Determination and inference of eukaryotic transcription factor sequence specificity.

Transcription factor (TF) DNA sequence preferences direct their regulatory activity, but are currently known for only ∼1% of eukaryotic TFs. Broadly sampling DNA-binding domain (DBD) types from multiple eukaryotic clades, we determined DNA sequence preferences for >1,000 TFs encompassing 54 different DBD classes from 131 diverse eukaryotes. We find that closely related DBDs almost always have very similar DNA sequence preferences, enabling inference of motifs for ∼34% of the ∼170,000 known or predicted eukaryotic TFs. Sequences matching both measured and inferred motifs are enriched in chromatin immunoprecipitation sequencing (ChIP-seq) peaks and upstream of transcription start sites in diverse eukaryotic lineages. SNPs defining expression quantitative trait loci in Arabidopsis promoters are also enriched for predicted TF binding sites. Importantly, our motif "library" can be used to identify specific TFs whose binding may be altered by human disease risk alleles. These data present a powerful resource for mapping transcriptional networks across eukaryotes.

Experiences of People with Aphasia Receiving Enhanced Community-Based Speech-Language Pathology Provided by Speech-Language Pathology Students.

INTRODUCTION: Treatment intensity for people with aphasia (PWA) is a significant factor in enhancing recovery. Personal factors such as fatigue, physical endurance, and motivation as well as clinician availability have been described as barriers to increased intensity. The use of student therapists has been shown to assist with addressing service gaps. METHODS: The aim of the study was to explore the experiences of PWA who received enhanced community-based treatment delivered by speech-language pathology (SLP) students. Enhanced community-based treatment was defined as three or more treatment sessions per week targeting communication. Semi-structured interviews were conducted with 10 PWA living in New Zealand. Interview data were analysed with reflexive thematic analysis. RESULTS: Two themes and six sub-themes were developed. Theme 1 related to intensity and included more treatment is better than less (sub-theme 1), there's a "right time" for more intensive treatment (sub-theme 2), the hard work is worth the effort (sub-theme 3). Theme 2 related to working with SLP students; it didn't feel like they were students (sub-theme 4), we just got on so well (sub-theme 5), and they listened to what I wanted (sub-theme 6). CONCLUSIONS: The findings confirm that PWA value access to more intensive treatment and desire involvement in decisions about flexible treatment schedules. PWA have positive experiences when treatment is provided by SLP students. Implications for clinical practice and future research directions are discussed.

Written reflective practice abilities of SLT students across the degree programme.

BACKGROUND: Written reflective practice (WRP) is a teaching tool used across speech-language therapy (SLT) clinical education programmes. The process aims to support the development of reflective skills required for the workplace (e.g., problem-solving and self-evaluation). AIMS: This cross-sectional and repeated-measures study design investigated students' demonstration of breadth of WRP across the clinical education programme. METHODS & PROCEDURES: The participants were 77 undergraduate SLT students in their first, second or final professional year of the clinical programme. Participants wrote critical reflections following an interaction with a client/s as part of their clinical education experiences. Formative feedback was provided after each written reflection (WR). In total four WRs per participant were coded for breadth of WRP using a modification of Plack et al.'s coding schema from 2005. This was completed for each of the four time points across the academic year for each professional year. OUTCOMES & RESULTS: There was a statistically significant association between time (i.e., professional year of the programme) and likelihood of demonstration of breadth of reflection for the lower level reflective element of 'attend' and higher level reflective element of 're-evaluate'. A positive trend between time and likelihood of demonstration of breadth of reflection was seen for the lower level element of 'reflection-for-action'. Final-professional-year students exhibited significant enhancements in the higher level elements (e.g., 'premise') compared with first- and second-professional-year students. CONCLUSIONS & IMPLICATIONS: This group of SLT students exhibited significant change in breadth of WRP across the degree programme. This finding has positive implications for facilitating WRP with students and using the current coding framework in clinical programmes. WHAT THIS PAPER ADDS: What is already known on this subject WRP is one form of reflective practice (RP) used in SLT, allied health, medical and nursing clinical education programmes. Researchers have suggested that RP skills develop over time for students. Previously, studies examining WRP have focused on one off assessment of skill or over a timeframe of 6-10 weeks. Here, we examine SLT students' WRP skills across the degree programme. What this paper adds to existing knowledge SLT students exhibited significant positive change in breadth of WRP across the degree programme as their clinical experience increased. Our results provide quantitative information in support of using RP as a learning tool throughout clinical education programmes for SLT. What are the potential or actual clinical implications of this work? This study offers support for educators of SLT students; for example, how educators can assess WRP, and how educators can foster SLT student skill development with formative feedback and reflective questioning. This study also offers support for student SLT, for example, describing how WRP can be part of their individualized learning approach and provide a purposeful examination of self and clinical skill development.

