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Constructing an ultrasensitive CRISPR/Cas-based biosensing strategy is highly significant for the detection of trace targets. Here we presented a dual-amplified biosensing method based on CRISPR/Cas13a-triggered Cas12a, namely, Cas13a-12a amplification. As proof-of-principle, the developed strategy was used for miRNA-155 detection. The target bound to the Cas13a-crRNA complex and activated the cleavage activity of Cas13a for cleaving uracil ribonucleotides (rU) in the bulge structure of blocker strand (BS), resulting in the release of primer strand (PS) from the BS modified on magnetic beads. Then, the released PS activated the cleavage activity of Cas12a to cleave single-strand DNA reporter probes, producing a significantly increased fluorescent signal. The detection limit of the Cas13a-12a amplification using synthetic miRNA-155 was as low as 0.35 fM, which was much lower than that of the only Cas13a-based assay. The applied performance of this amplification strategy was verified by accurately quantifying miRNA-155 expression levels in different cancer patients. Therefore, the developed strategy offers a supersensitive and highly specific miRNAs sensing platform for clinical application. [Display omitted] • A method based on CRISPR/Cas13a-triggered Cas12a for miRNA-155 detection was developed for the first time. • The detection limit of the method was much lower than that of the reported Cas13a-based strategies. • This amplification strategy was able to can accurately quantifying miRNA-155 in clinical samples. [ABSTRACT FROM AUTHOR]

The ribonucleotide reductase inhibitor and radiosensitizer triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), NSC 663249) is clinically being evaluated via the intravenous (IV) route for the treatment of cervical and vulvar cancer in combination with primary cisplatin chemoradiation. The need for a 2-h infusion and frequent administration of triapine is logistically challenging, prompting us to pursue oral (PO) administration. In support of the clinical trial investigating oral triapine in combination with chemoradiation, we developed and validated a novel LC–MS/MS assay for the quantification of triapine in 50 μL human plasma. After protein precipitation, chromatographic separation of the supernatant was achieved with a Shodex ODP2 column and an isocratic acetonitrile-water mobile phase with 10% ammonium acetate. Detection with an ABI 4000 mass spectrometer utilized electrospray positive mode ionization. The assay was linear from 3 to 3,000 ng/mL and proved to be accurate (97.1–103.1%) and precise (<7.4% CV), and met the U.S. FDA guidance for bioanalytical method validation. This LC–MS/MS assay will be an essential tool to further define the pharmacokinetics and oral bioavailability of triapine. [ABSTRACT FROM AUTHOR]

Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange. [ABSTRACT FROM AUTHOR]

New Findings What is the central question of this study? We investigated whether 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) could prevent acute increases in body fat and changes in omental and subcutaneous adipose tissue following the sudden transition from physical activity to physical inactivity., What is the main finding and its importance? AICAR prevented fat gains following the transition from physical activity to inactivity to levels comparable to rats that remained physically active. AICAR and continuous physical activity produced depot-specific changes in cyclin A1 mRNA and protein that were associated with the prevention of fat gain. These findings suggest that targeting AMP-activated protein kinase signalling could oppose rapid adipose mass growth., The transition from physical activity to inactivity is associated with drastic increases in 'catch-up' fat that in turn foster the development of many obesity-associated maladies. We tested whether 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) treatment would prevent gains in body fat following the sudden transition from a physically active state to an inactive state by locking a voluntary running wheel. Male Wistar rats were either sedentary (SED) or given wheel access for 4 weeks, at which time rats with wheels continued running (RUN), had their wheel locked (WL) or had WL with daily AICAR injection (WL + AICAR) for 1 week. RUN and WL + AICAR prevented gains in body fat compared with SED and WL ( P < 0.001). Cyclin A1 mRNA, a marker of cell proliferation, was decreased in omental, but not subcutaneous adipose tissue, in RUN and WL + AICAR compared with SED and WL groups ( P < 0.05). Both cyclin A1 mRNA and protein were positively associated with gains in fat mass ( P < 0.05). Cyclin A1 mRNA in omental, but not subcutaneous, adipose tissue was negatively correlated with p-AMPK levels ( P < 0.05). Differences in fat gain and omental mRNA and protein levels were independent of changes in food intake and in differences in select hypothalamic mRNAs. These findings suggest that AICAR treatment prevents acute gains in adipose tissue following physical inactivity to levels of rats that continuously run, and that together, continuous physical activity and AICAR could, at least initially in these conditions, exert similar inhibitory effects on adipogenesis in a depot-specific manner. [ABSTRACT FROM AUTHOR]

Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpolη is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpolκ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2η-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of humanDNApolymerases and indicates potential new functions of hpol η in vivo. [ABSTRACT FROM AUTHOR]

