Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

Amiodarone disrupts cholesterol biosynthesis pathway and causes accumulation of circulating desmosterol by inhibiting 24-dehydrocholesterol reductase

Background We have earlier reported that amiodarone, a potent and commonly used antiarrhythmic drug increases serum desmosterol, the last precursor of cholesterol, in 20 cardiac patients by an unknown mechanism. Objective Here, we extended our study to a large number of cardiac patients of heterogeneous diagnoses, evaluated the effects of combining amiodarone and statins (inhibitors of cholesterol synthesis at the rate-limiting step of hydroxy-methyl-glutaryl CoA reductase) on desmosterol levels and investigated the mechanism(s) by which amiodarone interferes with the metabolism of desmosterol using in vitro studies. Methods and Results We report in a clinical case-control setting of 236 cardiac patients (126 with and 110 without amiodarone treatment) that amiodarone medication is accompanied by a robust increase in serum desmosterol levels independently of gender, age, body mass index, cardiac and other diseases, and the use of statins. Lipid analyses in patient samples taken before and after initiation of amiodarone therapy showed a systematic increase of desmosterol upon drug administration, strongly arguing for a direct causal link between amiodarone and desmosterol accumulation. Mechanistically, we found that amiodarone resulted in desmosterol accumulation in cultured human cells and that the compound directly inhibited the 24-dehydrocholesterol reductase (DHCR24) enzyme activity. Conclusion These novel findings demonstrate that amiodarone blocks the cholesterol synthesis pathway by inhibiting DHCR24, causing a robust accumulation of cellular desmosterol in cells and in the sera of amiodarone-treated patients. It is conceivable that the antiarrhythmic potential and side effects of amiodarone may in part result from inhibition of the cholesterol synthesis pathway.Peer reviewe

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

Psalms for Organ, Volume 11: Ulster

Pages 23-27 in Psalms for Organ, Volume 11

Advancing the use of passive sampling in risk assessment and management of contaminated sediments: Results of an international passive sampling inter-laboratory comparison

This work presents the results of an international interlaboratory comparison on ex situ passive sampling in sediments. The main objectives were to map the state of the science in passively sampling sediments, identify sources of variability, provide recommendations and practical guidance for standardized passive sampling, and advance the use of passive sampling in regulatory decision making by increasing confidence in the use of the technique. The study was performed by a consortium of 11 laboratories and included experiments with 14 passive sampling formats on 3 sediments for 25 target chemicals (PAHs and PCBs). The resulting overall interlaboratory variability was large (a factor of ∼10), but standardization of methods halved this variability. The remaining variability was primarily due to factors not related to passive sampling itself, i.e., sediment heterogeneity and analytical chemistry. Excluding the latter source of variability, by performing all analyses in one laboratory, showed that passive sampling results can have a high precision and a very low intermethod variability

Healthy people in healthy premises: the Finnish Indoor Air and Health Programme 2018-2028

Clean and fresh indoor air supports health and well-being. However, indoor air can contain pollutants that can cause a variety of symptoms and reduce well-being. Individual exposure agents can also increase the risk of certain diseases. Finns have taken major steps to improve the quality of indoor air for several decades. The primary focus of these activities has been the prevention and reduction of exposure to poor indoor air quality through guidance and regulation directing remediation of damaged buildings. Nevertheless, reported symptoms related to poor indoor air quality are common in Finland. In addition to exposure to indoor air pollutants, this may be partly due to the lively public discussion on the health risks caused by poor indoor air quality, conflicting views between experts, and mistrust towards public authorities, building owners and builders. Because of the scale of the indoor air problems in Finland, people's needs for reliable information and support, and the major costs involved, there is a call for new evidence-based methods, perspectives and solutions. Therefore, the Finnish Institute for Health and Welfare initiated the Finnish Indoor Air and Health Programme 2018-2028 together with a number of collaborators and stakeholders. The primary, long-term objective of the programme is to reduce hazards to health and well-being linked to indoor environments in Finland. To fulfill this objective, the programme will focus on the promotion of human health and well-being, the prevention of hazards, improved communication and engage the whole health-care sector to manage better patients ' symptoms and complaints. The 10-year Finnish Indoor Air and Health Programme consists of four areas that aim (1) to increase understanding of the effects of indoor environments on health and well-being; (2) to develop the management of problems linked to indoor environments; (3) to improve the treatment and working and functional capacity of people with symptoms and illnesses; and (4) to strengthen the competence in matters related to indoor environments. The progress of the programme and reaching the predefined, quantitative goals will be monitored throughout the programme

