Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism.

CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.

Peatland pools are tightly coupled to the contemporary carbon cycle.

Peatlands are globally important stores of soil carbon (C) formed over millennial timescales but are at risk of destabilization by human and climate disturbance. Pools are ubiquitous features of many peatlands and can contain very high concentrations of C mobilized in dissolved and particulate organic form and as the greenhouses gases carbon dioxide (CO2 ) and methane (CH4 ). The radiocarbon content (14 C) of these aquatic C forms tells us whether pool C is generated by contemporary primary production or from destabilized C released from deep peat layers where it was previously stored for millennia. We present novel 14 C and stable C (δ13 C) isotope data from 97 aquatic samples across six peatland pool locations in the United Kingdom with a focus on dissolved and particulate organic C and dissolved CO2 . Our observations cover two distinct pool types: natural peatland pools and those formed by ditch blocking efforts to rewet peatlands (restoration pools). The pools were dominated by contemporary C, with the majority of C (~50%-75%) in all forms being younger than 300 years old. Both pool types readily transform and decompose organic C in the water column and emit CO2 to the atmosphere, though mixing with the atmosphere and subsequent CO2 emissions was more evident in natural pools. Our results show little evidence of destabilization of deep, old C in natural or restoration pools, despite the presence of substantial millennial-aged C in the surrounding peat. One possible exception is CH4 ebullition (bubbling), with our observations showing that millennial-aged C can be emitted from peatland pools via this pathway. Our results suggest that restoration pools formed by ditch blocking are effective at preventing the release of deep, old C from rewetted peatlands via aquatic export.

Single cell variability of CRISPR-Cas interference and adaptation.

While CRISPR-Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time-lapse microscopy and mathematical modelling, we studied invader clearance in Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell-to-cell variability, which originates from primed CRISPR adaptation. Faster growth and cell division and higher levels of Cascade increase the chance of clearance by interference, while slower growth is associated with increased chances of clearance by priming. Our findings suggest that Cascade binding to the mutated invader DNA, rather than spacer integration, is the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond quickly to invading threats. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.

Engaging advanced practice nurses in evidence-based practice: An e-mentoring program.

BACKGROUND: Mentoring is highly valued in the nursing profession and essential to building an evidence-based practice (EBP) culture. However, many organizations have a limited number of EBP mentors, who have limited non-clinical time to engage in mentoring. AIMS: This project aimed to test whether an e-mentoring approach to nursing inquiry could enhance EBP beliefs (EBPB), increase EBP Implementation (EBPI), and improve Organizational Culture and Readiness for System-Wide Implementation of EBP (OCRSIEP). METHODS: A pre-experimental pilot intervention project was implemented utilizing a pretest-posttest design with Advanced Practice Registered Nurses' (APRNs) in clinical practice. The OCRSIEP, EBPB, and EBPI scales were used to measure organizational readiness for EBP implementation, individual beliefs regarding the value of EBP, and the extent to which nurses integrate scientific evidence into their clinical practice, respectively. The Wilcoxon-Signed Rank test was used to analyze the difference between pretest and posttest scores of an EBP E-mentoring program. A post-hoc analysis was performed to calculate effect sizes. [Correction added on 13 May 2022, after first online publication: The Methods section was revised to add additional details.] RESULTS: Eleven APRNs completed the pretest and posttest surveys. When comparing the pre-and post-intervention scores, the median EBPB scores increased from 61 (IQR: 56-69) to 70 (IQR: 64-73), median EBPI scores increased from 13 (IQR: 7-33) to 20 (IQR: 13-31), and median OCRSIEP scores increased from 88 (IQR: 73-97) to 99 (IQR: 90-113). LINKING EVIDENCE TO ACTION: A 12-week Nurse Inquiry E-mentoring Program can leverage the small number of EBP mentors in an organization to improve EBPB. A program lasting longer in duration may also significantly improve EBPI and OCRSIEP scores. By utilizing technology and leveraging economies of scale, exponentially more nurses can be mentored to create and enhance an EBP culture.

An educational guide for nanopore sequencing in the classroom.

The last decade has witnessed a remarkable increase in our ability to measure genetic information. Advancements of sequencing technologies are challenging the existing methods of data storage and analysis. While methods to cope with the data deluge are progressing, many biologists have lagged behind due to the fast pace of computational advancements and tools available to address their scientific questions. Future generations of biologists must be more computationally aware and capable. This means they should be trained to give them the computational skills to keep pace with technological developments. Here, we propose a model that bridges experimental and bioinformatics concepts using the Oxford Nanopore Technologies (ONT) sequencing platform. We provide both a guide to begin to empower the new generation of educators, scientists, and students in performing long-read assembly of bacterial and bacteriophage genomes and a standalone virtual machine containing all the required software and learning materials for the course.

