Ribonucleic acids of human milk

The milk feeding is the most essential process laying the foundation of human health at the postnatal development. However little is known about nucleic acids secreted into mother's milk during lactation. In order to investigate the composition and abundance of human milk NA we adapted the conventional isolation method to achieve high yield of total nucleic acids from milk samples. Concentration of total NA in milk samples of different donors varies from 20 to 68 mkg/ml at early stages of lactation. The average concentration tends to fall down to the end of lactation. The chain length of the major forms of NA varies from mononucleotides up to approximately 100 bases. Compositions of milk oligonucleotides are similar in samples of different donors. Major milk oligonucleotides are formed of RNA. Human milk contains the set of long-chain oligonucleotides with a developed secondary structure. Sequences of some oligo-RNAs correspond to the 3'-part of 5.8 S human ribosomal RNA and to the 3'-parts of tRNAVal and tRNATyr Primary structures of some others oligo-RNAs were related to fragments of human 18S and 28S rRNAs.     

Nucleotide sequence of 4.5S RNA (C8 or hY5) from HeLa cells

The nucleotide sequence of C8 RNA, one of the 4.5S RNAs of HeLa cells, was determined. C8 RNA consists of 83 or 84 nucleotide residues containing pppA at its 5′-terminus and oligo U at its 3′-terminus. This RNA is rich in uridylate residues (about 38%) and does not have any modified nucleosides. C8 RNA is present mainly in the cytoplasm, with some in the nucleus and the C8 RNAs from the nucleus and cytoplasm have the same sequence.     

Linear polyethylenimine as a tool for comparative studies of antisense and short double-stranded RNA oligonucleotides

Despite the recently enlarged field of available RNA knock-down technologies, e.g., antisense oligonucleotides (ASOs) and duplexes of synthetic 21 nucleotides RNAs (siRNAs), no versatile transfection reagent has been reported to deliver different nucleic acids formats at high rates of efficiency. We have evaluated the versatility and efficacy of linear PEI in transfecting and properly delivering a broad panel of nucleic acids such as short oligonucleotides and double-stranded RNA into cells in culture.     

Linear Polyethylenimine as a Tool for Comparative Studies of Antisense and Short Double-Stranded RNA Oligonucleotides

Abstract

Despite the recently enlarged field of available RNA knock-down technologies, e.g., antisense oligonucleotides (ASOs) and duplexes of synthetic 21 nucleotides RNAs (siRNAs), no versatile transfection reagent has been reported to deliver different nucleic acids formats at high rates of efficiency. We have evaluated the versatility and efficacy of linear PEI in transfecting and properly delivering a broad panel of nucleic acids such as short oligonucleotides and double-stranded RNA into cells in culture.     

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control.     

Novel Method for the Synthesis of 2′ -Phosphorylated Oligonucleotides

We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2′-O-methylribonucleotides) that contain a 2′-phosphorylated ribonucleoside residue, and optimized it to avoid 2′ -3′ -isomerization and chain cleavage. Structures of the 2′ -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2′-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.     

Fractionation with ethanol of nucleic acids from viroid-infected plants

A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 5S, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids.     

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control.     

Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation.

Specific human milk oligosaccharides, especially fucosylated neutral oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral oligosaccharides during lactation, a total of 84 milk samples were obtained from 12 women at 7 time periods during weeks 1-49 postpartum. The neutral oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced milk fucosyloligosaccharides that were essentially devoid of alpha1,2 linkages (but contained alpha1,3- and alpha1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In milk samples from the remaining 11 donors, fucosyloligosaccharides containing alpha1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosyloligosaccharides devoid of alpha1,2-linked fucose. The ratio of alpha1,2-linked oligosaccharide concentrations to oligosaccharides devoid of alpha1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of milk oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human milk could also vary among individuals and during lactation.     

