A heterozygous variant in the human cardiac miR-133 gene, MIR133A2, alters miRNA duplex processing and strand abundance

BACKGROUND MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Sequential cleavage of miRNA precursors results in a ~22 nucleotide duplex of which one strand, the mature miRNA, is typically loaded into the RNA-induced silencing complex (RISC) while the passenger strand is degraded. Very little is known about how genetic variation might affect miRNA biogenesis and function. RESULTS We re-sequenced the MIR1-1, MIR1-2, MIR133A1, MIR133A2, and MIR133B genes, that encode the cardiac-enriched miRNAs, miR-1 and miR-133, in 120 individuals with familial atrial fibrillation and identified 10 variants, including a novel 79T > C MIR133A2 substitution. This variant lies within the duplex at the 3' end of the mature strand, miR-133a-3p, and is predicted to prevent base-pairing and weaken thermostability at this site, favoring incorporation of the passenger strand, miR-133a-5p, into RISC. Genomic DNA fragments containing miR-133a-2 precursor sequences with 79T and 79C alleles were transfected into HeLa cells. On Northern blotting the 79T allele showed strong expression of miR-133a-3p with weak expression of miR-133a-5p. In contrast, the 79C allele had no effect on miR-133a-3p but there was a significant increase (mean 3.6-fold) in miR-133a-5p levels. Deep sequencing of small RNA libraries prepared from normal human and murine atria confirmed that nearly all the mature miR-133a was comprised of miR-133a-3p and that levels of miR-133a-5p were very low. A number of isomiRs with variations at 5' and 3' ends were identified for both miR-133a-3p and miR-133a-5p, with 2 predominant miR-133a-3p isomiRs and one predominant miR-133a-5p isomiR. Bioinformatics analyses indicate that the major miR-133a-3p and 5p isomiRs have numerous predicted target mRNAs, only a few of which are in common. CONCLUSIONS Multiple miR-133a isomiRs with potential different mRNA target profiles are present in the atrium in humans and mice. We identified a human 79T > C MIR133A2 variant that alters miRNA processing and results in accumulation of the miR-133a-5p strand that is usually degraded

Environmental and ecological citizenship in civil society

Drawing from the work of Andrew Dobson, two notions of citizenship in civil society can be distinguished: environmental citizenship, which focuses on environmental rights and seeks to redefine the relationship between the state and the citizen; and ecological citizenship, which goes beyond a rights-based notion of citizenship to advocate the fair usage of ecological space across international borders. Using civil society initiatives to conserve forests, this article argues that these two notions of citizenship should be seen as overlapping in that civil society groups seek to work through national and international law to reduce the ecological footprint of some countries on others. The article concludes by drawing a distinction between the environmental state and the ecological state

From both sides now: Crossover effects influence navigation in patients with unilateral neglect

Unilateral neglect is a challenging disorder that pervades a range of behaviours following stroke and hampers recovery. Although a preponderance of clinical studies measure performance on a range of bedside assessments, including line bisection and cancellation tasks, there have been calls for studies to embrace more relevant functional measures. Here, for the first time, we present data from two separate tasks that characterise the performance of seven patients with unilateral neglect when navigating a power wheelchair. The tasks involved negotiating an obstacle course and steering a central path between gaps of different sizes. Results from the obstacle course confirmed the clinical observation and predicted bias of contralesional errors. However, the second task revealed a robust "crossover" effect. Patients deviated to the ipsilesional side for large gaps but deviated increasingly contralesionally when steering through small gaps in behaviour that was analogous to that previously shown on line bisection tasks. Contrary to being seen as an unintuitive finding, further analysis of these errors suggests that patients are giving disproportionate weight to the location of the ipsilesional object when plotting a midline course between two objects. Our results provide a platform for further studies to investigate the modulation and rehabilitation of this important skill

Electrodeposited NiCu bimetal on carbon paper as stable non-noble anode for efficient electrooxidation of ammonia

Electrochemical remediation of ammonia-containing wastewater at low cell voltage is an energy-effective technology which can simultaneously recover energy via hydrogen evolution reaction. One of the main challenges is to identify a robust, highly active and inexpensive anode for ammonia electrooxidation. Here we present an alternative anode, prepared by electrochemical co-deposition of Ni and Cu onto carbon paper. This NiCu bimetallic catalyst is characterised by scanning electron microscope, scanning transmission electron microscope, X-ray diffraction, x-ray photoelelectron spectroscopy, cyclic voltammetry, linear sweep voltammetry and chronoamperometry techniques. The stability and activity of NiCu bimetallic catalyst are largely improved in comparison with Ni or Cu catalyst. Moreover this noble-metal-free NiCu catalyst even performs better than Pt/C catalyst, as NiCu is not poisoned by ammonia. An ammonia electrolysis cell is fabricated with NiCu/carbon paper as anode for ammonia electrolysis. The influences of pH value, applied cell voltages and initial ammonia concentration on cell current density, ammonia removal and energy efficiency are tested. An ammonia removal efficiency of ∼80% and coulombic efficiency up to ∼92% have been achieved. Ni-Cu bimetal on carbon paper is a stable non-noble anode for efficient electrooxidation of ammonia

Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function

microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ∼40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5' start but common at the 3' end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5' isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5' isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5' most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.This work was supported by by the Victor Chang Cardiac Research Institute and grants 573726, 573731 and 514904 from the National Health & Medical Research Council awarded to TP

Impact of periconceptional and preimplantation undernutrition on factors regulating myogenesis and protein synthesis in muscle of singleton and twin fetal sheep.