Measuring significant changes in chromatin conformation with ACCOST.

Chromatin conformation assays such as Hi-C cannot directly measure differences in 3D architecture between cell types or cell states. For this purpose, two or more Hi-C experiments must be carried out, but direct comparison of the resulting Hi-C matrices is confounded by several features of Hi-C data. Most notably, the genomic distance effect, whereby contacts between pairs of genomic loci that are proximal along the chromosome exhibit many more Hi-C contacts that distal pairs of loci, dominates every Hi-C matrix. Furthermore, the form that this distance effect takes often varies between different Hi-C experiments, even between replicate experiments. Thus, a statistical confidence measure designed to identify differential Hi-C contacts must accurately account for the genomic distance effect or risk being misled by large-scale but artifactual differences. ACCOST (Altered Chromatin COnformation STatistics) accomplishes this goal by extending the statistical model employed by DEseq, re-purposing the 'size factors,' which were originally developed to account for differences in read depth between samples, to instead model the genomic distance effect. We show via analysis of simulated and real data that ACCOST provides unbiased statistical confidence estimates that compare favorably with competing methods such as diffHiC, FIND and HiCcompare. ACCOST is freely available with an Apache license at https://bitbucket.org/noblelab/accost.

Predicting gene expression in the human malaria parasite Plasmodium falciparum using histone modification, nucleosome positioning, and 3D localization features.

Empirical evidence suggests that the malaria parasite Plasmodium falciparum employs a broad range of mechanisms to regulate gene transcription throughout the organism's complex life cycle. To better understand this regulatory machinery, we assembled a rich collection of genomic and epigenomic data sets, including information about transcription factor (TF) binding motifs, patterns of covalent histone modifications, nucleosome occupancy, GC content, and global 3D genome architecture. We used these data to train machine learning models to discriminate between high-expression and low-expression genes, focusing on three distinct stages of the red blood cell phase of the Plasmodium life cycle. Our results highlight the importance of histone modifications and 3D chromatin architecture in Plasmodium transcriptional regulation and suggest that AP2 transcription factors may play a limited regulatory role, perhaps operating in conjunction with epigenetic factors.

A compendium of RNA-binding motifs for decoding gene regulation.

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

RNAcompete-S: Combined RNA sequence/structure preferences for RNA binding proteins derived from a single-step in vitro selection.

RNA-binding proteins recognize RNA sequences and structures, but there is currently no systematic and accurate method to derive large (>12base) motifs de novo that reflect a combination of intrinsic preference to both sequence and structure. To address this absence, we introduce RNAcompete-S, which couples a single-step competitive binding reaction with an excess of random RNA 40-mers to a custom computational pipeline for interrogation of the bound RNA sequences and derivation of SSMs (Sequence and Structure Models). RNAcompete-S confirms that HuR, QKI, and SRSF1 prefer binding sites that are single stranded, and recapitulates known 8-10bp sequence and structure preferences for Vts1p and RBMY. We also derive an 18-base long SSM for Drosophila SLBP, which to our knowledge has not been previously determined by selections from pure random sequence, and accurately discriminates human replication-dependent histone mRNAs. Thus, RNAcompete-S enables accurate identification of large, intrinsic sequence-structure specificities with a uniform assay.

