Thousands of long non-coding RNAs (lncRNAs) have been identified in mammalian cells. Some have important functions and their dysregulation can contribute to a variety of disease states. However, most ...lncRNAs have not been functionally characterized. Complicating their study, lncRNAs have widely varying subcellular distributions: some reside predominantly in the nucleus, the cytoplasm or in both compartments. One method to query function is to suppress expression and examine the resulting phenotype. Methods to suppress expression of mRNAs include antisense oligonucleotides (ASOs) and RNA interference (RNAi). Antisense and RNAi-based gene-knockdown methods vary in efficacy between different cellular compartments. It is not known if this affects their ability to suppress lncRNAs. To address whether localization of the lncRNA influences susceptibility to degradation by either ASOs or RNAi, nuclear lncRNAs (MALAT1 and NEAT1), cytoplasmic lncRNAs (DANCR and OIP5-AS1) and dual-localized lncRNAs (TUG1, CasC7 and HOTAIR) were compared for knockdown efficiency. We found that nuclear lncRNAs were more effectively suppressed using ASOs, cytoplasmic lncRNAs were more effectively suppressed using RNAi and dual-localized lncRNAs were suppressed using both methods. A mixed-modality approach combining ASOs and RNAi reagents improved knockdown efficacy, particularly for those lncRNAs that localize to both nuclear and cytoplasmic compartments.
Using captured waste carbon dioxide (CCU) as a chemical reagent is an attractive means to add value to carbon capture and storage (CCS) and is a high-priority target for manufacturing. One promising ...route is to copolymerize carbon dioxide and epoxides, to prepare aliphatic polycarbonates. In this study, three homogeneous dinuclear Zn and Mg catalysts, previously reported by our group (see Kember M. R. ; Knight P. D. ; Reung P. T. R. ; Williams C. K Angew. Chem., Int. Ed. 2009, 48, 931–933 and Kember M. R. ; Williams C. K. J. Am. Chem. Soc. 2012, 134, 15676–15679 ) have been investigated using captured and contaminated carbon dioxide, with cyclohexene oxide, to produce polymers. Carbon dioxide captured from the carbon capture demonstrator plant at Ferrybridge Power Station, U.K., is applied for the efficient production of poly(cyclohexylene carbonate). Remarkably, the dinuclear Zn and Mg catalysts display nearly equivalent turnover numbers (TON) and turnover frequencies (TOF) using captured CO2 versus those using purified CO2. The tolerance of the catalysts to reactions contaminated with known quantities of exogenous water, nitrogen, SO2, amine, and octadecanethiol are reported. The catalyst activities, productivities, and selectivities are presented, together with the polymers’ number-average molecular weights (M n), dispersities (Đ), and end-group analyses. The catalysts show high tolerance to protic impurities, including the addition of amine, thiol, and water. In particular, under certain conditions, efficient polymerization can be conducted in the presence of up to 400 equiv of water without compromising catalytic activity/productivity or selectivity. Furthermore, the catalysts can selectively produce polycarbonate polyols with molecular weights in the range of 600−9000 g/mol and disperities <1.10.
Exploratory qualitative.
The aim of this study was to describe the experiences of bowel and bladder dysfunction on social activities and relationships in people with spinal cord injury living in the ...community.
People living with spinal cord injury experiencing bowel and bladder dysfunction.
Participants were recruited through the Australian Quadriplegic Association Victoria. Semi-structured in-depth interviews were undertaken with purposively selected participants to ensure representation of age, gender, spinal cord injury level and compensation status. A thematic analysis was performed to interpret patient experiences.
Twenty-two participants took part in the study. Bladder and bowel dysfunction altered relationships because of issues with intimacy, strained partner relationships and role changes for family and friends. A lack of understanding from friends about bladder and bowel dysfunction caused frustration, as this impairment was often responsible for variable attendance at social activities. Issues with the number, location, access and cleanliness of bathrooms in public areas and in private residences negatively affected social engagement. Social activities were moderated by illness, such as urinary tract infections, rigid and unreliable bowel routines, stress and anxiety about incontinence and managing the public environment, and due to continuous changes in plans related to bowel and bladder issues. Social support and adaptation fostered participation in social activities.
