Rapid and Robust Polysome Isolation and Fraction RNA Extraction for Studying the Seed Translatome.

Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed-specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time-consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size-exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high-quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Robust polysome extraction for seeds Basic Protocol 2: Rapid fraction total RNA extraction.

Is the Norwegian stepped care model for allocation of patients with mental health problems working as intended? A cross-sectional study.

OBJECTIVE: Stepped care models are frameworks for mental health care systems in several countries. According to Norwegian guidelines, individuals with mental health problems of mild severity should be treated in community mental health services, moderate severity in specialist mental health services, while complex/severe problems are often a shared responsibility. This study investigated whether patients are allocated as intended. METHODS: In a cross-sectional study, 4061 outpatients recruited from community- and specialist mental health services reported demographic variables, symptoms of anxiety/depression, functional impairment, health status, and sick leave status. The community sample consisted of two subsamples: mild/moderate problems and complex problems. RESULTS: There was substantial overlap (80%-99%) of symptoms, impairment, and health between community- and specialist mental health services. More impairment, worse health, lower age, and being male were associated with treatment at specialist level compared to community mild/moderate. Better health, being in a relationship, and lower age were associated with specialized treatment compared to community complex group. CONCLUSION: The limited association between treatment level and symptoms and functional impairment reveals inconsistencies between treatment guidelines and clinical practice. How the existing organization affects patient outcomes and satisfaction should be investigated further.

Provider perceptions of the anticipated benefits, barriers, and facilitators associated with implementing a stepped care model for the delivery of addiction and mental health services in New Brunswick: a mixed-methods observational implementation study.

BACKGROUND: Providers who work within addiction and mental health (A&MH) services in New Brunswick (NB), Canada completed training in Stepped Care 2.0 and One-at-a-Time (OAAT) therapy as part of a provincial practice change initiative to implement a provincial stepped care model. The present study aimed to identify: (1) the perceived acceptability and feasibility of the SC2.0 model; (2) the perceived benefits, barriers, and facilitators to implement SC2.0 in practice; and (3) perceived impacts on clinical practice. METHODS: This is a mixed-methods observational implementation study. Quantitative surveys were completed after training courses. Open-ended responses were collected after completion of SC2.0 training. A subset of providers who completed surveys were asked to participate in semi-structured interviews. Descriptive statistics were used to describe results from surveys. Open-ended responses and semi-structured interviews were compiled and thematically synthesized in an iterative process using a grounded theory framework. Quantitative and qualitative data were triangulated to build an in-depth understanding of provider perceptions. RESULTS: 316 providers completed surveys and responded to open-ended prompts. Interviews were completed with 28 of those providers. SC2.0 was deemed to be acceptable, a suitable fit, and feasible to implement. Perceived benefits included: (1) timely access to services; (2) increased practice efficiency; and (3) increased availability of services. Perceived barriers included: (1) insufficient availability of resources to populate a SC2.0 continuum of care; (2) provider complacency with their current practice; and (3) difficulty for clients to accept and adjust to change. CONCLUSIONS: Identifying the perceived benefits, facilitators, and barriers to adopting stepped care in practice can lead to targeted implementation strategies and the collection of data that can inform continuous improvement cycles.

Implementing One-at-a-Time Therapy in community addiction and mental health centres: a retrospective exploration of the implementation process and initial outcomes.

