can the fossil record actually inform conservation?'

Inter-Rater Reliability and Impact of Disagreements on Acute Physiology and Chronic Health Evaluation IV Mortality Predictions.

Acute Physiology and Chronic Health Evaluation is a well-validated method to risk-adjust ICU patient outcomes. However, predictions may be affected by inter-rater reliability for manually entered elements. We evaluated inter-rater reliability for Acute Physiology and Chronic Health Evaluation IV manually entered elements among clinician abstractors and assessed the impacts of disagreements on mortality predictions. DESIGN: Cross-sectional. SETTING: Academic medical center. SUBJECTS: Patients admitted to five adult ICUs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Acute Physiology and Chronic Health Evaluation IV manually entered elements were abstracted from a selection of charts (n = 41) by two clinician "raters" trained in Acute Physiology and Chronic Health Evaluation IV methodology. Rater agreement (%) was determined for each manually entered element, including Acute Physiology and Chronic Health Evaluation diagnosis, Glasgow Coma Scale score, admission source, chronic conditions, elective/emergency surgery, and ventilator use. Cohen's kappa (K) or intraclass correlation coefficient was calculated for nominal and continuous manually entered elements, respectively. The impacts of manually entered element choices on Acute Physiology and Chronic Health Evaluation IV mortality predictions were computed using published Acute Physiology and Chronic Health Evaluation IV equations, and observed to expected hospital mortality ratios were compared between rater groups. The majority of manually entered element inconsistency was due to disagreement in choice of Glasgow Coma Scale (63.8% agreement, 0.83 intraclass correlation coefficient), Acute Physiology and Chronic Health Evaluation diagnosis (68.3% agreement, 0.67 kappa), and admission source (90.2% agreement, 0.85 kappa). The difference in predicted mortality between raters related to Glasgow Coma Scale disagreements was significant (observed to expected mortality ratios for Rater 1 [1.009] vs Rater 2 [1.134]; p < 0.05). Differences related to Acute Physiology and Chronic Health Evaluation diagnosis or admission source disagreements were negligible. The new "unable to score" choice for Glasgow Coma Scale was used for 18% of Glasgow Coma Scale measurements but accounted for 63% of "major" Glasgow Coma Scale disagreements, and 50% of the overall difference in Acute Physiology and Chronic Health Evaluation-predicted mortality between raters. CONCLUSIONS: Inconsistent use among raters of the new "unable to score" choice for Glasgow Coma Scale introduced in Acute Physiology and Chronic Health Evaluation IV was responsible for important decreases in both Glasgow Coma Scale and Acute Physiology and Chronic Health Evaluation IV mortality prediction reliability in our study. A Glasgow Coma Scale algorithm we developed after the study to improve reliability related to use of this new "unable to score" choice is presented.

Diagnosing and treating Enquiry Based Learning fatigue in Graduate Entry Nursing students.

The use of student directed study approaches such as Enquiry Based Learning (EBL) in the design and implementation of Graduate Entry Nursing Circular is well established. The rational relates to the maximisation of graduate attributes such as motivation to learn, the ability to identify, search and assimilate relevant literature and the desire to take ownership of the direction and pace of learning. Existing alongside this however, is the observation that students remain under confident in the application of knowledge to a clinical context and frustrated with learning approaches which do not appear directly related to improving their competence in this area. We suggest the result of this is a gradual disengagement and dissatisfaction the learning forum amongst students and faculty, which we have defined as EBL fatigue. The symptoms and consequences of EBL fatigue amongst students and faculty are discussed alongside strategies which we suggest may act as preventative measures in reducing the risk of a local epidemic.

Efficacy, dosage, and duration of action of branched chain amino Acid therapy for traumatic brain injury.