Tension exists between managing bowel and bladder dysfunction and the desire to participate in social activities. Multiple intersecting factors negatively affected the social relationships and activities of people with spinal cord injury and bowel and bladder dysfunction.
ABSTRACT
Purpose
Cataloguing endogenous miRNA targets by inhibiting miRNA function is fundamental to understanding the biological importance of each miRNA in gene regulatory pathways. Methods to ...down-regulate miRNA activity may help treat diseases where over-expression of miRNAs relates to the underlying pathophysiology. This study objectively evaluates the
in vitro
potency of different anti-miRNA oligonucleotides (AMOs) using various design and modification strategies described in the literature as well as some novel modification strategies.
Methods
MiR21 and miR16 AMOs, containing chemical modifications such as 2′-
O
-methyl RNA, locked nucleic acid and 2′-Fluoro bases with or without phosphorothioate linkages, were directly compared by transfection into HeLa cells using a dual-luciferase reporter assay to quantify miRNA inhibition.
Results
Potency for the various AMOs ranged from inactive at high dose (50 nM) to strongly inhibitory at both high and low dose (1 nM). Including phosphorothioate linkages improved nuclease stability and generally increased functional potency.
Conclusions
Incorporating high binding affinity modifications, such as LNA and 2′F bases, increases AMO potency while maintaining specificity; nevertheless, use of low dose is preferred when using high potency reagents to minimize the potential for cross reactivity. 2′OMe/LNA chimeras with PS modifications were the most potent constructs tested for miRNA inhibition
in vitro
.
MicroRNAs (miRNAs) are RNA sequences of approximately 22 nucleotides that mediate post-transcriptional regulation of specific mRNAs. miRNA sequences are dispersed throughout the genome and are ...classified as intergenic (between genes) or intronic (embedded into a gene). Intergenic miRNAs are expressed by their own promoter, and until recently, it was supposed that intronic miRNAs are transcribed from their host gene. Here, we performed a genomic analysis of currently known intronic miRNA regions and observed that approximately 35% of intronic miRNAs have upstream regulatory elements consistent with promoter function. Among all intronic miRNAs, 30% have associated Pol II regulatory elements, including transcription start sites, CpG islands, expression sequence tags, and conserved transcription factor binding sites, while 5% contain RNA Pol III regulatory elements (A/B box sequences). We cloned intronic regions encompassing miRNAs and their upstream Pol II (miR-107, miR-126, miR-208b, miR-548f-2, miR-569, and miR-590) or Pol III (miR-566 and miR-128-2) sequences into a promoterless plasmid, and confirmed that miRNA expression occurs independent of host gene transcription. For miR-128-2, a miRNA overexpressed in acute lymphoblastic leukemia, ChIP analysis suggests dual regulation by both intronic (Pol III) and host gene (Pol II) promoters. These data support complex regulation of intronic miRNA expression, and have relevance to disregulation in disease settings.
Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune ...cell infiltrate at the dermal-epidermal junction and upper dermis. Here, we explore the role of the immune cell-secreted serine protease, granzyme B, in pemphigoid disease pathogenesis using three independent murine models. In all models, granzyme B knockout or topical pharmacological inhibition significantly reduces total blistering area compared to controls. In vivo and in vitro studies show that granzyme B contributes to blistering by degrading key anchoring proteins in the dermal-epidermal junction that are necessary for dermal-epidermal adhesion. Further, granzyme B mediates IL-8/macrophage inflammatory protein-2 secretion, lesional neutrophil infiltration, and lesional neutrophil elastase activity. Clinically, granzyme B is elevated and abundant in human pemphigoid disease blister fluids and lesional skin. Collectively, granzyme B is a potential therapeutic target in pemphigoid diseases.
The physiological barriers of the brain impair drug delivery for treatment of many neurological disorders. One delivery approach that has not been investigated for their ability to penetrate the ...brain is RNA-based aptamers. These molecules can impart delivery to peripheral tissues and circulating immune cells, where they act as ligand mimics or can be modified to carry payloads. We developed a library of aptamers and an in vivo evolution protocol to determine whether specific aptamers could be identified that would home to the brain after injection into the peripheral vasculature. Unlike biopanning with recombinant bacteriophage libraries, we found that the aptamer library employed here required more than 15 rounds of in vivo selection for convergence to specific sequences. The aptamer species identified through this approach bound to brain capillary endothelia and penetrated into the parenchyma. The methods described may find general utility for targeting various payloads to the brain.
Objective
Melatonin has been widely studied in the treatment of sleep disorders and evidence is accumulating on a possible role for melatonin influencing mood. Our aim was to determine the efficacy ...and acceptability of melatonin for mood disorders.
Method
We conducted a comprehensive systematic review of randomized clinical trials on patients with mood disorders, comparing melatonin to placebo.
Results
Eight clinical trials were included; one study in bipolar, three in unipolar depression and four in seasonal affective disorder. We have only a small study on patients with bipolar disorder, while we have more studies testing melatonin as an augmentation strategy for depressive episodes in major depressive disorder and seasonal affective disorder. The acceptability and tolerability were good. We analyzed data from three trials on depressive episodes and found that the evidence for an effect of melatonin in improving mood symptoms is not significant (SMD = 0.37; 95% CI −0.05, 0.37; P = 0.09). The small sample size and the differences in methodology of the trials suggest that our results are based on data deriving from investigations occurring early in this field of study.
Conclusion
There is no evidence for an effect of melatonin on mood disorders, but the results are not conclusive and justify further research.
Anti-microRNA oligonucleotides (AMOs) are steric blocking antisense reagents that inhibit microRNA (miRNA) function by hybridizing and repressing the activity of a mature miRNA. First generation AMOs ...employed 2′-O-Methyl RNA nucleotides (2′OMe) with phosphorothioate (PS) internucleotide linkages positioned at both ends to block exonuclease attack. Second generation AMOs improved potency through the use of chemical modifications that increase binding affinity to the target, such as locked nucleic acid (LNA) residues. However, this strategy can reduce specificity as high binding affinity compounds can bind to and suppress function of related sequences even if one or more mismatches are present. Further, unnatural modified nucleic acid residues can have toxic side effects. In the present study, a variety of non-nucleotide modifiers were screened for utility in steric blocking antisense applications. A novel compound, N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (“ZEN”), was discovered that increased binding affinity and blocked exonuclease degradation when placed at or near each end of a single-stranded oligonucleotide. This new modification was combined with the 2′OMe RNA backbone to make ZEN-AMOs. The new ZEN-AMOs have high potency and can effectively inhibit miRNA function in vitro at low nanomolar concentrations, show high specificity, and have low toxicity in cell culture.
Genome-encoded microRNAs (miRNAs) provide a post-transcriptional regulatory layer that is important for pancreas development. However, how specific miRNAs are intertwined into the transcriptional ...network, which controls endocrine differentiation, is not well understood. Here, we show that microRNA-7 (miR-7) is specifically expressed in endocrine precursors and in mature endocrine cells. We further demonstrate that Pax6 is an important target of miR-7. miR-7 overexpression in developing pancreas explants or in transgenic mice led to Pax6 downregulation and inhibition of α- and β-cell differentiation, resembling the molecular changes caused by haploinsufficient expression of Pax6. Accordingly, miR-7 knockdown resulted in Pax6 upregulation and promoted α- and β-cell differentiation. Furthermore, Pax6 downregulation reversed the effect of miR-7 knockdown on insulin promoter activity. These data suggest a novel miR-7-based circuit that ensures precise control of endocrine cell differentiation.
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