Dicot wood is mainly composed of cellulose, xylan and lignin, and its formation requires the coordinated regulation of their biosynthesis. In this report, we demonstrate that the poplar ...wood-associated MYB transcriptional activators, PtrMYB3 and PtrMYB20, activate the biosynthetic pathways of cellulose, xylan and lignin when overexpressed in Arabidopsis and they are also able to activate the promoter activities of poplar wood biosynthetic genes. We also show that PtrMYB3 and PtrMYB20 are functional orthologs of Arabidopsis MYB46 and MYB83, and their expression is directly activated by poplar PtrWND2, suggesting their involvement in the regulation of wood formation in poplar.
Mass cytometry presents an exceptional opportunity to interrogate the biology of highly heterogeneous cell populations, owing to the ability to collect highly parametric proteomic data at a single ...cell level. However, sample-to-sample variability, due to antibody staining and/or instrument sensitivity, can introduce substantial artifacts into the data, which can in turn lead to erroneous conclusions. This variability can be eliminated by sample barcoding which enables samples to be pooled, stained and run simultaneously. Existing mass cytometry barcoding approaches require time intensive labeling, reduce the number of biologically meaningful parameters and/or rely on expensive reagents. We present an approach utilizing monoisotopic cisplatin to perform cell barcoding that does not require cell permeabilization, can be completed in 10 minutes and can be utilized in combination with existing barcoding techniques to greatly increase the number of samples which can be multiplexed to improve throughput and consistency.
To determine how different pioneer transcription factors form a targeted, accessible nucleosome within compacted chromatin and collaborate with an ATP-dependent chromatin remodeler, we generated ...nucleosome arrays in vitro with a central nucleosome containing binding sites for the hematopoietic E-Twenty Six (ETS) factor PU.1 and Basic Leucine Zipper (bZIP) factors C/EBPα and C/EBPβ. Our long-read sequencing reveals that each factor can expose a targeted nucleosome on linker histone-compacted arrays, but with different nuclease sensitivity patterns. The DNA binding domain of PU.1 binds mononucleosomes, but requires an additional intrinsically disordered domain to bind and open compacted chromatin. The canonical mammalian SWI/SNF (cBAF) remodeler was unable to act upon two forms of locally open chromatin unless cBAF was enabled by a separate transactivation domain of PU.1. cBAF potentiates the PU.1 DNA binding domain to weakly open chromatin in the absence of the PU.1 disordered domain. Our findings reveal a hierarchy by which chromatin is opened and show that pioneer factors can provide specificity for action by nucleosome remodelers.
The biosynthesis of secondary walls in vascular plants requires the coordinated regulation of a suite of biosynthetic genes, and this coordination has recently been shown to be executed by the ...secondary wall NAC (SWN)-mediated transcriptional network. In Arabidopsis, five SWNs, including SND1, NST1/2 and VND6/7, function as master transcriptional switches to activate their common targets and consequently the secondary wall biosynthetic program. A recent report by Zhong et al.1 revealed that SWNs bind to a common cis-acting element, namely secondary wall NAC binding element (SNBE), which is composed of an imperfect palindromic 19-bp consensus sequence, (T/A)NN(C/T)(T/C/G)TNNNNNNNA(A/C)GN(A/C/T)(A/T). Genome-wide analysis of direct targets of SWNs showed that SWNs directly activate the expression of not only many transcription factors but also a battery of genes involved in secondary wall biosynthesis, cell wall modification and programmed cell death, the promoters of which all contain multiple SNBE sites. The functional significance of the SNBE sites is further substantiated by our current in planta expression study demonstrating that representative SNBE sequences from several SWN direct target promoters are sufficient to drive the expression of the GUS reporter gene in secondary wall-forming cells. The identification of the SWN DNA binding element (SNBE) and the SWN direct targets marks an important step forward toward the dissection of the transcriptional network regulating the biosynthesis of secondary walls, the most abundant biomass produced by land plants.
This protocol provides a two-parameter analysis of single-molecule tracking (SMT) trajectories of Halo-tagged histones in living adherent cell lines and unveils a chromatin mobility landscape ...composed of five chromatin types, ranging from low to high mobility. When the analysis is applied to Halo-tagged, chromatin-binding proteins, it associates chromatin interaction properties with known functions in a way that previously used SMT parameters did not.
For complete information on the use and execution of this protocol, please refer to Lerner et al. (2020).
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•Single-molecule imaging to assay/compare motions of chromatin and nuclear factors•Protocol walks the user through imaging, detection, and tracking of single molecules•Two-parameter analysis allows assay of motions of chromatin-interacting molecules•Translates motions into interaction with functionally diverse chromatin domains
This protocol provides a two-parameter analysis of single-molecule tracking (SMT) trajectories of Halo-tagged histones in living adherent cell lines and unveils a chromatin mobility landscape composed of five chromatin types, ranging from low to high mobility. When the analysis is applied to Halo-tagged, chromatin-binding proteins, it associates chromatin interaction properties with known functions in a way that previously used SMT parameters did not.
Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible ...with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.