Replicative DNA polymerases misincorporate ribonucleoside triphosphates (rNTPs) into DNA approximately once every 2,000 base pairs synthesized. Ribonucleotide excision repair (RER) removes ribonucleoside monophosphates (rNMPs) from genomic DNA, replacing the error with the appropriate deoxyribonucleoside triphosphate (dNTP). Ribonucleotides represent a major threat to genome integrity with the potential to cause strand breaks. Furthermore, it has been shown in the bacterium Bacillus subtilis that loss of RER increases spontaneous mutagenesis. Despite the high rNTP error rate and the effect on genome integrity, the mechanism underlying mutagenesis in RER-deficient bacterial cells remains unknown. We performed mutation accumulation lines and genomewide mutational profiling of B. subtilis lacking RNase HII, the enzyme that incises at single rNMP residues initiating RER. We show that loss of RER in B. subtilis causes strand and sequence-context- dependent GC → AT transitions. Using purified proteins, we show that the replicative polymerase DnaE is mutagenic within the sequence context identified in RER-deficient cells. We also found that DnaE does not perform strand displacement synthesis. Given the use of nucleotide excision repair (NER) as a backup pathway for RER in RNase HII-deficient cells and the known mutagenic profile of DnaE, we propose that misincorporated ribonucleotides are removed by NER followed by error-prone resynthesis with DnaE. [ABSTRACT FROM AUTHOR]

High levels of transcription stimulate mutation rates in microorganisms, and this occurs primarily through an enhanced accumulation of DNA damage. The major source of transcription-associated damage in yeast is Topoisomerase I (Top1), an enzyme that removes torsional stress that accumulates when DNA strands are separated. Top1 relieves torsional stress by nicking and resealing one DNA strand, and some Top1-dependent mutations are due to trapping and processing of the covalent cleavage intermediate. Most, however, reflect enzyme incision at ribonucleotides, which are the most abundant noncanonical component of DNA. In either case, Top1 generates a distinctive mutation signature composed of short deletions in tandem repeats; in the specific case of ribonucleotide-initiated events, mutations reflect sequential cleavage by the enzyme. Top1-dependent mutations do not require highly activated transcription, but their levels are greatly increased by transcription, which partially reflects an interaction of Top1 with RNA polymerase. Recent studies have demonstrated that Top1-dependent mutations exhibit a strand bias, with the nature of the bias differing depending on the transcriptional status of the underlying DNA. Under low-transcription conditions, most Top1-dependent mutations arise in the context of replication and reflect incision at ribonucleotides incorporated during leading-strand synthesis. Under high-transcription conditions, most Top1-dependent events arise when the enzyme cleaves the non-transcribed strand of DNA. In addition to increasing genetic instability in growing cells, Top1 activity in transcriptionally active regions may be a source of mutations in quiescent cells. [ABSTRACT FROM AUTHOR]

5′-Phosphodiesterase (5′-PDE) catalyzes the hydrolysis of ribonucleic acid to obtain a mixture of ribonucleotides, such as 5′-guanosine monophosphate and 5′-adenosine monophosphate. In this study, a 5'-PDE was newly isolated and purified from Aspergillus fumigatus. Following purification, this enzyme exhibited a specific activity of 1036.76 U/mg protein, a molecular weight of 9.5 kDa, and an optimal temperature and pH for enzyme activity of 60°C and 5.0, respectively. However, its activity was partially inhibited by Fe3+, Cu2+, and Zn2+, but slightly improved by the presence of K+ and Na+. Additionally, chemical-modification experiments were also applied to investigate the structural information of 5'-PDE, in which the residues containing carboxyl and imidazole groups were essential for enzyme activity based on their localization in the 5′-PDE active site. Furthermore, purified 5′-PDE could specifically catalyze the synthesis of ribonucleotides with a Vmax 0.71 mmol/mg·min and a KM of 13.60 mg/mL. [ABSTRACT FROM AUTHOR]

TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role ofTBC1D1in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role forTBC1D1in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle. [ABSTRACT FROM AUTHOR]

Eukaryotic class I ribonucleotide reductases (RRs) generate deoxyribonucleotides for DNA synthesis. Binding of dNTP effectors is coupled to the formation of active dimers and induces conformational changes in a short loop (loop 2) to regulate RR specificity among its nucleoside diphosphate substrates. Moreover, ATPand dATP bind at an additional allosteric site 40 Å away from loop 2 and thereby drive formation of activated or inactive hexamers, respectively. To better understand how dNTP binding influences specificity, activity, and oligomerization of human RR, we aligned >300 eukaryotic RR sequences to examine natural sequence variation in loop 2. We found that most amino acids in eukaryotic loop 2 were nearly invariant in this sample; however, two positions co-varied as nonconservative substitutions (N291G and P294K; human numbering). We also found that the individual N291G and P294K substitutions in human RR additively affect substrate specificity. The P294K substitution significantly impaired effector-induced oligomerization required for enzyme activity, and oligomerization was rescued in the N291G/P294K enzyme. None of the other mutants exhibited altered ATP-mediated hexamerization; however, certain combinations of loop 2 mutations and dNTP effectors perturbed ATP's role as an allosteric activator. Our results demonstrate that the observed compensatory covariation of amino acids in eukaryotic loop 2 is essential for its role in dNTP-induced dimerization. In contrast, defects in substrate specificity are not rescued in the double mutant, implying that functional sequence variation elsewhere in the protein is necessary. These findings yield insight into loop 2's roles in regulating RR specificity, allostery, and oligomerization. [ABSTRACT FROM AUTHOR]

