RNA promotes the formation of spatial compartments in the nucleus.

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.

Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus.

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.

Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo.

Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.

Genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent Pre-mRNAs and chromatin sites.

Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5' splice sites and 5' splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.

Suitability of multifunction devices Myah and Myopia Master for monitoring myopia progression in children and adults.

PURPOSE: To assess the feasibility of using multifunction instruments to measure axial length for monitoring myopia progression in children and adults. METHODS: Axial length was measured in 60 children (aged 6-18 years) and 60 adults (aged 19-50 years) with multifunction instruments (Myah and Myopia Master) and stand-alone biometers (Lenstar LS900 and IOLMaster 700). Repeatability (measurements by the same examiner) and reproducibility (measurements by different examiners) were computed as the within-subject standard deviation (Sw) and 95% limits of agreement (LoA). Inter-instrument agreement was computed as intraclass correlation coefficients. The threshold for detecting myopic progression was taken as 0.1 mm. Measures were repeated only in children following the administration of 1% tropicamide to determine the impact of cycloplegia on axial length. RESULTS: Overall, the IOLMaster 700 had the best repeatability in children (0.014 mm) and adults (0.009 mm). Repeatability Sw values for all devices ranged from 0.005 to 0.021 mm (children) and 0.003 to 0.016 mm (adults). In children, reproducibility fell within 0.1 mm 95% of the time for the Myah, Myopia Master and IOLMaster 700. Agreement among all devices was classified as excellent (ICC 0.999; 95% CI 0.998-0.999), but the 95% LoA among the Myah, Myopia Master and Lenstar LS900 was ≥0.1 mm. Cycloplegia had no statistically significant effect on axial length (all p > 0.13). CONCLUSIONS: The Myah and Myopia Master multifunction instruments demonstrated good repeatability and reproducibility, and their accuracy was comparable to stand-alone biometers. Axial length measurements using different instruments can be considered interchangeable but should be compared with some caution. Accurate axial length measurements can be obtained without cycloplegia. The multifunction instruments Myah and Myopia Master are as well suited for monitoring myopia progression in children as the stand-alone biometers IOLMaster 700 and Lenstar LS900.

Influencing factors of nurses' short-term bereavement reactions after patient death.

An online cross-sectional survey was performed among 181 nurses in mainland China who experienced their most recent patient death within the last month. Multivariate linear regressions were used following bivariate analysis to identify influencing factors for their short-term professional bereavement reactions. More intensive reactions were associated with the nurse's fewer experiences of patient death; the nurse's employment in the intensive care unit rather than the emergency, oncology, geriatrics, or internal medicine departments; and the patient experiencing more pain in the last few days. Higher reaction scores were also reported by nurses who lost the patient more than 1 week prior.

The role of the social environment on dementia caregivers' pre-death grief: A mixed- methods systematic review.

This systematic review examined the role of social environment in pre-death grief experiences of dementia caregivers. Ninety-three Chinese and English articles were included from a comprehensive search of empirical studies using nine databases. Six social environment domains were generated: the person with dementia, dyadic relationship, family members and the wider community, health and social care services, place of care, and social-cultural contexts. A complex interplay between caregivers and their social environments that aggravate and attenuate pre-death grief experiences is evident. Research has focused mainly on the effects of people with dementia and dyadic relationships and has paid modest attention to the effects of family, relatives, and health and social care services. Caregivers' experiences with their friends, fellow caregivers, the wider community, and social-cultural norms are influential but understudied. Future research could adopt a systems thinking approach with sociological perspectives to generate a comprehensive and nuanced understanding of pre-death grief experiences.

What makes advance care planning discussion so difficult? A systematic review of the factors of advance care planning in healthcare settings.