"Take Us Into Account": Perspectives of Family Members of People With Parkinson's Disease Regarding Speech-Language Pathology Intervention.

PURPOSE: While communication changes associated with Parkinson's disease (PD) have been documented, research on the impact of these changes on family members is just beginning to emerge. With this new focus on family, questions arise as to how well speech-language pathology services address their needs communicating with their loved one with PD. The purpose of this study was to explore the experiences of family members of people with PD (PwPD) and their recommendations for speech-language pathology services that incorporated their needs. METHOD: Seventeen spouses/partners of PwPD participated in focus groups that were recorded, transcribed, and analyzed using thematic analyses. RESULTS: Three themes emerged, all focusing around the central tenet that the experiences of family members, and hence their need for speech-language pathology support, transitioned through the stages of PD progression. Theme 1 summarized increasing burdens on family to manage communication as PD progressed beyond a brief period of independent strategy use by PwPD. Theme 2 highlighted multifactorial contributors to communication burdens on families, with cognitive impairments being the most underrecognized. Theme 3 illustrated how families wanted more intervention options from speech-language pathologists (SLPs) that included them, but with a tailored approach for PD stages and personal preferences. CONCLUSIONS: When SLPs provide families with either generic communication strategies or strategies that do not fit the individualized needs of PwPD and their families, we may inadvertently be increasing the burden on families. There is a need for systematic, evidence-based, family-centered interventions that include, but go beyond, current speech-focused interventions to meet the shared communication needs of PwPD and their families.

Transcriptome analysis in mouse skin after exposure to ultraviolet radiation from a canopy sunbed.

Exposure to ultraviolet radiation (UV-R), from both natural and artificial tanning, heightens the risk of skin cancer by inducing molecular changes in cells and tissues. Despite established transcriptional alterations at a molecular level due to UV-R exposure, uncertainties persist regarding UV radiation characterization and subsequent genomic changes. Our study aimed to mechanistically explore dose- and time-dependent gene expression changes, that may drive short-term (e.g., sunburn) and long-term actinic (e.g., skin cancer) consequences. Using C57BL/6N mouse skin, we analyzed transcriptomic expression following exposure to five erythemally weighted UV-R doses (0, 5, 10, 20, and 40 mJ/cm2) emitted by a UV-R tanning device. At 96 h post-exposure, 5 mJ/cm2 induced 116 statistically significant differentially expressed genes (DEGs) associated with structural changes from UV-R damage. The highest number of significant gene expression changes occurred at 6 and 48 h post-exposure in the 20 and 40 mJ/cm2 dose groups. Notably, at 40 mJ/cm2, 13 DEGs related to skin barrier homeostasis were consistently perturbed across all timepoints. UV-R exposure activated pathways involving oxidative stress, P53 signaling, inflammation, biotransformation, skin barrier maintenance, and innate immunity. This in vivo study's transcriptional data offers mechanistic insights into both short-term and potential non-threshold-dependent long-term health effects of UV-R tanning.

RBPDB: a database of RNA-binding specificities.

The RNA-Binding Protein DataBase (RBPDB) is a collection of experimental observations of RNA-binding sites, both in vitro and in vivo, manually curated from primary literature. To build RBPDB, we performed a literature search for experimental binding data for all RNA-binding proteins (RBPs) with known RNA-binding domains in four metazoan species (human, mouse, fly and worm). In total, RPBDB contains binding data on 272 RBPs, including 71 that have motifs in position weight matrix format, and 36 sets of sequences of in vivo-bound transcripts from immunoprecipitation experiments. The database is accessible by a web interface which allows browsing by domain or by organism, searching and export of records, and bulk data downloads. Users can also use RBPDB to scan sequences for RBP-binding sites. RBPDB is freely available, without registration at http://rbpdb.ccbr.utoronto.ca/.

A qualitative study of reflective practice in the workplace. Speech-language pathologists have their say.

