Molecular approaches to the study of marine cyanophages

Cyanophages are thought to play an important role in the mortality and clonal composition of marine Synechococcus spp., and have been shown to be widespread throughout the world's oceans. However, relatively little research has been made into the molecular analysis of marine cyanophages. This study continued previous research to develop molecular probes (PCR primers) which would specifically detect cyanophages which infect marine Synechococcus spp., and be used to interrogate natural marine cyanophage populations. An attempt was made to develop a rapid technique for quantifying marine cyanophages, using competitive PCR (cPCR). For the development of cyanophage-specific PCR primers, several cyanophages which infected Synechococcus sp. strains WH7803 and WH80 18 were isolated from coastal Bermuda and the Sargasso Sea. A region of DNA had previously been found which showed homology amongst several marine cyanophages, and to T4 gene 20, which encodes a minor capsid protein. Homologues from three cyanophages were completely sequenced, and two, potentially cyanophage-specific, PCR primers were designed. The primers detected only marine cyanophages which belonged to the family Myoviridae, regardless of the geographical location of their isolation. They also detected cyanophages which infected different marine Synechococcus spp. strains, and therefore provide a more comprehensive tool than infective methods. The primers were able to detect as few as 190 cyanophages Ilr1, which would correspond to an in situ concentration of 103 PFU mH. The PCR should therefore detect most natural concentrations of marine cyanophages in surface waters, especially with prior concentration from seawater. Preliminary experiments showed that PCR products could be obtained from as little as I III of un concentrated seawater. PCR therefore provides a sensitive method for the detection of marine cyanophages, which is far more rapid than traditional infection techniques. Quantification by cPCR was attempted. An internal competitor was constructed, and a calibration curve was drawn for three cyanophages, with a loglinear relationship over ca. three orders of magnitude of cyanophage numbers. This demonstrates that rapid quantification of a known marine cyanophage is possible. However, cPCR of the three different cyanophages resulted in three different calibration curves. Hence, quantification of a marine sample containing a mixture of cyanophages was not yet possible. The cyanophage-specific primers were then applied to marine samples which were collected whilst on the AMT-2 cruise, from Port Stanley (Falkland Islands) to Plymouth (UK). Cyanophages were concentrated by tangential flow filtration, and PCR products were obtained from most of the surface samples throughout the Atlantic Ocean. Products from some of the stations were sequenced, providing novel genetic information of natural marine cyanophage populations. The results showed that cyanophage populations were highly diverse, with at least twelve genetically different cyanomyoviruses in one sample. Some sequences obtained from the same sample were clearly very similar to each other, whilst others within a sample could be as diverse as those isolated from different oceans. However, very similar sequences were obtained from some samples separated by thousands of miles, in different hemispheres, or even in different oceans

Voices from Istria

The book summarizes the historical research of the UE project Shared Culture between Italy and Slovenia (2007-2013). A research addressed to the petitions which were sent to the Venetian Signoria from the Istrian region in 16th and 17th centuries

High-resolution global maps of tidal flat ecosystems from 1984 to 2019

Assessments of the status of tidal flats, one of the most extensive coastal ecosystems, have been hampered by a lack of data on their global distribution and change. Here we present globally consistent, spatially-explicit data of the occurrence of tidal flats, defined as sand, rock or mud flats that undergo regular tidal inundation. More than 1.3 million Landsat images were processed to 54 composite metrics for twelve 3-year periods, spanning four decades (1984–1986 to 2017–2019). The composite metrics were used as predictor variables in a machine-learning classification trained with more than 10,000 globally distributed training samples. We assessed accuracy of the classification with 1,348 stratified random samples across the mapped area, which indicated overall map accuracies of 82.2% (80.0–84.3%, 95% confidence interval) and 86.1% (84.2–86.8%, 95% CI) for version 1.1 and 1.2 of the data, respectively. We expect these maps will provide a means to measure and monitor a range of processes that are affecting coastal ecosystems, including the impacts of human population growth and sea level rise

