Alternative splicing and protein diversity: plants versus animals

Plants, unlike animals, exhibit a very high degree of plasticity in their growth and development and employ diverse strategies to cope with the variations during diurnal cycles and stressful conditions. Plants and animals, despite their remarkable morphological and physiological differences, share many basic cellular processes and regulatory mechanisms. Alternative splicing (AS) is one such gene regulatory mechanism that modulates gene expression in multiple ways. It is now well established that AS is prevalent in all multicellular eukaryotes including plants and humans. Emerging evidence indicates that in plants, as in animals, transcription and splicing are coupled. Here, we reviewed recent evidence in support of co-transcriptional splicing in plants and highlighted similarities and differences between plants and humans. An unsettled question in the field of AS is the extent to which splice isoforms contribute to protein diversity. To take a critical look at this question, we presented a comprehensive summary of the current status of research in this area in both plants and humans, discussed limitations with the currently used approaches and suggested improvements to current methods and alternative approaches. We end with a discussion on the potential role of epigenetic modifications and chromatin state in splicing memory in plants primed with stresses

Does co-transcriptional regulation of alternative splicing mediate plant stress responses?

Plants display exquisite control over gene expression to elicit appropriate responses under normal and stress conditions. Alternative splicing (AS) of pre-mRNAs, a process that generates two or more transcripts from multi-exon genes, adds another layer of regulation to fine-tune condition-specific gene expression in animals and plants. However, exactly how plants control splice isoform ratios and the timing of this regulation in response to environmental signals remains elusive. In mammals, recent evidence indicate that epigenetic and epitranscriptome changes, such as DNA methylation, chromatin modifications and RNA methylation, regulate RNA polymerase II processivity, co-transcriptional splicing, and stability and translation efficiency of splice isoforms. In plants, the role of epigenetic modifications in regulating transcription rate and mRNA abundance under stress is beginning to emerge. However, the mechanisms by which epigenetic and epitranscriptomic modifications regulate AS and translation efficiency require further research. Dynamic changes in the chromatin landscape in response to stress may provide a scaffold around which gene expression, AS and translation are orchestrated. Finally, we discuss CRISPR/Cas-based strategies for engineering chromatin architecture to manipulate AS patterns (or splice isoforms levels) to obtain insight into the epigenetic regulation of AS

Differential nucleosome occupancy modulates alternative splicing in Arabidopsis thaliana

• Alternative splicing (AS) is a major gene regulatory mechanism in plants. Recent evidence supports co-transcriptional splicing in plants, hence the chromatin state can impact AS. However, how dynamic changes in the chromatin state such as nucleosome occupancy influence the cold-induced AS remains poorly understood. • Here, we generated transcriptome (RNA-Seq) and nucleosome positioning (MNase-Seq) data for Arabidopsis thaliana to understand how nucleosome positioning modulates cold-induced AS. • Our results show that characteristic nucleosome occupancy levels are strongly associated with the type and abundance of various AS events under normal and cold temperature conditions in Arabidopsis. Intriguingly, exitrons, alternatively spliced internal regions of protein-coding exons, exhibit distinctive nucleosome positioning pattern compared to other alternatively spliced regions. Likewise, nucleosome patterns differ between exitrons and retained introns pointing to their distinct regulation. • Collectively, our data show that characteristic changes in nucleosome positioning modulate AS in plants in response to cold

Healthcare professionals' intentions to use wiki-based reminders to promote best practices in trauma care: a survey protocol

