In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we ...show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA-driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans.
Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defence against parasitic nucleic acids and genome ...rearrangement. Many small regulatory RNAs are thought to function in nuclei. For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications. To understand further the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in Caenorhabditis elegans nuclei. Here we show that the gene nuclear RNAi defective-2 (nrde-2) encodes an evolutionarily conserved protein that is required for siRNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct an NRDE-2-dependent silencing of pre-messenger RNAs (pre-mRNAs) 3′ to sites of RNAi, an NRDE-2-dependent accumulation of RNA polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3′ to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation: siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.
Incomplete excision of malignant tissue is a major issue in breast-conserving surgery, with typically 20 - 30% of cases requiring a second surgical procedure arising from postoperative detection of ...an involved margin. We report advances in the development of a new intraoperative tool, optical coherence micro-elastography, for the assessment of tumor margins on the micro-scale. We demonstrate an important step by conducting whole specimen imaging in intraoperative time frames with a wide-field scanning system acquiring mosaicked elastograms with overall dimensions of ~50 × 50 mm, large enough to image an entire face of most lumpectomy specimens. This capability is enabled by a wide-aperture annular actuator with an internal diameter of 65 mm. We demonstrate feasibility by presenting elastograms recorded from freshly excised human breast tissue, including from a mastectomy, lumpectomies and a cavity shaving.
Abstract Regional manual volumetry is the gold standard of in vivo neuroanatomy, but is labor-intensive, can be imperfectly reliable, and allows for measuring limited number of regions. Voxel-based ...morphometry (VBM) has perfect repeatability and assesses local structure across the whole brain. However, its anatomic validity is unclear, and with its increasing popularity, a systematic comparison of VBM to manual volumetry is necessary. The few existing comparison studies are limited by small samples, qualitative comparisons, and limited selection and modest reliability of manual measures. Our goal was to overcome those limitations by quantitatively comparing optimized VBM findings with highly reliable multiple regional measures in a large sample ( N = 200) across a wide agespan (18–81). We report a complex pattern of similarities and differences. Peak values of VBM volume estimates (modulated density) produced stronger age differences and a different spatial distribution from manual measures. However, when we aggregated VBM-derived information across voxels contained in specific anatomically defined regions (masks), the patterns of age differences became more similar, although important discrepancies emerged. Notably, VBM revealed stronger age differences in the regions bordering CSF and white matter areas prone to leukoaraiosis, and VBM was more likely to report nonlinearities in age–volume relationships. In the white matter regions, manual measures showed stronger negative associations with age than the corresponding VBM-based masks. We conclude that VBM provides realistic estimates of age differences in the regional gray matter only when applied to anatomically defined regions, but overestimates effects when individual peaks are interpreted. It may be beneficial to use VBM as a first-pass strategy, followed by manual measurement of anatomically defined regions.
Campylobacter spp. cause mostly self-limiting enterocolitis, although a significant proportion of cases require hospitalisation highlighting potential for severe disease. Among people admitted, blood ...culture specimens are frequently collected and antibiotic treatment is initiated. We sought to understand clinical and host factors associated with bacteraemia, antibiotic treatment and isolate non-susceptibility among Campylobacter-associated hospitalisations. Using linked hospital microbiology and administrative data we identified and reviewed Campylobacter-associated hospitalisations between 2004 and 2013. We calculated population-level incidence for Campylobacter bacteraemia and used logistic regression to examine factors associated with bacteraemia, antibiotic treatment and isolate non-susceptibility among Campylobacter-associated hospitalisations. Among 685 Campylobacter-associated hospitalisations, we identified 25 admissions for bacteraemia, an estimated incidence of 0.71 cases per 100,000 population per year. Around half of hospitalisations (333/685) had blood culturing performed. Factors associated with bacteraemia included underlying liver disease (aOR 48.89, 95% CI 7.03-340.22, p < 0.001), Haematology unit admission (aOR 14.67, 95% CI 2.99-72.07, p = 0.001) and age 70-79 years (aOR 4.93, 95% CI 1.57-15.49). Approximately one-third (219/685) of admissions received antibiotics with treatment rates increasing significantly over time (p < 0.05). Factors associated with antibiotic treatment included Gastroenterology unit admission (aOR 3.75, 95% CI 1.95-7.20, p < 0.001), having blood cultures taken (aOR 2.76, 95% CI 1.79-4.26, p < 0.001) and age 40-49 years (aOR 2.34, 95% CI 1.14-4.79, p = 0.02). Non-susceptibility of isolates to standard antimicrobials increased significantly over time (p = 0.01) and was associated with overseas travel (aOR 11.80 95% CI 3.18-43.83, p < 0.001) and negatively associated with tachycardia (aOR 0.48, 95%CI 0.26-0.88, p = 0.02), suggesting a healthy traveller effect. Campylobacter infections result in considerable hospital burden. Among those admitted to hospital, an interplay of factors involving clinical presentation, presence of underlying comorbidities, complications and increasing age influence how a case is investigated and managed.