Tracking the rapid loss of tidal wetlands in the Yellow Sea

In the Yellow Sea region of East Asia, tidal wetlands are the frontline ecosystem protecting a coastal population of more than 60 million people from storms and sea-level rise. However, unprecedented coastal development has led to growing concern about the status of these ecosystems. We developed a remote-sensing method to assess change over ∼4000 km of the Yellow Sea coastline and discovered extensive losses of the region's principal coastal ecosystem - tidal flats - associated with urban, industrial, and agricultural land reclamations. Our analysis revealed that 28% of tidal flats existing in the 1980s had disappeared by the late 2000s (1.2% annually). Moreover, reference to historical maps suggests that up to 65% of tidal flats were lost over the past five decades. With the region forecast to be a global hotspot of urban expansion, development of the Yellow Sea coastline should pursue a course that minimizes the loss of remaining coastal ecosystems

Aromatase Is a Direct Target of FOXL2: C134W in Granulosa Cell Tumors via a Single Highly Conserved Binding Site in the Ovarian Specific Promoter

BACKGROUND: Granulosa cell tumors (GCT) of the ovary often express aromatase and synthesize estrogen, which in turn may influence their progression. Recently a specific point mutation (C134W) in the FOXL2 protein was identified in >94% of adult-type GCT and it is likely to contribute to their development. A number of genes are known to be regulated by FOXL2, including aromatase/CYP19A1, but it is unclear which are direct targets and whether the C134W mutation alters their regulation. Recently, it has been reported that FOXL2 forms a complex with steroidogenic factor 1 (SF-1) which is a known regulator of aromatase in granulosa cells. METHODOLOGY/PRINCIPAL FINDINGS: In this work, the human GCT-derived cell lines KGN and COV434 were heterozygous and wildtype for the FOXL2:C134W mutation, respectively. KGN had abundant FOXL2 mRNA expression but it was not expressed in COV434. Expression of exogenous FOXL2:C134W in COV434 cells induced higher expression of a luciferase reporter for the ovarian specific aromatase promoter, promoter II (PII) (-516bp) than expression of wildtype FOXL2, but did not alter induction of a similar reporter for the steroidogenic acute regulatory protein (StAR) promoter (-1300bp). Co-immunoprecipitation confirmed that FOXL2 bound SF-1 and that it also bound its homologue, liver receptor homologue 1 (LRH-1), however, the C134W mutation did not alter these interactions or induce a selective binding of the proteins. A highly conserved putative binding site for FOXL2 was identified in PII. FOXL2 was demonstrated to bind the site by electrophoretic mobility shift assays (EMSA) and site-directed mutagenesis of this element blocked its differential induction by wildtype FOXL2 and FOXL2:C134W. CONCLUSIONS/SIGNIFICANCE: These findings suggest that aromatase is a direct target of FOXL2:C134W in adult-type GCT via a single distinctive and highly conserved binding site in PII and therefore provide insight into the pathogenic mechanism of this mutation

Using citizen science to identify Australia’s least known birds and inform conservation action

Citizen science is a popular approach to biodiversity surveying, whereby data that are collected by volunteer naturalists may help analysts to understand the distribution and abundance of wild organisms. In Australia, birdwatchers have contributed to two major citizen science programs, eBird (run by the Cornell Lab of Ornithology) and Birdata (run by Birdlife Australia), which collectively hold more than 42 million records of wild birds from across the country. However, these records are not evenly distributed across space, time, or taxonomy, with particularly significant variation in the number of records of each species in these datasets. In this paper, we explore this variation and seek to determine which Australian bird species are least known as determined by rates of citizen science survey detections. We achieve this by comparing the rates of survey effort and species detection across each Australian bird species? range, assigning all 581 species to one of the four groups depending on their rates of survey effort and species observation. We classify 56 species into a group considered the most poorly recorded despite extensive survey effort, with Coxen?s Fig Parrot Cyclopsitta coxeni, Letter-winged Kite Elanus scriptus, Night Parrot Pezoporus occidentalis, Buff-breasted Buttonquail Turnix olivii and Red-chested Buttonquail Turnix pyrrhothorax having the very lowest numbers of records. Our analyses provide a framework to identify species that are poorly represented in citizen science datasets. We explore the reasons behind why they may be poorly represented and suggest ways in which targeted approaches may be able to help fill in the gaps.Publisher PDFPeer reviewe