Analysis of ribosomal inter‐subunit sites as targets for complementary oligonucleotides

The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2′‐O‐methyl oligoribonucleotides (2′‐OMe RNAs). Further, the inhibition efficiencies of the designed ribosome‐interfering 2′‐OMe RNAs were tested using a β‐galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC₅₀ values below 1 μM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2′‐OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double‐filter retention assay. Further, we applied heat‐shock chemical transformation to introduce 2′‐OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC₅₀ in the cell‐free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter‐subunit bridges can help design efficient antisense oligomers to probe the ribosome function.     

Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts.

An abrupt concommitant increase in total cellular RNA and protein was observed as cultured human diploid fibroblasts entered the senescent phase of their in vitro lifespan. DNA content remained stable from early to final passages. Fractionation of cellular RNAs by polyacrylamide gel electrophoresis demonstrated an increase in both 28S and 18S ribosomal and 4S transfer RNAs in these senescent cells. Separation of poly(A) RNA (mRNA) by oligo(dT)-cellulose chromatography suggests an increase in this group of RNAs. However, the ratios of 28S to 18S rRNAs, tRNA to rRNA, and mRNA to total cellular RNA were not significantly different in cells before and after senescence, indicating that the overall increases in total cellular RNA was not due to an accumulation of a single RNA class.     

绿豆子叶衰老期间核酸代谢的变化

萌发绿豆的子叶自然衰老期间,核酸含量降低,RNA降低的幅度比DNA大。电泳分析结果表明,子叶衰老期间细胞核主带DNA明显降低;而迁移慢的卫星带DNA变化不大。在RNA各组分中,18S rRNA从衰老前期就开始降低;25S rRNA和4～5S小分子RNA到衰老后期才缓慢下降。DNase和RNase活性在子叶整个衰老期间都明显升高,是导致核酸含量下降的主要原因。~3H-核苷掺入试验表明,核酸的合成速率在子叶衰老前期有所上升,到衰老后期又降低。poly(A)~ -mRNA含量在子叶开始衰老时明显上升。     

Characterization of extracellular RNAs produced by the marine photosynthetic bacterium Rhodovulum sulfidophilum

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids that are involved in its flocculation. These were found to be produced concomitantly with cell growth. The RNA fraction of these extracellular nucleic acids was subjected to cDNA analysis by applying a micro RNA cloning method and found to contain mainly fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Analyses of modified bases and genes of the RNAs revealed no structural difference between the intracellular and extracellular RNAs. This is the first report of structural analyses of bacterial extracellular RNAs.     

Effects of calcium salts of fatty acids and calcium salt of methionine hydroxy analogue on plasma prostaglandin F2alpha metabolite and milk fatty acid profiles in late lactation Holstein-Friesian cows

Effects of a dietary lipid supplement containing calcium salts of fatty acids and methionine hydroxy analogue on plasma prostaglandin F2alpha (PGF2alpha) metabolite (PGFM) and milk fatty acid profiles were examined in 40 late lactation, nonpregnant, Holstein-Friesian cows for a period of 70 days. Effects on milk production, milk composition, and blood metabolites were also examined. Cows were paired on the basis of lactation number (first lactation, n = 8; second lactation, n = 32) and randomly assigned from within pairs to one of two dietary treatments: unsupplemented control (C) or 400 g per cow per day of the lipid supplement (S). Cows receiving the supplement had higher (P < 0.05) total milk production, total fat production (kg), and total lactose production (kg). Plasma cholesterol was significantly higher (P < 0.01) after 30 days of treatment in cows receiving the supplement. Cows receiving the supplement had lower (P < 0.01) concentrations of short chain milk fatty acids (C4:0 to C14:1) and higher concentrations of long chain fatty acids (C18:1 and C18:2; P < 0.01) than control animals. Oxytocin-induced prostaglandin release on Day 16 postovulation was increased (P < 0.01) in cows receiving the supplement. In conclusion, supplementation with calcium salts of fatty acids and methionine hydroxy analogue significantly increased milk yield and plasma PGFM.     

TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site

Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G‐rich sequence, and with noncoding RNA, Telomeric repeat‐containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2′‐OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G‐quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate – the 3′‐end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3′‐end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Evidence of Aberrant Immune Response by Endogenous Double‐Stranded RNAs: Attack from Within

Many innate immune response proteins recognize foreign nucleic acids from invading pathogens to initiate antiviral signaling. These proteins mostly rely on structural characteristics of the nucleic acids rather than their specific sequences to distinguish self and nonself. One feature utilized by RNA sensors is the extended stretch of double‐stranded RNA (dsRNA) base pairs. However, the criteria for recognizing nonself dsRNAs are rather lenient, and hairpin structure of self‐RNAs can also trigger an immune response. Consequently, aberrant activation of RNA sensors has been reported in numerous human diseases. Yet, in most cases, the activating antigens remain unknown. Recent studies have developed sequencing techniques tailored to specifically capture dsRNAs and identified that various noncoding elements in the nuclear and the mitochondrial genome can generate dsRNAs. Here, the identity of endogenous dsRNAs, their recognition by dsRNA sensors, and their implications in the pathogenesis of human diseases ranging from inflammatory to degenerative are presented.     

Molecular detection and characterisation of black raspberry necrosis virus and raspberry bushy dwarf virus isolates in wild raspberries

Virus‐derived small interfering RNAs (siRNAs) were extracted from leaves of wild raspberries (Rubus idaeus) sampled from three different regions in Finland and subjected to deep sequencing. Assembly of the siRNA reads to contigs and their comparison to sequences in databases revealed the presence of the bipartite positive‐sense single‐stranded RNA viruses, raspberry bushy dwarf virus (RBDV, genus Idaeovirus), and black raspberry necrosis virus (BRNV, family Secoviridae) in 19 and 26 samples, respectively, including 15 plants coinfected with both viruses. Coverage with siRNA reads [21 and 22 nucleotides (nt)] was higher in BRNV‐FI (Finland) RNA1 (79%) than RNA2 (45%). In RBDV, the coverage of siRNA reads was 89% and 90% for RNA1 and RNA2, respectively. Average depth of coverage was 1.6–4.9 for BRNV and 16.5–36.5 for RBDV. PCR primers designed for RBDV and BRNV based on the contigs were used for screening wild raspberry and a few cultivated raspberry samples from different regions. Furthermore, the sequences of BRNV RNA1 and RNA2 were determined by amplification and sequencing of overlapping contigs (length 1000–1200 nt) except for the 3′ and 5′ ends of RNA1 and RNA2 covered by primers. RNA1 of the Finnish BRNV isolate (BRNV‐FI) was 80% and 86% identical to BRNV‐NA (USA) and BRNV‐Alyth (UK), respectively, whereas the identity of NA and Alyth was 79%. RNA2 of BRNV‐FI was 84% and 80% identical to BRNV‐NA and BRNV‐Alyth, respectively, whereas NA and Alyth were 82% identical. Hence, the strains detected in Finland differ from those reported in the UK and USA. Our results reveal the presence of BRNV in Finland for the first time. The virus is common in wild raspberries and nearly identical isolates are found in cultivated raspberries as well. The results show that wild raspberries in Finland are commonly infected with RBDV or BRNV or both viruses and thus are likely to serve as reservoirs of RBDV and BRNV for cultivated Rubus spp.     

Rapid communication capturing the destabilizing effect of dihydrouridine through molecular simulations

The structural effects of the commonly occurring modified nucleoside dihydrouridine (D) observed experimentally in model oligonucleotides include a strong destabilization of the C3′‐endo sugar conformation of D, the disruption of stacking interactions of neighboring residues with D and a possible destabilization of the C3′‐endo sugar pucker of the 5′‐neighboring nucleoside. Our simulations with a combination of a set of parameters for modified RNA residues with the recently developed AMBER FF99χ force field having reoptimized glycosidic torsion angle parameters for standard nucleosides was found to reproduce the destabilizing effect of dihydrouridine better than with the AMBER FF99 force field for nucleic acids for which the parameters for the modified residues were originally developed. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 985–991, 2014.