Campylobacter spp. infections are a globally important cause of enterocolitis, causing substantial morbidity. Capturing accurate information on hospitalisations is challenging and limited ...population-level data exist to describe the clinico-epidemiological characteristics of hospitalised cases.
Hospital administrative and laboratory datasets were linked to identify Campylobacter-associated hospitalisations between 2004 and 2013. Accuracy of morbidity coding was assessed using laboratory diagnosis as a gold standard, with health department surveillance data used to calculate population-based rates. Additional patient-level data were collected via review of medical records. Descriptive statistics were used to assess changes in rates and proportions and to assess relationships between key variables including age, length of stay, comorbidity and complications.
In total 685 Campylobacter-associated hospital admissions were identified, with the sensitivity of morbidity coding 52.8% (95% CI 48.9-56.7%). The mean annual rate of hospitalisation was 13.6%. Hospitalisation rates were higher for females across most age-groups, while for both genders marked increases were observed for those aged ≥60 years. Median admission age was 39.5 years, with an average length of stay of 3.5 days. Comorbidities were present in 34.5% (237/685) of admissions, with these patients more likely to develop electrolyte disturbances, hypotension, renal impairment or acute confusion (all p < 0.001). Bacteraemia and acute kidney injury were observed in 4.1% (28/685) and 3.6% (23/685) of admissions, respectively. Inpatient mortality was low (0.15%).
Under reporting of Campylobacter-associated hospitalisations is substantial but can be improved through data linkage. We observed demographic differences among those hospitalised but further work is needed to determine risk factors and predictors for hospitalisation.
Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational ...epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys 9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, which direct nuclear gene silencing in germ cells. In hrde-1- or nuclear RNAi-deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.
Ribonucleoprotein complexes consisting of Argonaute-like proteins and small regulatory RNAs function in a wide range of biological processes. Many of these small regulatory RNAs are predicted to act, ...at least in part, within the nucleus. We conducted a genetic screen to identify factors essential for RNA interference (RNAi) in nuclei of Caenorhabditis elegans and identified the Argonaute protein NRDE-3. In the absence of small interfering RNAs (siRNAs), NRDE-3 resides in the cytoplasm. NRDE-3 binds siRNAs generated by RNA-dependent RNA polymerases acting on messenger RNA templates in the cytoplasm and redistributes to the nucleus. Nuclear redistribution of NRDE-3 requires a functional nuclear localization signal, is required for nuclear RNAi, and results in NRDE-3 association with nuclear-localized nascent transcripts. Thus, specific Argonaute proteins can transport specific classes of small regulatory RNAs to distinct cellular compartments to regulate gene expression.
Self-management support forms a central aspect of chronic Illness management nationally and globally. Evidence for the success of self-management support has mainly focussed on individually-centred ...outcomes of behavioural change. While it is recognised that social network members play an important role there is currently a gap in knowledge regarding who provides what type of support and under what circumstances. This is relevant for understanding the division of labour and the meeting of needs for those living with a long-term condition. We therefore took a network approach to explore self-management support conceptualising it as types of illness 'work' undertaken within peoples' social networks. 300 people from deprived areas and with chronic illnesses took part in a survey conducted in 2010 in the North West of England. A concentric circles diagram was used as a research tool with which participants identified 2,544 network members who contributed to illness management. The results provide an articulation of how social network members are substantially involved in illness management. Whilst partners and close family make the highest contributions there is evidence of inputs from a wide range of relationships. Network member characteristics (type of relationship, proximity, frequency of contact) impact on the amount of illness work undertaken in peoples' networks. In networks with 'no partner' other people tend to contribute more in the way of illness related work than in networks with a partner. This indicates a degree of substitutability between differently constituted networks, and that the level and type of input by different members of a network might change according to circumstances. A network perspective offers an opportunity to redress the balance of an exclusively individual focus on self-management because it addresses the broader set of contributions and resources available to people in need of chronic illness management and support.
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