Traumatic brain injury (TBI) results in long-lasting cognitive impairments for which there is currently no accepted treatment. A well-established mouse model of mild to moderate TBI, lateral fluid percussion injury (FPI), shows changes in network excitability in the hippocampus including a decrease in net synaptic efficacy in area CA1 and an increase in net synaptic efficacy in dentate gyrus. Previous studies identified a novel therapy consisting of branched chain amino acids (BCAAs), which restored normal mouse hippocampal responses and ameliorated cognitive impairment following FPI. However, the optimal BCAA dose and length of treatment needed to improve cognitive recovery is unknown. In the current study, mice underwent FPI then consumed 100 mM BCAA supplemented water ad libitum for 2, 3, 4, 5, and 10 days. BCAA therapy ameliorated cognitive impairment at 5 and 10 days duration. Neither BCAA supplementation at 50 mM nor BCAAs when dosed 5 days on then 5 days off was sufficient to ameliorate cognitive impairment. These results suggest that brain injury causes alterations in hippocampal function, which underlie and contribute to hippocampal cognitive impairment, which are reversible with at least 5 days of BCAA treatment, and that sustaining this effect is dependent on continuous treatment. Our findings have profound implications for the clinical investigation of TBI therapy.

Multiplexed modulation of behavioral choice.

Stimuli in the environment, as well as internal states, influence behavioral choice. Of course, animals are often exposed to multiple external and internal factors simultaneously, which makes the ultimate determinants of behavior quite complex. We observed the behavioral responses of European leeches, Hirudo verbana, as we varied one external factor (surrounding water depth) with either another external factor (location of tactile stimulation along the body) or an internal factor (body distention following feeding). Stimulus location proved to be the primary indicator of behavioral response. In general, anterior stimulation produced shortening behavior, midbody stimulation produced local bending, and posterior stimulation usually produced either swimming or crawling but sometimes a hybrid of the two. By producing a systematically measured map of behavioral responses to body stimulation, we found wide areas of overlap between behaviors. When we varied the surrounding water depth, this map changed significantly, and a new feature - rotation of the body along its long axis prior to swimming - appeared. We found additional interactions between water depth and time since last feeding. A large blood meal initially made the animals crawl more and swim less, an effect that was attenuated as water depth increased. The behavioral map returned to its pre-feeding form after approximately 3 weeks as the leeches digested their blood meal. In summary, we found multiplexed impacts on behavioral choice, with the map of responses to tactile stimulation modified by water depth, which itself modulated the impact that feeding had on the decision to swim or crawl.

Systems biology of monovalent cation homeostasis in yeast: the translucent contribution.

Maintenance of monovalent cation homeostasis (mainly K(+) and Na(+)) is vital for cell survival, and cation toxicity is at the basis of a myriad of relevant phenomena, such as salt stress in crops and diverse human diseases. Full understanding of the importance of monovalent cations in the biology of the cell can only be achieved from a systemic perspective. Translucent is a multinational project developed within the context of the SysMO (System Biology of Microorganisms) initiative and focussed in the study of cation homeostasis using the well-known yeast Saccharomyces cerevisiae as a model. The present review summarize how the combination of biochemical, genetic, genomic and computational approaches has boosted our knowledge in this field, providing the basis for a more comprehensive and coherent vision of the role of monovalent cations in the biology of the cell.

Assessing the efficacy of oral immunotherapy for the desensitisation of peanut allergy in children (STOP II): a phase 2 randomised controlled trial.

BACKGROUND: Small studies suggest peanut oral immunotherapy (OIT) might be effective in the treatment of peanut allergy. We aimed to establish the efficacy of OIT for the desensitisation of children with allergy to peanuts. METHODS: We did a randomised controlled crossover trial to compare the efficacy of active OIT (using characterised peanut flour; protein doses of 2-800 mg/day) with control (peanut avoidance, the present standard of care) at the NIHR/Wellcome Trust Cambridge Clinical Research Facility (Cambridge, UK). Randomisation (1:1) was by use of an audited online system; group allocation was not masked. Eligible participants were aged 7-16 years with an immediate hypersensitivity reaction after peanut ingestion, positive skin prick test to peanuts, and positive by double-blind placebo-controlled food challenge (DBPCFC). We excluded participants if they had a major chronic illness, if the care provider or a present household member had suspected or diagnosed allergy to peanuts, or if there was an unwillingness or inability to comply with study procedures. Our primary outcome was desensitisation, defined as negative peanut challenge (1400 mg protein in DBPCFC) at 6 months (first phase). Control participants underwent OIT during the second phase, with subsequent DBPCFC. Immunological parameters and disease-specific quality-of-life scores were measured. Analysis was by intention to treat. Fisher's exact test was used to compare the proportion of those with desensitisation to peanut after 6 months between the active and control group at the end of the first phase. This trial is registered with Current Controlled Trials, number ISRCTN62416244. FINDINGS: The primary outcome, desensitisation, was recorded for 62% (24 of 39 participants; 95% CI 45-78) in the active group and none of the control group after the first phase (0 of 46; 95% CI 0-9; p<0·001). 84% (95% CI 70-93) of the active group tolerated daily ingestion of 800 mg protein (equivalent to roughly five peanuts). Median increase in peanut threshold after OIT was 1345 mg (range 45-1400; p<0·001) or 25·5 times (range 1·82-280; p<0·001). After the second phase, 54% (95% CI 35-72) tolerated 1400 mg challenge (equivalent to roughly ten peanuts) and 91% (79-98) tolerated daily ingestion of 800 mg protein. Quality-of-life scores improved (decreased) after OIT (median change -1·61; p<0·001). Side-effects were mild in most participants. Gastrointestinal symptoms were, collectively, most common (31 participants with nausea, 31 with vomiting, and one with diarrhoea), then oral pruritus after 6·3% of doses (76 participants) and wheeze after 0·41% of doses (21 participants). Intramuscular adrenaline was used after 0·01% of doses (one participant). INTERPRETATION: OIT successfully induced desensitisation in most children within the study population with peanut allergy of any severity, with a clinically meaningful increase in peanut threshold. Quality of life improved after intervention and there was a good safety profile. Immunological changes corresponded with clinical desensitisation. Further studies in wider populations are recommended; peanut OIT should not be done in non-specialist settings, but it is effective and well tolerated in the studied age group. FUNDING: MRC-NIHR partnership.

Student nurses in critical care: benefits and challenges of critical care as a learning environment for student nurses.

AIM: This article is a literature view of student nurses' experiences of critical care and the value of their experience to the nursing community. To explore the literature in relation to the value of critical care as a learning environment, the experiences and perceptions of student nurses in critical care and the impact of mentors on the experience. BACKGROUND: The shortage of critical care nurses and increasing numbers of acutely ill patients being cared for outside critical care areas has highlighted the importance of nurses being equipped with appropriate knowledge and skills to care for such patients. Student nurses have been experiencing critical care for many years and though it is accepted that it can be a stressful environment, there has been little empirical evidence of this. As anxiety has been said to interfere with learning, how much of an impact can this have and can anything be done about it? SEARCH STRATEGY: Using Medline, CINAHL and British Nursing Index (BNI) and search terms including nursing students or student nurses, critical or intensive care and perceptions, expectations or pre-conceptions, a literature search was carried out. Some articles explored registered nurses experiences, despite the titles, so this was set as an exclusion criteria. CONCLUSIONS: Critical care offers a wealth of learning opportunities which are only available in high acuity areas. Though students can be very anxious before commencing their placements, mentors and higher education play an important part in helping them overcome this. The challenge is how to work together to provide effective support systems for the benefit of the students.

Augmented Inhibition from Cannabinoid-Sensitive Interneurons Diminishes CA1 Output after Traumatic Brain Injury.

The neurological impairments associated with traumatic brain injury include learning and memory deficits and increased risk of seizures. The hippocampus is critically involved in both of these phenomena and highly susceptible to damage by traumatic brain injury. To examine network activity in the hippocampal CA1 region after lateral fluid percussion injury, we used a combination of voltage-sensitive dye, field potential, and patch clamp recording in mouse hippocampal brain slices. When the stratum radiatum (SR) was stimulated in slices from injured mice, we found decreased depolarization in SR and increased hyperpolarization in stratum oriens (SO), together with a decrease in the percentage of pyramidal neurons firing stimulus-evoked action potentials. Increased hyperpolarization in SO persisted when glutamatergic transmission was blocked. However, we found no changes in SO responses when the alveus was stimulated to directly activate SO. These results suggest that the increased SO hyperpolarization evoked by SR stimulation was mediated by interneurons that have cell bodies and/or axons in SR, and form synapses in stratum pyramidale and SO. A low concentration (100 nM) of the synthetic cannabinoid WIN55,212-2, restored CA1 output in slices from injured animals. These findings support the hypothesis that increased GABAergic signaling by cannabinoid-sensitive interneurons contributes to the reduced CA1 output following traumatic brain injury.

The Universal Form of Treatment Options (UFTO) as an alternative to Do Not Attempt Cardiopulmonary Resuscitation (DNACPR) orders: a mixed methods evaluation of the effects on clinical practice and patient care.

AIMS: To determine whether the introduction of the Universal Form of Treatment Options (the UFTO), as an alternative approach to Do Not Attempt Cardiopulmonary Resuscitation (DNACPR) orders, reduces harms in patients in whom a decision not to attempt cardiopulmonary resuscitation (CPR) was made, and to understand the mechanism for any observed change. METHODS: A mixed-methods before-and-after study with contemporaneous case controls was conducted in an acute hospital. We examined DNACPR (103 patients with DNACPR orders in 530 admissions) and UFTO (118 decisions not to attempt resuscitation in 560 admissions) practice. The Global Trigger Tool was used to quantify harms. Qualitative interviews and observations were used to understand mechanisms and effects. RESULTS: RATE OF HARMS IN PATIENTS FOR WHOM THERE WAS A DOCUMENTED DECISION NOT TO ATTEMPT CPR WAS REDUCED: Rate difference per 1000 patient-days was 12.9 (95% CI: 2.6-23.2, p-value=0.01). There was a difference in the proportion of harms contributing to patient death in the two periods (23/71 in the DNACPR period to 4/44 in the UFTO period (95% CI 7.8-36.1, p-value=0.006). Significant differences were maintained after adjustment for known confounders. No significant change was seen on contemporaneous case control wards. Interviews with clinicians and observation of ward practice revealed the UFTO helped provide clarity of goals of care and reduced negative associations with resuscitation decisions. CONCLUSIONS: Introducing the UFTO was associated with a significant reduction in harmful events in patients in whom a decision not to attempt CPR had been made. Coupled with supportive qualitative evidence, this indicates the UFTO improved care for this vulnerable group. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN85474986 UK Comprehensive Research Network Portfolio 7932.

C. elegans chemotaxis assay.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior. The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress (1-10). The method described here aims to address these issues by modifying the assay developed by Bargmann et al.(1). A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

Primordial enemies: fungal pathogens in thrips societies.