PURPOSE: Engaging in reflective practice (RP) and demonstrating reflective abilities is an essential graduate skill for speech-language pathologists (SLPs), yet limited studies have examined the perspectives of practicing SLPs and how and why they engage in RP. This qualitative study aimed to examine SLPs' experiences and perspectives of RP in diverse workplaces. METHOD: Individual semi-structured interviews were conducted with 30 SLPs working in health, education, or private practice sectors. Interviews were analysed using thematic analysis. RESULT: Three themes were developed from the data, describing what SLPs use RP for, what SLPs perceive as important in order to engage in RP in the workplace, as well as the barriers they have identified, and how SLPs have observed a change in engaging in RP as they have progressed in their careers. CONCLUSION: SLPs described that RP is valued in the workplace for supporting client focused care, problem-solving, and lifelong learning. SLPs wanted time to be protected for RP at all stages of their career and valued the relationships with others as contributing positively to RP. Perceptions of and engagement in RP changed in relation to SLPs' clinical experience. Implications for clinical practice are discussed.

Combining viral genomics and clinical data to assess risk factors for severe COVID-19 (mortality, ICU admission, or intubation) amongst hospital patients in a large acute UK NHS hospital Trust.

Throughout the COVID-19 pandemic, valuable datasets have been collected on the effects of the virus SARS-CoV-2. In this study, we combined whole genome sequencing data with clinical data (including clinical outcomes, demographics, comorbidity, treatment information) for 929 patient cases seen at a large UK hospital Trust between March 2020 and May 2021. We identified associations between acute physiological status and three measures of disease severity; admission to the intensive care unit (ICU), requirement for intubation, and mortality. Whilst the maximum National Early Warning Score (NEWS2) was moderately associated with severe COVID-19 (A = 0.48), the admission NEWS2 was only weakly associated (A = 0.17), suggesting it is ineffective as an early predictor of severity. Patient outcome was weakly associated with myriad factors linked to acute physiological status and human genetics, including age, sex and pre-existing conditions. Overall, we found no significant links between viral genomics and severe outcomes, but saw evidence that variant subtype may impact relative risk for certain sub-populations. Specific mutations of SARS-CoV-2 appear to have little impact on overall severity risk in these data, suggesting that emerging SARS-CoV-2 variants do not result in more severe patient outcomes. However, our results show that determining a causal relationship between mutations and severe COVID-19 in the viral genome is challenging. Whilst improved understanding of the evolution of SARS-CoV-2 has been achieved through genomics, few studies on how these evolutionary changes impact on clinical outcomes have been seen due to complexities associated with data linkage. By combining viral genomics with patient records in a large acute UK hospital, this study represents a significant resource for understanding risk factors associated with COVID-19 severity. However, further understanding will likely arise from studies of the role of host genetics on disease progression.

Multiple pathways of SARS-CoV-2 nosocomial transmission uncovered by integrated genomic and epidemiological analyses during the second wave of the COVID-19 pandemic in the UK.

Introduction: Throughout the global COVID-19 pandemic, nosocomial transmission has represented a major concern for healthcare settings and has accounted for many infections diagnosed within hospitals. As restrictions ease and novel variants continue to spread, it is important to uncover the specific pathways by which nosocomial outbreaks occur to understand the most suitable transmission control strategies for the future. Methods: In this investigation, SARS-CoV-2 genome sequences obtained from 694 healthcare workers and 1,181 patients were analyzed at a large acute NHS hospital in the UK between September 2020 and May 2021. These viral genomic data were combined with epidemiological data to uncover transmission routes within the hospital. We also investigated the effects of the introduction of the highly transmissible variant of concern (VOC), Alpha, over this period, as well as the effects of the national vaccination program on SARS-CoV-2 infection in the hospital. Results: Our results show that infections of all variants within the hospital increased as community prevalence of Alpha increased, resulting in several outbreaks and super-spreader events. Nosocomial infections were enriched amongst older and more vulnerable patients more likely to be in hospital for longer periods but had no impact on disease severity. Infections appeared to be transmitted most regularly from patient to patient and from patients to HCWs. In contrast, infections from HCWs to patients appeared rare, highlighting the benefits of PPE in infection control. The introduction of the vaccine at this time also reduced infections amongst HCWs by over four-times. Discussion: These analyses have highlighted the importance of control measures such as regular testing, rapid lateral flow testing alongside polymerase chain reaction (PCR) testing, isolation of positive patients in the emergency department (where possible), and physical distancing of patient beds on hospital wards to minimize nosocomial transmission of infectious diseases such as COVID-19.