<p>Abstract</p> <p>Background</p> <p>Healthcare professionals are increasingly using wikis as collaborative tools to create, synthesize, share, and disseminate knowledge in healthcare. Because wikis depend on collaborators to keep content up-to-date, healthcare professionals who use wikis must adopt behaviors that foster this collaboration. This protocol describes the methods we will use to develop and test the metrological qualities of a questionnaire that will assess healthcare professionals' intentions and the determinants of those intentions to use wiki-based reminders that promote best practices in trauma care.</p> <p>Methods</p> <p>Using the Theory of Planned Behavior, we will conduct semi-structured interviews of healthcare professionals to identify salient beliefs that may affect their future use of wikis. These beliefs will inform our questionnaire on intended behavior. A test-retest of the survey will verify the questionnaire's stability over time. We will interview 50 healthcare professionals (25 physicians and 25 allied health professionals) working in the emergency departments of three trauma centers in Quebec, Canada. We will analyze the content of the interviews and construct and pilot a questionnaire. We will then test the revised questionnaire with 30 healthcare professionals (15 physicians and 15 allied health professionals) and retest it two weeks later. We will assess the internal consistency of the questionnaire constructs using Cronbach's alpha coefficients and determine their stability with the intra-class correlation (ICC).</p> <p>Discussion</p> <p>To our knowledge, this study will be the first to develop and test a theory-based survey that measures healthcare professionals' intentions to use a wiki-based intervention. This study will identify professionals' salient beliefs qualitatively and will quantify the psychometric capacities of the questionnaire based on those beliefs.</p

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012

OBJECTIVE: To provide an update to the "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," last published in 2008. DESIGN: A consensus committee of 68 international experts representing 30 international organizations was convened. Nominal groups were assembled at key international meetings (for those committee members attending the conference). A formal conflict of interest policy was developed at the onset of the process and enforced throughout. The entire guidelines process was conducted independent of any industry funding. A stand-alone meeting was held for all subgroup heads, co- and vice-chairs, and selected individuals. Teleconferences and electronic-based discussion among subgroups and among the entire committee served as an integral part of the development. METHODS: The authors were advised to follow the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations as strong (1) or weak (2). The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasized. Recommendations were classified into three groups: (1) those directly targeting severe sepsis; (2) those targeting general care of the critically ill patient and considered high priority in severe sepsis; and (3) pediatric considerations. RESULTS: Key recommendations and suggestions, listed by category, include: early quantitative resuscitation of the septic patient during the first 6 h after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm a potential source of infection (UG); administration of broad-spectrum antimicrobials therapy within 1 h of the recognition of septic shock (1B) and severe sepsis without septic shock (1C) as the goal of therapy; reassessment of antimicrobial therapy daily for de-escalation, when appropriate (1B); infection source control with attention to the balance of risks and benefits of the chosen method within 12 h of diagnosis (1C); initial fluid resuscitation with crystalloid (1B) and consideration of the addition of albumin in patients who continue to require substantial amounts of crystalloid to maintain adequate mean arterial pressure (2C) and the avoidance of hetastarch formulations (1B); initial fluid challenge in patients with sepsis-induced tissue hypoperfusion and suspicion of hypovolemia to achieve a minimum of 30 mL/kg of crystalloids (more rapid administration and greater amounts of fluid may be needed in some patients (1C); fluid challenge technique continued as long as hemodynamic improvement is based on either dynamic or static variables (UG); norepinephrine as the first-choice vasopressor to maintain mean arterial pressure ≥65 mmHg (1B); epinephrine when an additional agent is needed to maintain adequate blood pressure (2B); vasopressin (0.03 U/min) can be added to norepinephrine to either raise mean arterial pressure to target or to decrease norepinephrine dose but should not be used as the initial vasopressor (UG); dopamine is not recommended except in highly selected circumstances (2C); dobutamine infusion administered or added to vasopressor in the presence of (a) myocardial dysfunction as suggested by elevated cardiac filling pressures and low cardiac output, or (b) ongoing signs of hypoperfusion despite achieving adequate intravascular volume and adequate mean arterial pressure (1C); avoiding use of intravenous hydrocortisone in adult septic shock patients if adequate fluid resuscitation and vasopressor therapy are able to restore hemodynamic stability (2C); hemoglobin target of 7-9 g/dL in the absence of tissue hypoperfusion, ischemic coronary artery disease, or acute hemorrhage (1B); low tidal volume (1A) and limitation of inspiratory plateau pressure (1B) for acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure (PEEP) in ARDS (1B); higher rather than lower level of PEEP for patients with sepsis-induced moderate or severe ARDS (2C); recruitment maneuvers in sepsis patients with severe refractory hypoxemia due to ARDS (2C); prone positioning in sepsis-induced ARDS patients with a PaO (2)/FiO (2) ratio of ≤100 mm Hg in facilities that have experience with such practices (2C); head-of-bed elevation in mechanically ventilated patients unless contraindicated (1B); a conservative fluid strategy for patients with established ARDS who do not have evidence of tissue hypoperfusion (1C); protocols for weaning and sedation (1A); minimizing use of either intermittent bolus sedation or continuous infusion sedation targeting specific titration endpoints (1B); avoidance of neuromuscular blockers if possible in the septic patient without ARDS (1C); a short course of neuromuscular blocker (no longer than 48 h) for patients with early ARDS and a PaO (2)/FI O (2) 180 mg/dL, targeting an upper blood glucose ≤180 mg/dL (1A); equivalency of continuous veno-venous hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1B); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding in patients with bleeding risk factors (1B); oral or enteral (if necessary) feedings, as tolerated, rather than either complete fasting or provision of only intravenous glucose within the first 48 h after a diagnosis of severe sepsis/septic shock (2C); and addressing goals of care, including treatment plans and end-of-life planning (as appropriate) (1B), as early as feasible, but within 72 h of intensive care unit admission (2C). Recommendations specific to pediatric severe sepsis include: therapy with face mask oxygen, high flow nasal cannula oxygen, or nasopharyngeal continuous PEEP in the presence of respiratory distress and hypoxemia (2C), use of physical examination therapeutic endpoints such as capillary refill (2C); for septic shock associated with hypovolemia, the use of crystalloids or albumin to deliver a bolus of 20 mL/kg of crystalloids (or albumin equivalent) over 5-10 min (2C); more common use of inotropes and vasodilators for low cardiac output septic shock associated with elevated systemic vascular resistance (2C); and use of hydrocortisone only in children with suspected or proven "absolute"' adrenal insufficiency (2C). CONCLUSIONS: Strong agreement existed among a large cohort of international experts regarding many level 1 recommendations for the best care of patients with severe sepsis. Although a significant number of aspects of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for this important group of critically ill patients