Lung-protective ventilation initiated in the emergency department (LOV-ED): A study protocol for a quasi-experimental, before-after trial aimed at reducing pulmonary complications

INTRODUCTION: In critically ill patients, acute respiratory distress syndrome (ARDS) and ventilator-associated conditions (VACs) are associated with increased mortality, survivor morbidity and healthcare resource utilisation. Studies conclusively demonstrate that initial ventilator settings in patients with ARDS, and at risk for it, impact outcome. No studies have been conducted in the emergency department (ED) to determine if lung-protective ventilation in patients at risk for ARDS can reduce its incidence. Since the ED is the entry point to the intensive care unit for hundreds of thousands of mechanically ventilated patients annually in the USA, this represents a knowledge gap in this arena. A lung-protective ventilation strategy was instituted in our ED in 2014. It aims to address the parameters in need of quality improvement, as demonstrated by our previous research: (1) prevention of volutrauma; (2) appropriate positive end-expiratory pressure setting; (3) prevention of hyperoxia; and (4) aspiration precautions. METHODS AND ANALYSIS: The lung-protective ventilation initiated in the emergency department (LOV-ED) trial is a single-centre, quasi-experimental before-after study testing the hypothesis that lung-protective ventilation, initiated in the ED, is associated with reduced pulmonary complications. An intervention cohort of 513 mechanically ventilated adult ED patients will be compared with over 1000 preintervention control patients. The primary outcome is a composite outcome of pulmonary complications after admission (ARDS and VACs). Multivariable logistic regression with propensity score adjustment will test the hypothesis that ED lung-protective ventilation decreases the incidence of pulmonary complications. ETHICS AND DISSEMINATION: Approval of the study was obtained prior to data collection on the first patient. As the study is a before-after observational study, examining the effect of treatment changes over time, it is being conducted with waiver of informed consent. This work will be disseminated by publication of full-length manuscripts, presentation in abstract form at major scientific meetings and data sharing with other investigators through academically established means. TRIAL REGISTRATION NUMBER: NCT02543554

Awareness with paralysis and symptoms of post-traumatic stress disorder among mechanically ventilated emergency department survivors (ED-AWARENESS-2 Trial): study protocol for a pragmatic, multicenter, stepped wedge cluster randomized trial.

BACKGROUND: Awareness with paralysis (AWP) is memory recall during neuromuscular blockade (NMB) and can cause significant psychological harm. Decades of effort and rigorous trials have been conducted to prevent AWP in the operating room, where prevalence is 0.1-0.2%. By contrast, AWP in mechanically ventilated emergency department (ED) patients is common, with estimated prevalence of 3.3-7.4% among survivors given NMB. Longer-acting NMB use is a critical risk for AWP, and we have shown an association between ED rocuronium use and increased AWP prevalence. As NMB are given to more than 90% of ED patients during tracheal intubation, this trial provides a platform to test an intervention aimed at reducing AWP. The overall objective is to test the hypothesis that limiting ED rocuronium exposure will significantly reduce the proportion of patients experiencing AWP. METHODS: This is a pragmatic, stepped wedge cluster randomized trial conducted in five academic EDs, and will enroll 3090 patients. Per the design, all sites begin in a control phase, under observational conditions. At 6-month intervals, sites sequentially enter a 2-month transition phase, during which we will implement the multifaceted intervention, which will rely on use of nudges and defaults to change clinician decisions regarding ED NMB use. During the intervention phase, succinylcholine will be the default NMB over rocuronium. The primary outcome is AWP, assessed with the modified Brice questionnaire, adjudicated by three independent, blinded experts. The secondary outcome is the proportion of patients developing clinically significant symptoms of post-traumatic stress disorder at 30 and 180 days after hospital discharge. We will also assess for symptoms of depression and anxiety, and health-related quality of life. A generalized linear model, adjusted for time and cluster interactions, will be used to compare AWP in control versus intervention phases, analyzed by intention-to-treat. DISCUSSION: The ED-AWARENESS-2 Trial will be the first ED-based trial aimed at preventing AWP, a critical threat to patient safety. Results could shape clinical use of NMB in the ED and prevent more than 10,000 annual cases of AWP related to ED care. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT05534243 . Registered 06, September 2022