Assessing and developing the written reflective practice skills of speech-language pathology students.

PURPOSE: Written reflective practice aims to support critical thinking and problem solving skills in speech-language pathology (SLP) clinical education programmes. Yet, there has been limited investigation of students' development of written reflective practice skills over time and during a real-time clinical experience. The purpose of this study was to investigate students' development of breadth and depth of written reflective practice across a six-week clinical experience. METHOD: Participants were 59 undergraduate and 14 postgraduate SLP students. Participants wrote critical reflections describing an interaction with a client/s at the conclusion of weeks two, four and six of their clinical experience. Formative feedback was provided after each submission. Breadth and depth of reflection were coded using a modification of Plack et al.'s coding schema. RESULT: There was a statistically significant association between time and likelihood of development of breadth of reflection for the elements process and content. Depth of reflection improved significantly across time. The majority of participants were classified as "reflectors" or critical reflector at the conclusion of the study. CONCLUSION: SLP students can make significant improvements in both breadth and depth of written reflective practice over a six-week period. Implications for clinical teaching are discussed.

Changes in genome organization of parasite-specific gene families during the Plasmodium transmission stages.

The development of malaria parasites throughout their various life cycle stages is coordinated by changes in gene expression. We previously showed that the three-dimensional organization of the Plasmodium falciparum genome is strongly associated with gene expression during its replication cycle inside red blood cells. Here, we analyze genome organization in the P. falciparum and P. vivax transmission stages. Major changes occur in the localization and interactions of genes involved in pathogenesis and immune evasion, host cell invasion, sexual differentiation, and master regulation of gene expression. Furthermore, we observe reorganization of subtelomeric heterochromatin around genes involved in host cell remodeling. Depletion of heterochromatin protein 1 (PfHP1) resulted in loss of interactions between virulence genes, confirming that PfHP1 is essential for maintenance of the repressive center. Our results suggest that the three-dimensional genome structure of human malaria parasites is strongly connected with transcriptional activity of specific gene families throughout the life cycle.

The dynamic three-dimensional organization of the diploid yeast genome.

The budding yeast Saccharomyces cerevisiae is a long-standing model for the three-dimensional organization of eukaryotic genomes. However, even in this well-studied model, it is unclear how homolog pairing in diploids or environmental conditions influence overall genome organization. Here, we performed high-throughput chromosome conformation capture on diverged Saccharomyces hybrid diploids to obtain the first global view of chromosome conformation in diploid yeasts. After controlling for the Rabl-like orientation using a polymer model, we observe significant homolog proximity that increases in saturated culture conditions. Surprisingly, we observe a localized increase in homologous interactions between the HAS1-TDA1 alleles specifically under galactose induction and saturated growth. This pairing is accompanied by relocalization to the nuclear periphery and requires Nup2, suggesting a role for nuclear pore complexes. Together, these results reveal that the diploid yeast genome has a dynamic and complex 3D organization.

High-throughput characterization of protein-RNA interactions.

RNA-binding proteins (RBPs) are important regulators of eukaryotic gene expression. Genomes typically encode dozens to hundreds of proteins containing RNA-binding domains, which collectively recognize diverse RNA sequences and structures. Recent advances in high-throughput methods for assaying the targets of RBPs in vitro and in vivo allow large-scale derivation of RNA-binding motifs as well as determination of RNA-protein interactions in living cells. In parallel, many computational methods have been developed to analyze and interpret these data. The interplay between RNA secondary structure and RBP binding has also been a growing theme. Integrating RNA-protein interaction data with observations of post-transcriptional regulation will enhance our understanding of the roles of these important proteins.

The Crystal Structure of the NHL Domain in Complex with RNA Reveals the Molecular Basis of Drosophila Brain-Tumor-Mediated Gene Regulation.