Cas4-Cas1 fusions drive efficient PAM selection and control CRISPR adaptation.

Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR-Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.

Eliminating international normalized ratio threshold for transfusion in pediatric patients with acute liver failure.

INTRODUCTION: Transfusion protocols are not well-studied for pediatric patients with acute liver failure (ALF). This study evaluates the utility of an international normalized ratio (INR)-based transfusion threshold for these patients. METHODS: Forty-four ALF pediatric patients from 2009 to 2018 were reviewed and divided into two groups: (a) a threshold group including patients between 2009 and 2015 who were transfused for an INR above 3.0, per institutional policy (n = 30), and (b) a post-threshold group including patients after 2015 through 2018 who were transfused based on clinical judgment (n = 14). Preoperative INRs, preoperative transfusions, intraoperative transfusions, early reoperation, renal function, graft function and deaths were compared. RESULTS: Liver failure severity was similar between threshold and post-threshold groups. Threshold patients had a lower average INR prior to transplantation, 2.8 (range 1.8-3.8) vs 4.4 (range 2.1-9.0), respectively (P = .01). Twenty-six threshold patients (87%) received preoperative FFP compared with seven post-threshold patients (50%, P = .0088). Two threshold patients (7%) received preoperative cryoprecipitate compared with five post-threshold patients (36%, P = .014). The incidence of pre-transplant bleeding, operative transfusions, and 1-year patient and graft survival did not differ significantly. CONCLUSION: Clinical judgment vs an INR-based threshold for transfusions did not increase perioperative complications in children with ALF.

Role of nucleotide identity in effective CRISPR target escape mutations.

Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR interference and interference-dependent priming, while cytosine-substitutions are more readily tolerated. Furthermore, we show that this effect is based on strongly decreased binding affinity of the effector complex Cascade for guanine-mismatched targets, while cytosine-mismatched targets only minimally affect target DNA binding. Structural modeling of Cascade-bound targets with mismatches shows that steric clashes of mismatched guanines lead to unfavorable conformations of the RNA-DNA duplex. This effect has strong implications for the natural selection of target site mutations that lead to effective escape from type I CRISPR-Cas systems.

Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR-Cas system.

CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity.

Transitions in Pediatric Gastroenterology: Results of a National Provider Survey.

OBJECTIVES: Transition and transfer to adult-oriented health care is an important yet challenging task for adolescents and young adults with chronic medical conditions. Transition practices vary widely, but a paucity of data makes determination of best practices difficult. We described North American pediatric gastroenterologists' preferences and present transition practice patterns and explored whether experience affected providers' perspectives. METHODS: An online survey was distributed via e-mail to members of the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition. Participation was voluntary and answers were anonymous. Quantitative and qualitative analysis was performed. RESULTS: Almost three quarters of the 175 respondents describe providing transition or self-care management education, but only 23% use structured readiness assessments. Most respondents (88%) report having age cutoffs above which they no longer accept new referrals, with the most common age being 18 years (57%). One third report the ability to provide age-appropriate care to patients older than 21 years. Only 6% indicate that their practice or institution should provide care for individuals older than 25 years. Many (63%) indicate that their practice or institution has a policy regarding age of transfer, but most (79%) are flexible. Provider preferences for triggers to transfer to adult care diverge widely between age, milestones, and comorbidities. Overall, parent (81%) and patient (74%) attachment to pediatric health care providers are cited as the most common barriers to transition. CONCLUSIONS: Preferences and practices surrounding transition preparation and transfer to adult care vary widely, reflecting continued uncertainty regarding optimal transition strategies.

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼ 350 new spacers acquired in priming events and identified a 5'-protospacer-GG-3' protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.

Novel protocol including liver biopsy to identify and treat CD8+ T-cell predominant acute hepatitis and liver failure.

In the majority of children with ALF, the etiology is unknown and liver transplantation is often needed for survival. A patient case prompted us to consider that immune dysregulation may be the cause of indeterminate acute hepatitis and liver failure in children. Our study includes nine pediatric patients treated under a multidisciplinary clinical protocol to identify and treat immune-mediated acute liver injury. Patients with evidence of inflammation and no active infection on biopsy received treatment with intravenous immune globulin and methylprednisolone. Seven patients had at least one positive immune marker before or after treatment. All patients had a CD8+ T-cell predominant liver injury that completely or partially responded to immune therapy. Five of the nine patients recovered liver function and did not require liver transplantation. Three of these patients subsequently developed bone marrow failure and were treated with either immunosuppression or stem cell transplant. This series highlights the importance of this tissue-based approach to diagnosis and treatment that may improve transplant-free survival. Further research is necessary to better characterize the immune injury and to predict the subset of patients at risk for bone marrow failure who may benefit from earlier and stronger immunosuppressive therapy.

mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles.

mRNA vaccines have recently generated significant interest due to their success during the COVID-19 pandemic. Their success is due to advances in mRNA design and encapsulation into ionizable lipid nanoparticles (iLNPs). This has highlighted the potential for the use of mRNA-iLNPs in other settings such as cancer, gene therapy, or vaccines for different infectious diseases. Here, we describe the production of mRNA-iLNPs using commercially available reagents that are suitable for use as vaccines and therapeutics. This article contains detailed protocols for the synthesis of mRNA by in vitro transcription with enzymatic capping and tailing and the encapsulation of the mRNA into iLNPs using the ionizable lipid DLin-MC3-DMA. DLin-MC3-DMA is often used as a benchmark for new formulations and provides an efficient delivery vehicle for screening mRNA design. The protocol also describes how the formulation can be adapted to other lipids. Finally, a stepwise methodology is presented for the characterization and quality control of mRNA-iLNPs, including measuring mRNA concentration and encapsulation efficiency, particle size, and zeta potential. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailing Basic Protocol 2: Encapsulation of mRNA into ionizable lipid nanoparticles Alternate Protocol: Small-scale encapsulation of mRNA using preformed vesicles Basic Protocol 3: Characterization and quality control of mRNA ionizable lipid nanoparticles.

Implementing a Standardized Communication Tool in an Intensive Care Unit.

BACKGROUND: Effective communication is essential in critical care settings. Use of the SBAR (Situation, Background, Assessment, Recommendation) tool has been shown to standardize and improve communication among health care providers. LOCAL PROBLEM: This quality improvement project was designed to improve communication in an intensive care unit that lacked a standardized communication protocol. Communication practices differed greatly between nurses and advanced practice providers. As a result, patient safety was put at risk owing to incomplete, inaccurate, or delayed information when clinical concerns were reported or escalated. METHODS: This project used a pre-post design in which surveys were used to gather information on staff perceptions of communication and collaboration between nurses and advanced practice providers before and after an educational intervention. The 2 groups received identical education on SBAR guidelines adapted for use in the intensive care unit setting and patient safety. RESULTS: Results showed improvement in all areas of communication. Significant improvements were found on the General Perceptions subscale among advanced practice providers (P = .04) and among nurses (P = .007). In the combined study population, improvements were observed on all subscales, with significant results for the Open Communication (P = .03) and General Perceptions (P = .002) subscales. A significant increase was found in the percentage of nurses using the SBAR tool after the intervention (95%) compared with before the intervention (66%; P < .001). CONCLUSION: Implementation of the SBAR communication tool significantly improved general perceptions of communication in this intensive care unit.

Developing bonds: An exploration of the development of bonds between mentors and young people.

The article describes a piece of research exploring young people's experience of a mentoring service (PROMISE). The scheme has been developed to offer vulnerable young people a supportive relationship to assist their lives. This article explores the nature of the mentoring relationship, including how mentors and mentees view its development. Conjoint interviews were conducted which also permitted an analysis of the nature of the conversational processes between the pairs, including how they constructed shared meanings of the development of their relationship. This provided a window into the emotional dynamics of their mentoring relationships. Implications for similar mentoring programmes are discussed alongside wider implications for assisting this group of young people.

Predictive action perception from explicit intention information in autism.

Social difficulties in autism spectrum disorder (ASD) may originate from a reduced top-down modulation of sensory information that prevents the spontaneous attribution of intentions to observed behaviour. However, although people with autism are able to explicitly reason about others' mental states, the effect of abstract intention information on perceptual processes has remained untested. ASD participants (n = 23) and a neurotypical (NT) control group (n = 23) observed a hand either reaching for an object or withdrawing from it. Prior to action onset, the participant either instructed the actor to "Take it" or "Leave it", or heard the actor state "I'll take it" or "I'll leave it", which provided an explicit intention that was equally likely to be congruent or incongruent with the subsequent action. The hand disappeared before completion of the action, and participants reported the last seen position of the tip of the index finger by touching the screen. NT participants exhibited a predictive bias in response to action direction (reaches perceived nearer the object, withdrawals perceived farther away), and in response to prior knowledge of the actor's intentions (nearer the object after "Take it", farther away after "Leave it"). However, ASD participants exhibited a predictive perceptual bias only in response to the explicit intentions, but not in response to the motion of the action itself. Perception in ASD is not immune from top-down modulation. However, the information must be explicitly presented independently from the stimulus itself, and not inferred from cues inherent in the stimulus.

Doing things differently: Exploring attachment patterns and parental intentions in families where a child has a diagnosis of autism.