Wood biomass is mainly made of secondary cell walls; hence, elucidation of the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis during wood formation will ...be instrumental to design strategies for genetic improvement of wood biomass. Here, we provide direct evidence demonstrating that the poplar (Populus trichocarpa) wood-associated NAC domain transcription factors (PtrWNDs) are master switches activating a suite of downstream transcription factors, and together, they are involved in the coordinated regulation of secondary wall biosynthesis during wood formation. We show that transgenic poplar plants with dominant repression of PtrWNDs functions exhibit a drastic reduction in secondary wall thickening in woody cells, and those with PtrWND overexpression result in ectopic deposition of secondary walls. Analysis of PtrWND2B overexpressors revealed up-regulation of the expression of a number of wood-associated transcription factors, the promoters of which were also activated by PtrWND6B and the Eucalyptus EgWND1. Transactivation analysis and electrophoretic mobility shift assay demonstrated that PtrWNDs and EgWND1 activated gene expression through direct binding to the secondary wall NAC-binding elements, which are present in the promoters of several wood-associated transcription factors and a number of genes involved in secondary wall biosynthesis and modification. The WND-regulated transcription factors PtrNAC150, PtrNAC156, PtrNAC157, PtrMYB18, PtrMYB74, PtrMYB75, PtrMYB121, PtrMYB128, PtrZF1, and PtrGATA8 were able to activate the promoter activities of the biosynthetic genes for all three major wood components. Our study has uncovered that the WND master switches together with a battery of their downstream transcription factors form a transcriptional network controlling secondary wall biosynthesis during wood formation.
Abstract
Background
Older adults frequently attend the Emergency Department (ED) and experience high rates of adverse outcomes following ED presentation including functional decline, ED ...re-presentation and unplanned hospital admission. The development of effective interventions to prevent such outcomes is a key priority for research and service provision. A presentation to an ED can be viewed as an opportunity to assess those at risk of adverse outcomes and initiate a care plan in those deemed as ‘high risk'. Our aim was to evaluate the feasibility of a physiotherapy led integrated care intervention for older adults discharged from the ED (ED-PLUS).
Methods
Older adults presenting to the ED with undifferentiated medical complaints and discharged within 72 hours were computer randomised in a ratio of 1:1:1 to deliver usual care, Comprehensive Geriatric Assessment (CGA) in the ED, or ED-PLUS (Trial registration: NCT04983602). ED-PLUS is an evidence-based and stakeholder-informed intervention to bridge the care transition between the ED and community by initiating a CGA in the ED and implementing a six-week, multi-component, self-management programme in the patient’s own home. Feasibility (recruitment and retention rates) and acceptability of the programme were assessed quantitatively and qualitatively. Functional decline was examined post-intervention using the Barthel Index. All outcomes were assessed by a research nurse blinded to group allocation.
Results
29 participants were recruited, indicating 97% of our recruitment target. 90% of participants completed the ED-PLUS intervention. All participants expressed positive feedback about the intervention. The incidence of functional decline at 6 weeks was 10% in the ED-PLUS group versus 70-89% in the usual care and CGA-only groups.
Conclusion
High adherence and retention rates were observed among participants and preliminary findings indicate a lower incidence of functional decline in the ED-PLUS group. Recruitment challenges existed in the context of COVID-19. Data collection is ongoing for six-month outcomes.
Background: Older adults frequently attend the emergency department (ED) and experience high rates of subsequent adverse outcomes including functional decline, ED re-presentation and unplanned ...hospital admission. The development of effective interventions to prevent such outcomes is a key priority for research and service provision. Our aim was to evaluate the feasibility of a physiotherapy-led integrated care intervention for older adults discharged from the ED (ED PLUS). Patients and Methods: Older adults presenting to the ED of a university teaching hospital with undifferentiated medical complaints and discharged within 72 hours were computer randomised in a ratio of 1:1:1 to deliver usual care, Comprehensive Geriatric Assessment (CGA) in the ED, or ED PLUS. ED PLUS is an evidence-based and stakeholder-informed intervention to bridge the care transition between the ED and community by initiating a CGA in the ED and implementing a six-week, multi-component, self-management programme in the patient's home. Feasibility and acceptability were assessed quantitatively and qualitatively. All clinical and process outcomes were assessed by a research nurse blinded to group allocation. Data analyses were primarily descriptive. Results: Twenty-nine participants were recruited indicating a 67% recruitment rate. At 6 months, there was 100% retention in the usual care group, 88% in the CGA group and 90% in the ED PLUS group. ED PLUS participants expressed positive feedback, and there was a trend towards improved function and quality of life and less ED revisits and unscheduled hospitalisations in the ED PLUS group. Conclusion: ED PLUS bridges the transition of care between the index visit to the ED and the community and is feasible using systematic recruitment strategies. Despite recruitment challenges in the context of COVID-19, the intervention was successfully delivered and well received by participants. There was a lower incidence of functional decline and improved quality of life in the ED PLUS group. Trial Registration: The trial was registered in Clinical Trials Protocols and Results System as of 21st July 2021, with registration number NCT04983602. Plain Language Summary: Older people need support when they are discharged from the Emergency Department (ED) to their home as a visit to the ED is linked to negative outcomes. This study reports on a trial to reduce negative outcomes in older people discharged from the ED by an assessment in the ED and treatment in the home. Twenty-nine patients attending the ED of University Hospital Limerick were randomly assigned to three groups as follows: One group got the normal care and treatment; Group two received a comprehensive assessment by a dedicated geriatric doctor in the ED and were referred to other health professionals as needed; a third group, called ED PLUS, also received a comprehensive assessment by a dedicated geriatric doctor in the ED and an additional six-week treatment program led by a physiotherapist. The physiotherapist visited the older adult in their own home within twenty-four hours of discharge to start treatment based on what the older adult needed to do to live safely in their own home and again at week four and week six. The same geriatric doctor who saw the older adult in the ED telephoned the older adult in week two to check medications and discussed the case with the physiotherapist. A dietitian and occupational therapist telephoned the older adults to check nutritional intake and if any aids were needed in the home. All older adults in the ED PLUS group had improved quality of their life, improved mobility and did not need to go back to the hospital or ED at six weeks and six months unlike the two other groups. The COVID-19 pandemic affected recruitment, but all ED PLUS patients were extremely satisfied. Keywords: emergencies, recruitment, integrated care, care transitions, public and patient involvement
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