In this study, we determined the effect of maternal undernutrition in the periconceptional (PCUN: ~80 days before to 6 days after conception) and preimplantation (PIUN: 0-6 days after conception) periods on the mRNA and protein abundance of key factors regulating myogenesis and protein synthesis, and on the relationship between the abundance of these factors and specific microRNA expression in the quadriceps muscle of singleton and twin fetal sheep at 135-138 days of gestation. PCUN and PIUN resulted in a decrease in the protein abundance of MYF5, a factor which determines the myogenic lineage, in singletons and twins. Interestingly, there was a concomitant increase in insulin-like growth factor-1 mRNA expression, a decrease in the protein abundance of the myogenic inhibitor, myostatin (MSTN), and an increase in the mRNA and protein abundance of the MSTN inhibitor, follistatin (FST), in the PCUN and PIUN groups in both singletons and twins. These promyogenic changes may compensate for the decrease in MYF5 protein abundance evoked by early embryonic undernutrition. PCUN and PIUN also increased the protein abundance of phosphorylated eukaryotic translation initiation factor binding protein 1 (EIF4EBP1; T70 and S65) in fetal muscle in singletons and twins. There was a significant inverse relationship between the expression of miR-30a-5p, miR-30d-5p, miR-27b-3p, miR106b-5p, and miR-376b and the protein abundance of mechanistic target of rapamycin (MTOR), FST, or MYF5 in singletons or twins. In particular, the expression of miR-30a-5p was increased and MYF5 protein abundance was decreased, in PCUN and PIUN twins supporting the conclusion that the impact of PCUN and PIUN is predominantly on the embryo

Estimation of Lower-Body Kinetics from Loading Profile and Kinematics Alone, Without Measured Ground Reaction Forces

Biomechanical models of human motion can estimate kinetic outcomes, such as joint moments, joint forces and muscle forces. Typically, one performs an inverse dynamics (ID) analysis to compute joint moments from joint angles and measured external forces. Sometimes it is impractical to measure ground reaction forces and moments (GRF&M). We devised an empirical method for performing ID analysis of resistance exercises without measured GRF&M. The method solves the multibody dynamics equations of motion with four key assumptions about the GRF&M that reduce the number of unknowns. The assumptions are 1) negligible ground reaction moments, 2) fixed lateral/medial location of the center of pressure (COP), 3) equal fore/aft location of the COP between the feet, and 4) constant angle of the GRF vector relative to the vertical axis in the frontal plane. We used evaluation trials from a spaceflight countermeasure resistance training device to test this approach. Four participants performed squat and deadlift exercises at various loads. We compared results from traditional ID analysis to results without measured GRF&M using our method. We found that joint moment trajectories in the sagittal plane were qualitatively similar in shape between the two methods, and the amount of root mean squared error (RMSE), measured by difference in joint moment impulse, was typically under 10 percent. Non-sagittal joint moment trajectories, which are much lower in overall magnitude, were not qualitatively similar in shape between the two methods. Non-sagittal moments displayed much higher RMSE, with typical values well over 50 percent. These findings were further supported by validation metrics (Sprague and Geers' P and M metrics, Pearson's r correlation coefficient). Based on these findings, we concluded that useful kinetic results are obtained from ID analysis of squat and deadlift exercises, even when GRF&M are not measured, as long as the outcomes of interest lie in the sagittal plane

Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell–mediated liver damage

Hepatitis B virus (HBV) causes chronic infection in more than 350 million people worldwide. It replicates in hepatocytes but is non-cytopathic; liver damage is thought to be immune mediated. Here, we investigated the role of innate immune responses in mediating liver damage in patients with chronic HBV infection. Longitudinal analysis revealed a temporal correlation between flares of liver inflammation and fluctuations in interleukin (IL)-8, interferon (IFN)-α, and natural killer (NK) cell expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) directly ex vivo. A cross-sectional study confirmed these findings in patients with HBV-related liver inflammation compared with healthy carriers. Activated, TRAIL-expressing NK cells were further enriched in the liver of patients with chronic HBV infection, while their hepatocytes expressed increased levels of a TRAIL death–inducing receptor. IFN-α concentrations found in patients were capable of activating NK cells to induce TRAIL-mediated hepatocyte apoptosis in vitro. The pathogenic potential of this pathway could be further enhanced by the ability of the IFN-α/IL-8 combination to dysregulate the balance of death-inducing and regulatory TRAIL receptors expressed on hepatocytes. We conclude that NK cells may contribute to liver inflammation by TRAIL-mediated death of hepatocytes and demonstrate that this non-antigen–specific mechanism can be switched on by cytokines produced during active HBV infection

Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent Apoptosis, but are insensitive to direct activation with exogenous fas ligand

Introduction Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma. Aims To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines. Results Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both. Conclusions 1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes