The long non-coding RNA Xist directs a remarkable instance of developmentally regulated, epigenetic change known as X Chromosome Inactivation (XCI). By spreading in cis across the X chromosome from which it is expressed, Xist RNA facilitates the creation of a heritably silent, heterochromatic nuclear territory that displays a three-dimensional structure distinct from that of the active X chromosome. How Xist RNA attaches to and propagates across a chromosome and its influence over the three-dimensional (3D) structure of the inactive X are aspects of XCI that have remained largely unclear. Here, we discuss studies that have made significant contributions towards answering these open questions.

Cetacean behaviour has long attracted scientific attention as humans endeavour to discover what makes these mammals so emotive and engaging. To date, much of this research has focussed on abundant and widely distributed cetacean species such as bottlenose dolphins ( Tursiops truncatus ) and humpback whales ( Megaptera novaeangliae ). As an endangered and often evasive species, research regarding Irrawaddy dolphin ( Orcaella brevirostris ) behaviour is limited. This study uses data collected by The Cambodian Marine Mammal Conservation Project, to investigate the behavioural responses of Irrawaddy dolphins towards a dead conspecific. During a routine boat survey of Cambodia’s Kep Archipelago, the carcass of an adult female Irrawaddy dolphin was recovered and attached to the stern of the research vessel and promptly towed to the research island for further examination. During this survey, there was a four-fold increase in the number of Irrawaddy dolphin groups observed compared to the seasonal average (post-monsoon), in addition to an atypically positive response towards the research vessel and an atypical increase in the number of behavioural events observed. These behavioural variations were believed to be in response to the towed dead conspecific. The authors propose future dedicated research to assess the complexity of wild Irrawaddy dolphin behaviour, cognition, and awareness, to robustly exemplify the species’ apparent sentience and intelligence.

Female eutherians transcriptionally silence one X chromosome to balance gene dosage between the sexes. X-chromosome inactivation (XCI) is initiated by the lncRNA Xist, which assembles many proteins within the inactive X chromosome (Xi) to trigger gene silencing and heterochromatin formation. It is well established that gene silencing on the Xi is maintained through repressive epigenetic processes, including histone deacetylation and DNA methylation. Recent studies revealed a new mechanism where RNA-binding proteins that interact directly with the RNA contribute to the maintenance of Xist localization and gene silencing. In addition, a surprising plasticity of the Xi was uncovered with many genes becoming upregulated upon experimental deletion of Xist. Intriguingly, immune cells normally lose Xist from the Xi, suggesting that thisXist dependence is utilized in vivo to dynamically regulate gene expression from the Xi. These new studies expose fundamental regulatory mechanisms for the chromatin association of RNAs, highlight the need for studying the maintenance of XCI and Xist localization in a gene- and cell-type-specific manner, and are likely to have clinical impact.

Background

In a large statewide initiative, New York State implemented collaborative care (CC) from 2012 to 2014 in 32 primary care settings where residents were trained and supported its sustainability through payment reforms implemented in 2015. Twenty-six clinics entered the sustainability phase and six opted out, providing an opportunity to examine factors predicting continued CC participation and fidelity.

Methods

We used descriptive statistics to assess implementation metrics in sustaining vs. opt-out clinics and trends in implementation fidelity 1 and 2 years into the sustainability phase among sustaining clinics. To characterize barriers and facilitators, we conducted 31 semi-structured interviews with psychiatrists, clinic administrators, primary care physicians, and depression care managers (24 at sustaining, 7 at opt-out clinics).

Results

At the end of the implementation phase, clinics opting to continue the program had significantly higher care manager full-time equivalents (FTEs) and achieved greater clinical improvement rates (46% vs. 7.5%, p = 0.004) than opt-out clinics. At 1 and 2 years into sustainability, the 26 sustaining clinics had steady rates of depression screening, staffing FTEs and treatment titration rates, significantly higher contacts/patient and improvement rates and fewer enrolled patients/FTE. During the sustainability phase, opt-out sites reported lower patient caseloads/FTE, psychiatry and care manager FTEs, and physician/psychiatrist CC involvement compared to sustaining clinics. Key barriers to sustainability noted by respondents included time/resources/personnel (71% of respondents from sustaining clinics vs. 86% from opt-out), patient engagement (67% vs. 43%), and staff/provider engagement (50% vs. 43%). Fewer respondents mentioned early implementation barriers such as leadership support, training, finance, and screening/referral logistics. Facilitators included engaging patients (e.g., warm handoffs) (79% vs. 86%) and staff/providers (71% vs. 100%), and hiring personnel (75% vs. 57%), particularly paraprofessionals for administrative tasks (67% vs. 0%).

Conclusions

Clinics that saw early clinical improvement and who invested in staffing FTEs were more likely to elect to enter the sustainability phase. Structural rules (e.g., payment reform) both encouraged participation in the sustainability phase and boosted long-term outcomes. While limited to settings with academic affiliations, these results demonstrate that patient and provider engagement and care manager resources are critical factors to ensuring sustainability.

Understanding the biologic role of N⁶-methyladenosine (m⁶A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m⁶A in exons but very rarely in introns. The m⁶A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m⁶A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m⁶As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m⁶A in wild-type mouse stem cells is spliced the same in cells lacking the major m⁶A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m⁶As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m⁶A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m⁶A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.

Many long non-coding RNAs (lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with Xist, three of these proteins--SHARP, SAF-A and LBR--are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA polymerase II (Pol II) from the inactive X. Both SMRT and HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome.