Test-retest repeatability of child's respiratory symptoms and perceived indoor air quality - comparing self-and parent-administered questionnaires

Background: Questionnaires can be used to assess perceived indoor air quality and symptoms in schools. Questionnaires for primary school aged children have traditionally been parent-administered, but self-administered questionnaires would be easier to administer and may yield as good, if not better, information. Our aim was to compare the repeatability of self- and parent-administered indoor air questionnaires designed for primary school aged pupils. Methods: Indoor air questionnaire with questions on child's symptoms and perceived indoor air quality in schools was sent to parents of pupils aged 7-12 years in two schools and again after two weeks. Slightly modified version of the questionnaire was administered to pupils aged 9-12 years in another two schools and repeated after a week. 351 (52%) parents and 319 pupils (86%) answered both the first and the second questionnaire. Test-retest repeatability was assessed with intra-class correlation (ICC) and Cohen's kappa coefficients (k). Results: Test-retest repeatability was generally between 0.4-0.7 (ICC; k) in both self-and parent-administered questionnaire. In majority of the questions on symptoms and perceived indoor air quality test-retest repeatability was at the same level or slightly better in self-administered compared to parent-administered questionnaire. Agreement of self-and parent administered questionnaires was generally <0.4 (ICC; k) in reported symptoms and 0.4-0.6 (ICC; k) in perceived indoor air quality. Conclusions: Children aged 9-12 years can give as, or even more, repeatable information about their respiratory symptoms and perceived indoor air quality than their parents. Therefore, it may be possible to use self-administered questionnaires in future studies also with children.Peer reviewe

Mechanism of the Very Efficient Quenching of Tryptophan Fluorescence in Human γD- and γS-Crystallins: The γ-Crystallin Fold May Have Evolved To Protect Tryptophan Residues from Ultraviolet Photodamage†

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated β-sheet Greek key domains of β- and γ-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human γD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) Biochemistry 45, 11552−11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human γS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n − 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates.National Eye Institute (Grant EY 015834

Cataract-Causing Defect of a Mutant γ-Crystallin Proceeds through an Aggregation Pathway Which Bypasses Recognition by the α-Crystallin Chaperone

Background: The transparency of the eye lens depends upon maintenance of the native state of the γ- and β-crystallins, which is aided by the abundant chaperones αA- and αB-crystallin. Mature onset cataract, the leading cause of blindness worldwide, involves the polymerization of covalently damaged or partially unfolded crystallins into light-scattering aggregates. A number of single amino acid substitutions and truncations of γ-crystallins result in congenital cataract in both humans and mice, though in many cases the coupling between the protein alterations and the accumulation of aggregates is poorly defined. Methodology/Principal Findings: We have studied the aggregation properties and chaperone interactions of human γD-crystallin carrying substitutions of two buried core mutants, I90F and V75D, which cause congenital cataract in mice. The in vitro aggregation pathway competing with productive refolding was not altered by either substitution. Furthermore, this aggregation pathway for both mutant proteins–originating from a partially folded intermediate–was efficiently suppressed by αB-crystallin. Thus the cataract pathology was unlikely to be associated with a direct folding defect. The native state of wild-type human γD-crystallin exhibited no tendency to aggregate under physiological conditions. However both I90F and V75D native-like proteins exhibited slow (days) aggregation to high molecular weight aggregates under physiological conditions. The perturbed conformation of I90F was recognized and bound by both αA and αB chaperones. In contrast, the aggregation derived from the perturbed state of V75D was not suppressed by either chaperone, and the aggregating species were not bound by the chaperone. Conclusions/Significance: The cataract phenotype of I90F in mice may be due to premature saturation of the finite α- crystallin pool. The V75D aggregation pathway and its escape from chaperone surveillance and aggregation suppression can account for the congenital cataract pathology of this mutant. Failure of chaperone recognition may be an important source of pathology for many other protein folding defects.National Eye Institute (Grant no. EY015834 )National Institutes of Health (U.S.) (Grant no. GM17980