BACKGROUND: The Department of Health of the Government of New Brunswick and Regional Health Authorities elected to implement Stepped Care 2.0 (SC2.0) in 2021, and began with One-at-a-Time (OAAT) therapy in Community Addiction and Mental Health Centres (CAMHCs) to facilitate rapid access to addiction and mental healthcare. This study: 1) explicated the process of implementing OAAT therapy as it aligned to evidence-based implementation frameworks and strategies; 2) assessed readiness for change among providers during the implementation; and 3) evaluated initial client and system outcomes. METHODS: The process of implementing OAAT therapy within CAMHCs was documented and retrospectively aligned with the Active Implementation Frameworks-Stages of Implementation, Consolidated Framework for Implementation Research, and incorporated strategies endorsed by the Expert Recommendations for Implementing Change. Providers working in CAMHCs completed online asynchronous courses in OAAT therapy and SC2.0, and were recruited to participate in research on perceptions of organizational readiness. Initial outcomes of the implementation were evaluated through client satisfaction surveys administered in CAMHCs and system performance indicators. RESULTS: Aligning with implementation stages, key strategies included: 1) continuously monitoring readiness and soliciting stakeholder feedback for iterative improvement; 2) building a representative implementation team with engaged leaders; 3) creating a comprehensive implementation plan on staff training, communication, and system changes; and 4) supporting sustainability. Providers who participated in research (N = 170, ~ 50% response rate) agreed that their organization was ready for implementation, and that OAAT therapy delivered within a SC2.0 framework was acceptable, appropriate, and feasible. More than 3,600 OAAT therapy sessions were delivered during the initial implementation stage, and waitlists were reduced by 64.1%. The majority of clients who completed surveys (N = 1240, ~ 35% response rate) reported that their OAAT therapy session was helpful, with a minority reporting that additional intervention was needed. CONCLUSIONS: Thoughtful planning and execution, aligned with evidence-based implementation frameworks and strategies, played an important role in this provincial change initiative. Implementation steps outlined can help inform others looking to enact large-scale change.

HEARTSMAP-U: Adapting a Psychosocial Self-Screening and Resource Navigation Support Tool for Use by Post-secondary Students.

Background: Mental health challenges are highly prevalent in the post-secondary educational setting. Screening instruments have been shown to improve early detection and intervention. However, these tools often focus on specific diagnosable conditions, are not always designed with students in mind, and lack resource navigational support. Objective: The aim of this study was to describe the adaptation of existing psychosocial assessment (HEARTSMAP) tools into a version that is fit-for-purpose for post-secondary students, called HEARTSMAP-U. Methods: We underwent a three-phase, multi-method tool adaptation process. First, a diverse study team proposed a preliminary version of HEARTSMAP-U and its conceptual framework. Second, we conducted a cross-sectional expert review study with Canadian mental health professionals (N = 28), to evaluate the clinical validity of tool content. Third, we conducted an iterative series of six focus groups with diverse post-secondary students (N = 54), to refine tool content and language, and ensure comprehensibility and relevance to end-users. Results: The adaptation process resulted in the HEARTSMAP-U self-assessment and resource navigational support tool, which evaluates psychosocial challenges across 10 sections. In Phase two, clinician experts expressed that HEARTSMAP-U's content aligned with their own professional experiences working with students. In Phase three, students identified multiple opportunities to improve the tool's end-user relevance by calling for more "common language," such as including examples, definitions, and avoiding technical jargon. Conclusions: The HEARTSMAP-U tool is well-positioned for further studies of its quantitative psychometric properties and clinical utility in the post-secondary educational setting.

Scoping review of stepped care interventions for mental health and substance use service delivery to youth and young adults.

AIMS: Many young people with mental health and/or substance use concerns do not have access to timely, appropriate, and effective services. Within this context, stepped care models (SCMs) have emerged as a guiding framework for care delivery, inspiring service innovations across the globe. However, substantial gaps remain in the evidence for SCMs as a strategy to address the current systemic challenges in delivering services for young people. This scoping review aims to identify where these gaps in evidence exist, and the next steps for addressing them. METHODS: A scoping review was conducted involving both peer-reviewed and grey literature. Eligible studies explored SCMs implemented in the various health care settings accessed by young people aged 12-24 seeking treatment for mental health and substance use challenges. After screening titles and abstracts, two reviewers examined full-text articles and extracted data to create a descriptive summary of the models. RESULTS: Of the 656 studies that were retrieved, 51 studies were included and grouped by study team for a final yield of 43 studies. Almost half of the studies were focused on the adult population (i.e., 18 and over), and most did not specify interventions for young people. Among the SCMs, substantial variability was found in almost every aspect of the models. CONCLUSIONS: Considering the current body of evidence, there is an urgent need for a consensus position on the definition, implementation, and outcome measures required for rigorously assessing the utility of SCMs for young people.