Microbial pathogens are ancient selective agents that have driven many aspects of multicellular evolution, including genetic, behavioural, chemical and immune defence systems. It appears that fungi specialised to attack insects were already present in the environments in which social insects first evolved and we hypothesise that if the early stages of social evolution required antifungal defences, then covariance between levels of sociality and antifungal defences might be evident in extant lineages, the defences becoming stronger with group size and increasing social organisation. Thus, we compared the activity of cuticular antifungal compounds in thrips species (Insecta: Thysanoptera) representing a gradient of increasing group size and sociality: solitary, communal, social and eusocial, against the entomopathogen Cordyceps bassiana. Solitary and communal species showed little or no activity. In contrast, the social and eusocial species killed this fungus, suggesting that the evolution of sociality has been accompanied by sharp increases in the effectiveness of antifungal compounds. The antiquity of fungal entomopathogens, demonstrated by fossil finds, coupled with the unequivocal response of thrips colonies to them shown here, suggests two new insights into the evolution of thrips sociality: First, traits that enabled nascent colonies to defend themselves against microbial pathogens should be added to those considered essential for social evolution. Second, limits to the strength of antimicrobials, through resource constraints or self-antibiosis, may have been overcome by increase in the numbers of individuals secreting them, thus driving increases in colony size. If this is the case for social thrips, then we may ask: did antimicrobial traits and microbes such as fungal entomopathogens play an integral part in the evolution of insect sociality in general?

The relationship between voltage-sensitive dye imaging signals and spiking activity of neural populations in primate V1.

Voltage-sensitive dye imaging (VSDI) is a powerful technique for measuring neural population responses from a large cortical region simultaneously with millisecond temporal resolution and columnar spatial resolution. However, the relationship between the average VSDI signal and the average spiking activity of neural populations is largely unknown. To better understand this relationship, we compared visual responses measured from V1 of behaving monkeys using VSDI and single-unit electrophysiology. We found large and systematic differences between position and orientation tuning properties obtained with these two techniques. We then determined that a simple computational model could explain these tuning differences. This model, together with our experimental results, allowed us to estimate the quantitative relationship between the average VSDI signal and local spiking activity. We found that this relationship is similar to the previously reported nonlinear relationship between average membrane potential and spike rate in single V1 neurons, suggesting that VSDI signals are dominated by subthreshold synaptic activity. This model, together with the VSDI measured maps for spatial position (retinotopy) and orientation, also allowed us to estimate the spatial integration area over which neural responses contribute to the VSDI signal at a given location. We found that the VSDI-integration area is consistent with a Gaussian envelope with a space constant of ∼230 µm. Finally, we show how this model and estimated parameters can be used to predict the pattern of population responses at the level of spiking activity from VSDI responses.

Uniform spatial spread of population activity in primate parafoveal V1.

What are the shape and size of the region in primate V1 that processes information from a single point in visual space? This region, a fundamental property termed cortical point image (CPI) (McIlwain 1986), represents the minimal population of V1 neurons that can be activated by a visual stimulus and therefore has important implications for population coding in the cortex. Previous indirect attempts to measure the CPI in macaque V1 using sparse microelectrode recordings resulted in conflicting findings. Whereas some early studies suggested that CPI size is constant throughout V1 (e.g., Hubel and Wiesel 1974), others have reported large changes in CPI size in parafoveal V1 (e.g., Van Essen et al. 1984). To resolve this controversy, we used voltage-sensitive dye imaging in V1 of fixating monkeys to directly measure the subthreshold CPI and several related properties across a range of parafoveal eccentricities. We found that despite large changes in other properties of the retinotopic map, the subthreshold CPI is approximately constant and extends over ∼6 × 8 mm(2). This large and invariant CPI ensures a uniform representation of each point in visual space, with a complete representation of all visual features in V1, as originally proposed by Hubel and Wiesel (1974). In addition, we found several novel and unexpected asymmetries and anisotropies in the shapes of the CPI and the population receptive field. These results expand our understanding of the representation of visual space in V1 and are likely to be relevant for the representations of stimuli in other sensory cortical areas.

Contextual modulation of behavioral choice.

We review the influence of context on behavioral choice. Context can refer to external (environmental) factors such as the season or presence of predators and it can also refer to the internal or behavioral state of an animal. Usually, animals make decisions in the midst of other ongoing behaviors. We discuss recent findings on the impact of both types of contexts, focusing on how context gets encoded at the intersection between the sensory and motor systems, emphasizing the role of neuromodulators. We also review recent technological advances that have made feasible the exploration of neural correlates of decision making in freely moving, behaving animals.