Understanding the epigenetics of alternative splicing in Arabidopsis thaliana

Being sessile and photosynthetic necessitates that plants must have a certain degree of predictability for ambient conditions during the daily cycles to maximise efficiency and survival. However, subtle or sudden changes in weather conditions alongside different growth and developmental phases necessitate that plants must continuously monitor different environmental cues and synchronise them with their physiology and metabolism in a time, growth phase and development-stage dependent manner. Plants use complex gene regulatory mechanisms to overcome environmental challenges. Alternative splicing (AS) of pre-mRNAs, a process that generates two or more transcripts from multi-exon genes, adds another layer of complexity to gene regulatory mechanisms to modulate transcriptome diversity in a tissue- and condition-dependent manner. In mammals, mounting evidence indicates that chromatin structure can regulate co-transcriptional AS. Recent evidence supports co-transcriptional regulation of AS in plants, but how dynamic changes in the chromatin influence the AS process upon cold stress remains poorly understood and is the subject of this study. In order to answer this question, four approaches were followed in parallel; (1) Arabidopsis thaliana (Arabidopsis) plants with identical DNA sequence but differential DNA methylation and nucleosome occupancy (Ctrl and AzadC plants with wild type DNA methylation and hypomethylation, respectively) were developed to perform (2) RNA sequencing (RNA-seq) and (3) Micrococcal Nuclease sequencing (MNase-seq) for Ctrl and AzadC grown at 22oC and at 4oC as cold treatment for 24 hours, and (4) whole genome bisulphite sequencing (WGBS) for AzadC grown at 22oC and at 4oC as cold treatment for 24 hours. This strategy allowed us to understand how epigenetic variations between AzadC treatment derived lines and Ctrl plants affect AS under cold conditions, without the confounding effects of sequence variation. Interestingly, RNA-seq, MNase-seq, and WGBS show a strong reprogramming of AS patterns upon cold stress associated with changes in epigenetic features (i.e. DNA methylation and nucleosome occupancy). To my knowledge, this is the first study in Arabidopsis that demonstrates that changes in transcriptional and AS patterns coincide with genome-wide changes in nucleosome occupancy and DNA methylation patterns upon temperature shift