OBJECTIVES: Existing systematic reviews related to advance care planning (ACP) largely focus on specific groups and intervention efficacy or are limited to contextual factors. This research aims to identify the modifiable factors perceived by different users of ACP in healthcare settings and inform healthcare professionals about the factors affecting ACP practice. METHODS: Five English-language databases (ProQuest, PubMed, CINAHL Plus, Scopus, and Medline) and two Chinese-language databases (CNKI and NCL) were searched up to November 2022. Empirical research identifying factors related to ACP in healthcare settings was included. ACP is defined as a discussion process on future end-of-life care. Thematic synthesis was performed on all included studies. RESULTS: A total of 1871 unique articles were screened; the full texts of 193 were assessed by 4 reviewers, and 45 articles were included for analysis. Twenty-two (54%) studies were qualitative, 15 (33%) were quantitative, and 6 (13%) used mixed methods. Foci varied from 28 (62%) studies on a single subject group (either patient, family, or physician), 11 (25%) on 2 subject groups (either patient and family or patient and healthcare professional), and 6 (13%) covered 3 subject groups (patient, family, and healthcare professional). Among the 17 studies involving more than 1 subject group, only 2 adopted a dyadic lens in analysis. Complex interwoven factors were categorized into (1) intrapersonal factors, (2) interpersonal factors, and (3) socio-environmental factors, with a total of 11 themes: personal belief, emotions, the burden on others, timing, responsiveness, relationship, family dynamics, experience, person taking the lead, culture, and support. SIGNIFICANCE OF RESULTS: Patients, families, and healthcare professionals are the essential stakeholders of ACP in healthcare settings. Factors are interweaved among the intrapersonal, interpersonal, and socio-environmental dimensions. Research is warranted to examine the dynamic interactions of the 3 essential stakeholders from a multidimensional perspective, and the mechanism of the interweaving of factors.

The connections of physical and psychosocial symptoms among patients with terminal illnesses: A network analysis.

BACKGROUND: Patients with terminal illnesses have different symptoms. The goal of palliative care is to alleviate the suffering induced by these symptoms. Available research on symptoms has focused on their prevalence or identification of clusters. Little is known about the central symptoms contributing to the maintenance of the whole system of symptoms or the mechanism of influence between symptoms, particularly between physical and psychosocial symptoms. AIM: The study used network analysis to explore symptom clusters among patients with terminal illnesses and identify the central symptoms, the bridge symptoms, and pathways between physical and psychosocial symptoms. DESIGN AND SETTING/PARTICIPANTS: This is a retrospective cohort study using baseline symptom assessment of 677 adults with terminal illnesses receiving care from the Integrated Community End-of-Life Care Support Teams. RESULTS: Three symptom clusters were identified: physical, psychological, and practical clusters. "Weakness or lack of energy" was identified as the crucial symptom affecting the changes of other symptoms, followed by depression and anxiety. Three connections between the psychological and physical symptoms were identified: anxiety-pain, depression-constipation, and perceived family anxiety-poor appetite pathways. Pathways of practical concern-peace and practical concern-depression were indicated between practical and psychological symptoms. CONCLUSIONS: This study is the first of its kind to investigate the connections and mechanism of influence between symptoms among patients with terminal illnesses. It offers clear pathways for intervention with the most influential symptoms of weakness, anxiety, and depression.

Trust and Access: Eye Information-seeking Practices and Preferences among Canadians.