DYNAMIN-RELATED PROTEIN DRP1A functions with DRP2B in plant growth, flg22-immune responses, and endocytosis.

Ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (FLS2) is critical for maintaining its proper abundance in the plasma membrane (PM) to initiate and subsequently down regulate cellular immune responses to bacterial flagellin or flg22-peptide. The molecular components governing PM abundance of FLS2, however, remain mostly unknown. Here, we identified Arabidopsis (Arabidopsis thaliana) DYNAMIN-RELATED PROTEIN1A (DRP1A), a member of a plant-specific family of large dynamin GTPases, as a critical contributor to ligand-induced endocytosis of FLS2 and its physiological roles in flg22-signaling and immunity against Pseudomonas syringae pv. tomato DC3000 bacteria in leaves. Notably, drp1a single mutants displayed similar flg22-defects as those previously reported for mutants in another dynamin-related protein, DRP2B, that was previously shown to colocalize with DRP1A. Our study also uncovered synergistic roles of DRP1A and DRP2B in plant growth and development as drp1a drp2b double mutants exhibited severely stunted roots and cotyledons, as well as defective cell shape, cytokinesis, and seedling lethality. Furthermore, drp1a drp2b double mutants hyperaccumulated FLS2 in the PM prior to flg22-treatment and exhibited a block in ligand-induced endocytosis of FLS2, indicating combinatorial roles for DRP1A and DRP1B in governing PM abundance of FLS2. However, the increased steady-state PM accumulation of FLS2 in drp1a drp2b double mutants did not result in increased flg22 responses. We propose that DRP1A and DRP2B are important for the regulation of PM-associated levels of FLS2 necessary to attain signaling competency to initiate distinct flg22 responses, potentially through modulating the lipid environment in defined PM domains.

The INternet ThERapy for deprESsion Trial (INTEREST): protocol for a patient-preference, randomised controlled feasibility trial comparing iACT, iCBT and attention control among individuals with comorbid chronic pain and depression.

INTRODUCTION: Approximately one-third of adults with chronic pain also report clinically relevant levels of depression. Internet-delivered psychological therapies such as Cognitive Behavioural Therapy (iCBT) and Acceptance and Commitment Therapy (iACT) have been developed to overcome barriers of access to services and ensure the timely delivery of care. The objective of this trial is to collect data on feasibility, acceptability and range of probable effect sizes for iCBT and iACT interventions tailored towards the treatment of depression and chronic pain using a randomised controlled patient-preference design. METHODS AND ANALYSIS: Community dwelling adults with chronic non-cancer pain (CNCP) and major depression will be recruited from pain clinics and primary care providers in Newfoundland and Labrador, Canada. The study is a randomised controlled patient-preference trial. Eligible patients will be randomly assigned to a 'preference' or 'no-preference' arm during the first step of randomisation and to intervention or control in the second step of randomisation. Two interventions (ie, iCBT or iACT) will be evaluated relative to attention control. iCBT and iACT involve the completion of 7-weekly online modules augmented with one session of motivational enhancement and weekly therapy sessions. Primary outcomes include (1) feasibility and acceptability parameters and (2) change in symptoms of depression. Secondary outcomes include pain, physical function, emotional function and quality of life. We will recruit 60 participants and examine the range of effect sizes obtained from the trial but will not conduct significance testing as per recommendations for behavioural trial development. ETHICS AND DISSEMINATION: Ethics was approved by the provincial Health Research Ethics Board. Dissemination of results will be published in a peer-reviewed academic journal and presented at scientific conferences. TRIAL REGISTRATION NUMBER: NCT04009135.