The article examines the experience of parenting a child with a diagnosis of autism with a focus on scripts and intentions in relation to the parents' own childhood experiences of being parented. Five parents participated in a multiple case study design involving in-depth interviews, Adult Attachment Interviews and a parenting intentions scaling task. The findings revealed that all of the parents had experienced significant adverse events in their own childhoods, including trauma and losses. They also expressed intentions to offer parenting that was 'corrective' in terms of providing a better emotional environment for their children. Their corrective attempts and also intentions to repeat positive aspects of being parented were moderated by unconscious aspects of their early childhood experiences and also by the autistic features of their children. The interplay between early embodied experiences, theories of autism, parenting experiences and intentions is discussed along with clinical implications.

SAFE, a new therapeutic intervention for families of children with autism: a randomised controlled feasibility trial.

OBJECTIVES: To establish the feasibility of a definitive randomised controlled trial of Systemic Autism-related Family Enabling (SAFE), an intervention for families of children with autism. DESIGN: A randomised, controlled, multicentred feasibility study. SETTING: Participants were identified from three National Health Service (NHS) diagnosing centres in Plymouth and Cornwall and a community pathway. PARTICIPANTS: 34 families of a child with a diagnosis of autism severity level 1 or 2 between 3 and 16 years. Four families were lost to follow-up. INTERVENTIONS: SAFE is a manualised five-session family therapy-based intervention delivered over 16 weeks and designed for families of children with autism. SAFE involves families attending five 3-hour sessions led by systemic practitioners. PRIMARY AND SECONDARY OUTCOME MEASURES: The proposed primary outcome measure was the Systemic CORE 15 (SCORE-15). Proposed secondary outcome measures: Patient Health Questionnaire-Somatic Anxiety Depressive Symptoms, the Coding of Attachment-Related Parenting for use with children with Autism, the Child Behaviour Checklist (CBCL), the Reflective Functioning Questionnaire (RFQ) and the Caregiving Helplessness Questionnaire. Outcome measures were collected at baseline and 24 weeks post randomisation. RESULTS: All primary caregivers retained in the study completed the SCORE-15 at both time points. 34 of the target of 36 families were recruited and 88% of families were retained. Training for therapists was effective. Feedback revealed willingness to undergo randomisation. There was 100% attendance at appropriate sessions for core family members. The SCORE-15 showed reduction in scores for families receiving SAFE compared with controls suggesting positive change. Qualitative data also revealed that families found the study acceptable and families receiving SAFE experienced positive change. Feedback indicated that the SCORE-15 should be retained as a primary measure in a future trial, but secondary measures should be reduced. CONCLUSIONS: This study indicates that a larger trial of SAFE is feasible. Findings suggest that SAFE can address current gaps in recommended care, can be confidently delivered by NHS staff and has potential as a beneficial treatment. TRIAL REGISTRATION NUMBERS: ISCTRN83964946 and IRAS213527.

Nutritional Issues in Food Allergy.

Diet and nutrition play an important role in the development and management of food allergy. The diet of expectant mothers can have an effect on their offspring in terms of allergic outcomes. A host of confounding factors may influence this, with a maternal diet rich in fruits and vegetables, fish, vitamin D-rich foods associated with a lower risk of allergic disease in their children. More surprisingly, the consumption of milk and butter has also been shown to have a protective effect, especially in a farm environment. Similarly, the diet of the infant can also be important, not only in terms of breast feeding, but also the timing of the introduction of complementary foods, the diversity of the diet and the effect of individual foods on the development of allergy. One factor which has clearly been shown not to influence the development of food allergy is allergen avoidance by expectant mothers. In the infant diet, the manipulation of the gut microbiome to prevent the development of atopic disease is clearly an area which promises much, although studies have yet to provide a breakthrough in the prevention of atopic dermatitis. More concrete evidence of the value of diet in prevention has come from studies evaluating infant eating patterns which may protect gut health, through the consumption of large amounts of home-processed fruits and vegetables. The consumption of fish during the first year of life has also been shown to be protective. The importance of nutritional issues in children and adults who have a food allergy has become much more accepted in recent years. The primary allergenic foods in infancy and childhood, milk, egg, wheat and soy are also ones which are present in many foods and thus their avoidance can be problematic from a nutritional perspective. Thus, children with a food allergy can have their growth compromised through avoidance, especially pre-diagnosis, when foods may be excluded without any expert nutritional input. The management of a food allergy largely remains the exclusion of the offending food(s), but it is now clear that in doing so, children in particular can be at nutritional risk if insufficient attention is paid to the rest of the diet. Adults with food allergy are often thought not to need nutritional counselling; however, many will exclude a wide range of foods due to anxiety about trace exposure, or similar foods causing reactions. The avoidance of staple foods such as milk and wheat are common, but substitute foods very often do not have comparable nutritional profiles. Adults may also be more susceptible to on-line promotion of extreme nutritional regimes which can be extremely harmful. All food allergic individuals, whatever their age, should have a nutrition review to ensure they are consuming a healthy, balanced diet, and are not avoiding food groups unnecessarily.