A dual-center cohort study on the association between early deep sedation and clinical outcomes in mechanically ventilated patients during the COVID-19 pandemic: The COVID-SED study

BACKGROUND: Mechanically ventilated patients have experienced greater periods of prolonged deep sedation during the coronavirus disease (COVID-19) pandemic. Multiple studies from the pre-COVID era demonstrate that early deep sedation is associated with worse outcome. Despite this, there is a lack of data on sedation depth and its impact on outcome for mechanically ventilated patients during the COVID-19 pandemic. We sought to characterize the emergency department (ED) and intensive care unit (ICU) sedation practices during the COVID-19 pandemic, and to determine if early deep sedation was associated with worse clinical outcomes. STUDY DESIGN AND METHODS: Dual-center, retrospective cohort study conducted over 6 months (March-August, 2020), involving consecutive, mechanically ventilated adults. All sedation-related data during the first 48 h were collected. Deep sedation was defined as Richmond Agitation-Sedation Scale of - 3 to - 5 or Riker Sedation-Agitation Scale of 1-3. To examine impact of early sedation depth on hospital mortality (primary outcome), we used a multivariable logistic regression model. Secondary outcomes included ventilator-, ICU-, and hospital-free days. RESULTS: 391 patients were studied, and 283 (72.4%) experienced early deep sedation. Deeply sedated patients received higher cumulative doses of fentanyl, propofol, midazolam, and ketamine when compared to light sedation. Deep sedation patients experienced fewer ventilator-, ICU-, and hospital-free days, and greater mortality (30.4% versus 11.1%) when compared to light sedation (p \u3c 0.01 for all). After adjusting for confounders, early deep sedation remained significantly associated with higher mortality (adjusted OR 3.44; 95% CI 1.65-7.17; p \u3c 0.01). These results were stable in the subgroup of patients with COVID-19. CONCLUSIONS: The management of sedation for mechanically ventilated patients in the ICU has changed during the COVID pandemic. Early deep sedation is common and independently associated with worse clinical outcomes. A protocol-driven approach to sedation, targeting light sedation as early as possible, should continue to remain the default approach

Lack of a functioning P2X7 receptor leads to increased susceptibility to toxoplasmic ileitis

Background: Oral infection of C57BL/6J mice with the protozoan parasite Toxoplasma gondii leads to a lethal inflammatory ileitis. Principal Findings: Mice lacking the purinergic receptor P2X7R are acutely susceptible to toxoplasmic ileitis, losing significantly more weight than C57BL/6J mice and exhibiting much greater intestinal inflammatory pathology in response to infection with only 10 cysts of T. gondii. This suscep-tibility is not dependent on the ability of P2X7R-deficient mice to control the parasite, which they accomplish just as efficiently as C57BL/6J mice. Rather, susceptibility is associated with elevated ileal concentrations of pro-inflammatory cytokines, reactive nitrogen interme-diates and altered regulation of elements of NFκB activation in P2X7R-deficient mice. Conclusions: Our data support the thesis that P2X7R, a well-documented activator of pro-inflammatory cytokine production, also plays an important role in the regulation of intestinal inflammation