TRIM-NHL proteins are conserved among metazoans and control cell fate decisions in various stem cell linages. The Drosophila TRIM-NHL protein Brain tumor (Brat) directs differentiation of neuronal stem cells by suppressing self-renewal factors. Brat is an RNA-binding protein and functions as a translational repressor. However, it is unknown which RNAs Brat regulates and how RNA-binding specificity is achieved. Using RNA immunoprecipitation and RNAcompete, we identify Brat-bound mRNAs in Drosophila embryos and define consensus binding motifs for Brat as well as a number of additional TRIM-NHL proteins, indicating that TRIM-NHL proteins are conserved, sequence-specific RNA-binding proteins. We demonstrate that Brat-mediated repression and direct RNA-binding depend on the identified motif and show that binding of the localization factor Miranda to the Brat-NHL domain inhibits Brat activity. Finally, to unravel the sequence specificity of the NHL domain, we crystallize the Brat-NHL domain in complex with RNA and present a high-resolution protein-RNA structure of this fold.

Brain tumor is a sequence-specific RNA-binding protein that directs maternal mRNA clearance during the Drosophila maternal-to-zygotic transition.

BACKGROUND: Brain tumor (BRAT) is a Drosophila member of the TRIM-NHL protein family. This family is conserved among metazoans and its members function as post-transcriptional regulators. BRAT was thought to be recruited to mRNAs indirectly through interaction with the RNA-binding protein Pumilio (PUM). However, it has recently been demonstrated that BRAT directly binds to RNA. The precise sequence recognized by BRAT, the extent of BRAT-mediated regulation, and the exact roles of PUM and BRAT in post-transcriptional regulation are unknown. RESULTS: Genome-wide identification of transcripts associated with BRAT or with PUM in Drosophila embryos shows that they bind largely non-overlapping sets of mRNAs. BRAT binds mRNAs that encode proteins associated with a variety of functions, many of which are distinct from those implemented by PUM-associated transcripts. Computational analysis of in vitro and in vivo data identified a novel RNA motif recognized by BRAT that confers BRAT-mediated regulation in tissue culture cells. The regulatory status of BRAT-associated mRNAs suggests a prominent role for BRAT in post-transcriptional regulation, including a previously unidentified role in transcript degradation. Transcriptomic analysis of embryos lacking functional BRAT reveals an important role in mediating the decay of hundreds of maternal mRNAs during the maternal-to-zygotic transition. CONCLUSIONS: Our results represent the first genome-wide analysis of the mRNAs associated with a TRIM-NHL protein and the first identification of an RNA motif bound by this protein family. BRAT is a prominent post-transcriptional regulator in the early embryo through mechanisms that are largely independent of PUM.

Paralogs hnRNP L and hnRNP LL exhibit overlapping but distinct RNA binding constraints.

HnRNP (heterogeneous nuclear ribonucleoprotein) proteins are a large family of RNA-binding proteins that regulate numerous aspects of RNA processing. Interestingly, several paralogous pairs of hnRNPs exist that exhibit similar RNA-binding specificity to one another, yet have non-redundant functional targets in vivo. In this study we systematically investigate the possibility that the paralogs hnRNP L and hnRNP LL have distinct RNA binding determinants that may underlie their lack of functional redundancy. Using a combination of RNAcompete and native gel analysis we find that while both hnRNP L and hnRNP LL preferentially bind sequences that contain repeated CA dinucleotides, these proteins differ in their requirement for the spacing of the CAs. Specifically, hnRNP LL has a more stringent requirement for a two nucleotide space between CA repeats than does hnRNP L, resulting in hnRNP L binding more promiscuously than does hnRNP LL. Importantly, this differential requirement for the spacing of CA dinucleotides explains the previously observed differences in the sensitivity of hnRNP L and LL to mutations within the CD45 gene. We suggest that overlapping but divergent RNA-binding preferences, as we show here for hnRNP L and hnRNP LL, may be commonplace among other hnRNP paralogs.