SIGNIFICANCE: This study highlights the value that the public places on obtaining trusted and accessible health-related information and their preference for obtaining it from their health care practitioners. Previous research has not been specific to Canadians or vision. Findings can be used to increase eye health literacy and eye care utilization. PURPOSE: Canadians underuse eye care and underestimate the occurrence of asymptomatic eye disease. This study explored eye information-seeking practices and preferences among a group of Canadians. METHODS: Using snowball sampling, a 28-item online survey collected respondent perceptions about their eye and health information-seeking practices and preferences. Questions examined electronic device access, information source use, and demographics. Two open-ended questions examined information-seeking practices and preferences. Respondents were at least 18 years old and living in Canada. Individuals working in eye care were excluded. Response frequencies and z scores were computed. Written comments were assessed using content analysis. RESULTS: Respondents searched for less eye than health information ( z scores ≥ 2.25, P < .05). For eye and health information, primary care providers were the used and preferred source, and reliance on Internet searches was greater than desired. Trust and access drove information-seeking practices. Respondent comments suggested that a hierarchy of trust operates across My Health Team, My Network, and My External Sources, with a persistent threat posed by Discredited Sources. Access to information sources seemed mediated by enablers (Convenience and Accessible Features) and barriers (Unreachable Health Team and Absent Systems). Eye information was seen as more specialized and harder to find. There was a high regard for health care practitioners who provide their patients with curated trusted information. CONCLUSIONS: These Canadians value trusted and accessible health-related information. They prefer eye and health information from their health care practitioners and value when their health team provides online curated information, particularly regarding eyes.

The meaning of patient deaths for professional caregivers: A quantitative construct validation.

The meaning of patient deaths is key to understanding professional bereavement. To validate its construct, we applied partial least square structural equation modeling (PLS-SEM) to online survey data from 563 Chinese physicians and nurses. Both the personal meaning and professional meaning contribute significantly while not interchangeably to the meaning of patient deaths. In addition to the loss of an acquaintance, the loss of a valuable life, and the loss of a professional goal, patient deaths can also mean trauma, bad luck, identity crisis, a warning, a learning opportunity, and a time to empathize with others' sufferings.

Redefining caregiver strain for family caregivers in end-of-life care in Hong Kong.

OBJECTIVES: Caregiving for family members at their end of life is stressful. Caregivers' strain, burden, or stress has been measured in various geographical and sociodemographic contexts. The concept of stress, burden, and strain are sometimes used interchangeably. By analysing the factor structure of the Chinese version of the Modified Caregiver Strain Index (C-M-CSI), this study aimed to examine the caregiving strain concept and its demographic correlates. METHODS: A sample of 453 family caregivers of patients with a terminal illness in Hong Kong was employed. Exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) were performed. In addition, generalized linear models (GLM) were used to examine the demographic correlates. RESULTS: The EFA yielded a 3-factor model termed "Perception of Caregiving," "Empathetic Strain," and "Adjustment Demand." This 3-factor model explained 50% of the variance and showed good internal consistency. The CFA confirmed the 3-factor construct with satisfactory internal reliability (χ2 [61, N = 226] = 108.86, p < 0.001, CFI = 0.96, TLI = 0.95, SRMR = 0.04, and RMSEA = 0.06). Inspired by this factor structure and concepts of stress and strain from engineering discipline, a new model of caregiver strain is proposed. Additionally, family caregivers of non-cancer patients, who were not living with the patient, or younger were associated negatively with different dimensions of caregiver strain. SIGNIFICANCE OF RESULTS: The results gave insights into the advancement of the conceptualization of caregiver strain, its multidimensional nature, and process of change, which inform directions for future research and practices.

Bereavement After Patient Deaths Among Chinese Physicians and Nurses: A Qualitative Description Study.

In order to achieve an in-depth understanding of professional caregivers' experiences of bereavement after patient deaths in Mainland China, qualitative description was employed. 24 physicians and nurses from hospitals in Nanjing, China, participated in one-to-one, semi-structured interviews. Thematic analysis was adopted for data analysis. Five themes were generated: the nature of professional bereavement experiences, the meaning of patient deaths, immediate bereavement reactions, long-term changes, and coping strategies. Each theme included personal and professional dimensions. Professional bereavement experiences in Mainland China were found to be influenced by workplace violence against professional caregivers, traditional Chinese medical ethics, the strong death taboo, and inadequacies of the healthcare system. Professional bereavement experiences are meaning-driven, comprehensive, and usually disenfranchised. They involve multidimensional reactions and have both short-term and long-lasting, both event-specific and accumulated impacts. Cultural and systemic factors could shape professional bereavement experiences.

Determinants of Organ Donation Registration Among University Students in Hong Kong.