Tuberculosis and parasitic diseases, such as giardiasis, amebiasis, leishmaniasis, and trypanosomiasis, all urgently require improved treatment options. Recently, it has been shown that antitubercular bicyclic nitroimidazoles such as pretomanid and delamanid have potential as repurposed therapeutics for the treatment of visceral leishmaniasis. Here, we show that pretomanid also possesses potent activity against Giardia lamblia and Entamoeba histolytica, thus expanding the therapeutic potential of nitroimidazooxazines. Synthetic analogues with a novel nitroimidazopyrazin-one/-e bicyclic nitroimidazole chemotype were designed and synthesized, and structure-activity relationships were generated. Selected derivatives had potent antiparasitic and antitubercular activity while maintaining drug-like properties such as low cytotoxicity, good metabolic stability in liver microsomes and high apparent permeability across Caco-2 cells. The kinetic solubility of the new bicyclic derivatives varied and was found to be a key parameter for future optimization. Taken together, these results suggest that promising subclasses of bicyclic nitroimidazoles containing different core architectures have potential for further development.

Background

Insufficient data exist on population-based trends in morbidity and mortality to determine the success of prevention strategies and improvements in health care delivery in stroke. The aim of this study was to determine trends in incidence and outcome (1-year mortality, 28-day case-fatality) in relation to management and risk factors for stroke in the multi-ethnic population of Auckland, New Zealand (NZ) over 30-years.

Methods

Four stroke incidence population-based register studies were undertaken in adult residents (aged ≥15 years) of Auckland NZ in 1981-1982, 1991-1992, 2002-2003 and 2011-2012. All used standard World Health Organization (WHO) diagnostic criteria and multiple overlapping sources of case-ascertainment for hospitalised and non-hospitalised, fatal and non-fatal, new stroke events. Ethnicity was consistently self-identified into four major groups. Crude and age-adjusted (WHO world population standard) annual incidence and mortality with corresponding 95% confidence intervals (CI) were calculated per 100,000 people, assuming a Poisson distribution.

Results

5400 new stroke patients were registered in four 12 month recruitment phases over the 30-year study period; 79% were NZ/European, 6% Māori, 8% Pacific people, and 7% were of Asian or other origin. Overall stroke incidence and 1-year mortality decreased by 23% (95% CI 5%-31%) and 62% (95% CI 36%-86%), respectively, from 1981 to 2012. Whilst stroke incidence and mortality declined across all groups in NZ from 1991, Māori and Pacific groups had the slowest rate of decline and continue to experience stroke at a significantly younger age (mean ages 60 and 62 years, respectively) compared with NZ/Europeans (mean age 75 years). There was also a decline in 28-day stroke case fatality (overall by 14%, 95% CI 11%-17%) across all ethnic groups from 1981 to 2012. However, there were significant increases in the frequencies of pre-morbid hypertension, myocardial infarction, and diabetes mellitus, but a reduction in frequency of current smoking among stroke patients.

Conclusions

In this unique temporal series of studies spanning 30 years, stroke incidence, early case-fatality and 1-year mortality have declined, but ethnic disparities in risk and outcome for stroke persisted suggesting that primary stroke prevention remains crucial to reducing the burden of this disease.

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear¹. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus² and binding diverse proteins^3-5 to achieve X-chromosome inactivation (XCI)^6,7. The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off⁸. Here we show that the Xist RNA-binding proteins PTBP1⁹, MATR3¹⁰, TDP-43¹¹ and CELF1¹² assemble on the multivalent E-repeat element of Xist⁷ and, via self-aggregation and heterotypic protein-protein interactions, form a condensate¹ in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase⁸, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.

Background

Dengue is the most prevalent arboviral infection causing an estimated 50-60 million cases of febrile illness globally per year, exacting considerable disease burden. Few instruments exist to assess the patient illness experience, with most based on healthcare provider assessment, lacking standardization in timepoints and symptom assessment. This study aimed to evaluate the content validity of the novel 'Dengue Virus Daily Diary (DENV-DD)', designed to measure symptom intensity and disease burden within outpatient infant to adult populations.

Methods

The Dengue Illness Index Report Card was used as a foundation to create the DENV-DD, consisting of patient- and observer-reported outcome (PRO/ObsRO) instruments. In two South American dengue-endemic communities, qualitative combined concept elicitation and cognitive debriefing interviews were conducted among individuals and caregivers of children with symptomatic laboratory-confirmed dengue. Interviews were conducted across two rounds allowing DENV-DD modifications. A small-scale quantitative assessment of the DENV-DD was also conducted with data from an independent Dengue Human Infection Model (DHIM) to generate early evidence of feasibility of DENV-DD completion, instrument performance and insight into the sign/symptom trajectory over the course of illness.

Results

Forty-eight participants were interviewed (20 adults, 20 older children/adolescents with their caregivers, 8 caregivers of younger children). A wide spectrum of signs/symptoms lasting 3-15 days were reported with fever, headache, body ache/pain, loss of appetite, and body weakness each reported by > 70% participants. DENV-DD instructions, items and response scales were understood, and items were considered relevant across ages. DHIM data supported feasibility of DENV-DD completion.

Conclusions

Findings demonstrate content validity of the DENV-DD (PRO/ObsRO instruments) in dengue-endemic populations. Psychometric and cultural validity studies are ongoing to support use of the DENV-DD in clinical studies.