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses.

The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis (Arabidopsis thaliana) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1's impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1, impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes.

Comparing the effectiveness of individual and group therapy for students with symptoms of anxiety and depression: A randomized pilot study.

Objective: To examine the effectiveness of individual versus group therapy for anxiety and depression among university students. Participants: Forty-one university students experiencing moderate to severe symptoms of anxiety and/or depression participated during one of three academic semesters from 2015 to 2016. Methods: Participants were randomly assigned to either 6-weeks of individual or group therapy and completed outcome measures at pre-and-post-treatment. Results: Significant reductions in both depression and anxiety scores were found across time, with no significant difference between group and individual therapy outcomes. Exploratory analysis of attitudes toward therapy found that while individual therapy was rated more favorably than group therapy overall, attitudes toward therapy became more favorable from pre to post-treatment for all participants. An interaction showed differences in attitudes toward individual and group therapy according to participants' randomly assigned treatment. Conclusions: These findings support the increased usage of group therapy within university counseling centers, with implications for stepped care discussed.

Implementation of the Ottawa Hospital Pain Clinic stepped care program: A preliminary report.

BACKGROUND: Access to multidisciplinary pain management treatment in Canada is limited, with wait times up to 4 years. Stepped care approaches to mental health treatment have led to substantial reduction and elimination of wait times and may be applicable to chronic pain settings. There is no unifying framework for stepped care chronic pain programs. A systematic review of the efficacy of stepped care in chronic pain management conducted by the Canadian Agency for Drugs and Technologies reported varied results that may be due to heterogeneous stepped care models across facilities. AIM: We propose a unifying framework for multidisciplinary stepped care chronic pain programs and present its application at The Ottawa Hospital Pain Clinic. The Ottawa Hospital stepped care framework is an eight-tiered approach that allows patients the opportunity to decide collaboratively with a health care professional which treatment program will best suit their needs for the management of chronic pain. As levels of stepped care increase, the time and resource commitment to each step will also increase. Treatment is stepped up or down, depending on patient needs. METHOD: This is a descriptive case study. RESULTS: Implementing the interprofessional model of care with the stepped care program has eliminated wait times for access to The Ottawa Hospital Pain Clinic Interprofessional Chronic Pain Management Program and has improved communication between professions of the interprofessional team, resulting in better care for patients. CONCLUSION: More research is needed to further develop and evaluate the clinical efficacy of stepped care to manage chronic pain.

Contexte: L'accès à la prise en charge multidisciplinaire de la douleur au Canada est limité, avec des délais d'attente pouvant aller jusqu'à quatre ans. Les approches de soins de santé mentale par paliers ont donné lieu à une réduction et une élimination des temps d'attente et peut être applicable aux contextes de soins pour la douleur chronique. Il n'existe pas de cadre unificateur pour les programmes de soins par paliers pour la douleur chronique. Un examen systématique de l'efficacit' des soins par paliers dans la prise en charge de la douleur chronique menée par l'Agence canadienne des médicaments et des technologies de la santé a fait état de résultats variés qui peuvent être attribuables à l'hétérogénéité des modèles de soins par paliers dans les vtablissements.Objectifs: Nous proposons un cadre unificateur pour les programmes de soins multidisciplinaires par paliers pour la douleur chronique et présentons son application à la Clinique de la douleur de l'Hôpital d'Ottawa. Le cadre de soins par paliers de l'Hôpital d'Ottawa est une approche à huit niveaux qui donne aux patients la possibilité de décider, en collaboration avec un professionnel de la santé, du programme de traitement qui répondra le mieux à leurs besoins pour la prise en charge de leur douleur chronique. À mesure que les niveaux de soins par paliers augmentent, le temps et les ressources nécessaires à chaque palier augmentent également. Le traitement est intensifié ou réduit, en fonction des besoins du patient.Méthodes: Il s'agit d'une étude de cas descriptive.Résultats: La mise en œuvre du modèle interprofessionnel de soins avec le programme de soins par paliers a éliminé les délais d'attente pour l'accès au programme de prise en charge interprofessionnelle de la douleur chronique de la clinique de la douleur de l'Hôpital d'Ottawa et a amélioré la communication entre les professions de l'équipe interprofessionnelle, ce qui a donné lieu à une meilleure prise en charge des patients.Conclusions: Des recherches supplémentaires sont nécessaires pour développer et évaluer davantage l'efficacité clinique des soins par paliers pour la prise en charge de la douleur chronique.