The present study explored the determinants of registration to facilitate the promotion of posthumous organ donation based on the theory of reasoned actions. A total of 211 university students participated in the study. Logistic regression, multiple regressions, and path analysis were conducted. The logistic regression model correctly predicted nearly 80% of registration status, and a good fit model of registration for posthumous organ donation (MRPOD) was proposed. Findings suggested that affective attitude, subjective norm of family, self-rated knowledge, distance from death, and age were predictors of registration. Worries about organ removal and postmortem mutilation anxiety were also mediated through bodily integrity, which had an indirect effect on the registration through affective attitude. Policymakers are suggested to address the negative emotions associated with challenges towards bodily integrity, encourage parents to initiate discussions with children, instill correct knowledge of brain death, and intervene the bystander effect on organ donation.

A Tale of Two Surveys: Life-Affirming Strategy of Mortuary Improves the Quality of Bereavement Care After Hospital Death.

Background: The mortuary plays an important, under-recognized role in end-of-life care. A 'Life-affirming strategy' was introduced in the mortuary of a university hospital to enhance respect for the deceased and next-of-kin (NOK). Design: NOK who collected bodies in the mortuary of a university hospital participated in a survey. The satisfaction scores, needs and expectations were compared with a similar survey from 2015. Results: The overall experience for NOK improved significantly compared with 2015. The greatest improvement was achieved in 'mortuary environment', 'attitude of mortuary staff' and 'body viewing arrangement in the mortuary'. The perceived need for additional psychosocial support was significantly reduced. Conclusions: Results demonstrate success of the life-affirming strategy in enhancing end-of-life care for bereaved families. The person-centered approach modernizes and professionalizes mortuary services, with a positive impact on the deceased, NOK, mortuary staff, hospital administration and wider community.

Extracellular Secretion and Simple Purification of Bacterial Collagen from Escherichia coli.

Because of structural similarities with type-I animal collagen, recombinant bacterial collagen-like proteins have been progressively used as a source of collagen for biomaterial applications. However, the intracellular expression combined with current costly and time-consuming chromatography methods for purification makes the large-scale production of recombinant bacterial collagen challenging. Here, we report the use of an adapted secretion pathway, used natively byEscherichia colito secrete curli fibers, for extracellular secretion of the bacterial collagen. We confirmed that a considerable fraction of expressed collagen (∼70%) is being secreted freely into the extracellular medium, with an initial purity of ∼50% in the crude culture supernatant. To simplify the purification of extracellular collagen, we avoided cell lysis and used cross-flow filtration or acid precipitation to concentrate the voluminous supernatant and separate the collagen from impurities. We confirmed that the secreted collagen forms triple helical structures, using Sirius Red staining and circular dichroism. We also detected collagen biomarkers via Raman spectroscopy, further supporting that the recombinant collagen forms a stable triple helical conformation. We further studied the effect of the isolation methods on the morphology and secondary structure, concluding that the final collagen structure is process-dependent. Overall, we show that the curli secretion system can be adapted for extracellular secretion of the bacterial collagen, eliminating the need for cell lysis, which simplifies the collagen isolation process and enables a simple cost-effective method with potential for scale-up.

m(6)A RNA methylation promotes XIST-mediated transcriptional repression.

The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues-a reversible base modification of unknown function in long non-coding RNAs. We show that m6A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m6A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m6A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m6A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression.

Grief literacy: A call to action for compassionate communities.

The compassionate communities movement challenges the notion that death and dying should be housed within clinical and institutional contexts, and works to normalize conversations about death and dying by promoting death literacy and dialogue in public spaces. Community-based practices and conversations about grief remain marginal in this agenda. We aimed to theorize how grief could be better conceptualized and operationalized within the compassionate communities movement. We develop the concept of Grief Literacy and present vignettes to illustrate a grief literate society. Grief literacy augments the concept of death literacy, thereby further enhancing the potential of the compassionate communities approach.