Operative Binding of Class I Release Factors and YaeJ Stabilizes the Ribosome in the Nonrotated State.

During translation, the small subunit of the ribosome rotates with respect to the large subunit primarily between two states as mRNA is being translated into a protein. At the termination of bacterial translation, class I release factors (RFs) bind to a stop codon in the A-site and catalyze the release of the peptide chain from the ribosome. Periodically, mRNA is truncated prematurely, and the translating ribosome stalls at the end of the mRNA forming a nonstop complex requiring one of several ribosome rescue factors to intervene. One factor, YaeJ, is structurally homologous with the catalytic region of RFs but differs by binding to the ribosome directly through its C-terminal tail. Structures of the ribosome show that the ribosome adopts the nonrotated state conformation when these factors are bound. However, these studies do not elucidate the influence of binding to cognate or noncognate codons on the dynamics of intersubunit rotation. Here, we investigate the effects of wild-type and mutant forms of RF1, RF2, and YaeJ binding on ribosome intersubunit rotation using single-molecule Förster resonance energy transfer. We show that both RF1 binding and RF2 binding are sufficient to shift the population of posthydrolysis ribosome complexes from primarily the rotated to the nonrotated state only when a cognate stop codon is present in the A-site. Similarly, YaeJ binding stabilizes nonstop ribosomal complexes in the nonrotated state. Along with previous studies, these results are consistent with the idea that directed conformational changes and binding of subsequent factors to the ribosome are requisite for efficient termination and ribosome recycling.

Meeting the mental health needs of today's college student: Reinventing services through Stepped Care 2.0.

A new stepped care model developed in North America reimagines the original United Kingdom model for the modern university campus environment. It integrates a range of established and emerging online mental health programs systematically along dimensions of treatment intensity and associated student autonomy. Program intensity can be either stepped up or down depending on level of client need. Because monitoring is configured to give both provider and client feedback on progress, the model empowers clients to participate actively in care options, decisions, and delivery. Not only is stepped care designed to be more efficient than traditional counseling services, early observations suggest it improves outcomes and access, including the elimination of service waitlists. This paper describes the new model in detail and outlines implementation experiences at 3 North American universities. While the experiences implementing the model have been positive, there is a need for development of technology that would facilitate more thorough evaluation. (PsycINFO Database Record

Preparation of ribosomes for smFRET studies: A simplified approach.

During the past decade, single-molecule studies of the ribosome have significantly advanced our understanding of protein synthesis. The broadest application of these methods has been towards the investigation of ribosome conformational dynamics using single-molecule Förster resonance energy transfer (smFRET). The recent advances in fluorescently labeled ribosomes and translation components have resulted in success of smFRET experiments. Various methods have been employed to target fluorescent dyes to specific locations within the ribosome. Primarily, these methods have involved additional steps including subunit dissociation and/or full reconstitution, which could result in ribosomes of reduced activity and translation efficiency. In addition, substantial time and effort are required to produce limited quantities of material. To enable rapid and large-scale production of highly active, fluorescently labeled ribosomes, we have developed a procedure that combines partial reconstitution with His-tag purification. This allows for a homogeneous single-step purification of mutant ribosomes and subsequent integration of labeled proteins. Ribosomes produced with this method are shown to be as active as ribosomes purified using classical methods. While we have focused on two labeling sites in this report, the method is generalizable and can in principle be extended to any non-essential ribosomal protein.

Single molecule studies of RNA-RNA interactions.

Non-coding RNAs including microRNAs, siRNAs, and snoRNAs interact with their targets directly through RNA-RNA interactions by base-paring (van Himbergen et al., Nucleic Acids Res 21(8):1713-1717, 1993). RNA-RNA interactions play important roles in gene transcription and protein translation, which can be investigated with several experimental techniques including single molecule methods. Here, we describe how single molecule Förster resonance energy transfer (FRET) can be used to study RNA-RNA interactions in vitro by either surface immobilization or vesicle encapsulation.

Interprofessional mental health training in rural primary care: findings from a mixed methods study.

The benefits of interprofessional care in providing mental health services have been widely recognized, particularly in rural communities where access to health services is limited. There continues to be a need for more continuing interprofessional education in mental health intervention in rural areas. There have been few reports of rural programs in which mental health content has been combined with training in collaborative practice. The current study used a sequential mixed-method and quasi-experimental design to evaluate the impact of an interprofessional, intersectoral education program designed to enhance collaborative mental health capacity in six rural sites. Quantitative results reveal a significant increase in positive attitudes toward interprofessional mental health care teams and self-reported increases in knowledge and understanding about collaborative mental health care delivery. The analysis of qualitative data collected following completion of the program, reinforced the value of teaching mental health content within the context of collaborative practice and revealed practice changes, including more interprofessional and intersectoral collaboration. This study suggests that imbedding explicit training in collaborative care in content focused continuing professional education for more complex and chronic health issues may increase the likelihood that professionals will work together to effectively meet client needs.

Single-Molecule Detection in Nanogap-Embedded Plasmonic Gratings.

We introduce nanogap-embedded silver plasmonic gratings for single-molecule (SM) visualization using an epifluorescence microscope. This silver plasmonic platform was fabricated by a cost-effective nano-imprint lithography technique, using an HD DVD template. DNA/ RNA duplex molecules tagged with Cy3/Cy5 fluorophores were immobilized on SiO2-capped silver gratings. Light was coupled to the gratings at particular wavelengths and incident angles to form surface plasmons. The SM fluorescence intensity of the fluorophores at the nanogaps showed approximately a 100-fold mean enhancement with respect to the fluorophores observed on quartz slides using an epifluorescence microscope. This high level of enhancement was due to the concentration of surface plasmons at the nanogaps. When nanogaps imaged with epifluorescence mode were compared to quartz imaged using total internal reflection fluorescence (TIRF) microscopy, more than a 30-fold mean enhancement was obtained. Due to the SM fluorescence enhancement of plasmonic gratings and the correspondingly high emission intensity, the required laser power can be reduced, resulting in a prolonged detection time prior to photobleaching. This simple platform was able to perform SM studies with a low-cost epifluorescence apparatus, instead of the more expensive TIRF or confocal microscopes, which would enable SM analysis to take place in most scientific laboratories.

Loss of Arabidopsis thaliana Dynamin-Related Protein 2B reveals separation of innate immune signaling pathways.

Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC-, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC-. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.

Ribosome structure and dynamics by smFRET microscopy.

Composed of both RNA and protein components, the ribosome is one of the largest macromolecular machines in life responsible for the production of all protein. Interestingly, the major catalytic center of the ribosome (the peptidyl transferase center) and much of the binding regions for both mRNA and tRNA are composed of RNA making the ribosome one of the most complex and widely studied ribozymes. Further, large-scale conformational rearrangements throughout the ribosome are required for proper function making the ribosome a riboswitch as well. Recent advances in single-molecule biophysics have significantly augmented our understanding of ribosome function as both a ribozyme and riboswitch. Here, we discuss single-molecule Förster resonance energy transfer and its application to the study of the ribosome. Also, we describe how these experiments are designed from sample preparation to data acquisition and analysis. The general approach and methods described here can be generally applied to many other biological systems.

RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation.

Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.