A new metal-organic quadrangular macrocycle Co(III) complex was achieved via self-assembly by comprising anthracene moieties as fluorophores, while the tridentate N 2 O units containing amide groups as guest binding sites and communicators, for specific responses to adenosine-5′-triphosphate disodium over the other disodium 5′-ribonucleotides and inorganic phosphate anions. The M 4 L 4 structure of macrocycle Co(III) complex was further confirmed by the crystal analysis of comparison. [ABSTRACT FROM AUTHOR]

Aims: The aim of this short study was to test the combinations of RNA extracts (both the connective tissue extracts-cartilage and synovia along with yeast extract) found in natural ribonucleotide extract Osteochondrin S (OST) on human osteoclast formation and activity in vitro. Methods: In vitro human osteoclasts were treated with the RNA extracts (cartilage, synovia and yeast) at concentrations equivalent to those in OST starting from day 7 of the culture. A tartrate resistant acid phosphatase stain (TRAP) was used to indicate osteoclast formation and activity assessed by determining area of dentine resorption. Results: The combination of all components as is found in OST suppressed both osteoclast formation and activity. The yeast extract suppressed osteoclast activity at similar levels to that observed with all components combined. Conclusions: Our findings indicate that yeast RNA extracts found in OST may be the key component responsible for suppression of osteoclast activity. [ABSTRACT FROM AUTHOR]

The rnhAB mutant Escherichia coli , deficient in two RNase H enzymes that remove both R-loops and incorporated ribonucleotides (rNs) from DNA, grow slowly, suggesting accumulation of rN-containing DNA lesions (R-lesions). We report that the rnhAB mutants have reduced viability, form filaments with abnormal nucleoids, induce SOS, and fragment their chromosome, revealing replication and/or segregation stress. R-loops are known to interfere with replication forks, and sensitivity of the double rnhAB mutants to translation inhibition points to R-loops as precursors for R-lesions. However, the strict specificity of bacterial RNase HII for RNA–DNA junctions indicates that R-lesions have rNs integrated into DNA. Indeed, instead of relieving problems of rnhAB mutants, transient inhibition of replication from oriC kills them, suggesting that oriC -initiated replication removes R-loops instead of compounding them to R-lesions. Yet, replication from an R-loop-initiating plasmid origin kills the double rnhAB mutant, revealing generation of R-lesions by R-loop-primed DNA synthesis. These R-lesions could be R-tracts, contiguous runs of ≥ 4 RNA nucleotides within DNA strand and the only common substrate between the two bacterial RNase H enzymes. However, a plasmid relaxation test failed to detect R-tracts in DNA of the rnhAB mutants, although it readily detected R-patches (runs of 1–3 rNs). Instead, we detected R-gaps, single-strand gaps containing rNs, in the chromosomal DNA of the rnhAB mutant. Therefore, we propose that RNase H-deficient mutants convert some R-loops into R-tracts, which progress into R-gaps and then to double-strand breaks—explaining why R-tracts do not accumulate in RNase H-deficient cells, while double-strand breaks do. [ABSTRACT FROM AUTHOR]

Genomic DNA is transiently contaminated with ribonucleotide residues during the process of DNA replication through misincorporation by the replicative DNA polymerases α, δ and ε, and by the normal replication process on the lagging strand, which uses RNA primers. These ribonucleotides are efficiently removed during replication by RNase H enzymes and the lagging strand synthesis machinery. However, when ribonucleotides remain in DNA they can distort the DNA helix, affect machineries for DNA replication, transcription and repair, and can stimulate genomic instabilities which are manifest as increased mutation, recombination and chromosome alterations. The genomic instabilities associated with embedded ribonucleotides are considered here, along with a discussion of the origin of the lesions that stimulate particular classes of instabilities. [ABSTRACT FROM AUTHOR]

A new virus was isolated from a bellflower ( Campanula takesimana) plant showing veinal mottle symptoms, and its complete genome sequence was determined. The viral genome consists of a positive-sense single-stranded RNA of 8,259 ribonucleotides. Electron microscopic observation revealed that the viral genome is packaged as a filamentous particle with an average length of approximately 760 nm. BLAST searches of protein databases showed that the encoded polyprotein has a maximum amino acid sequence identity of 34.1% (with 95% coverage) to that of the isolate AD of Chinese yam necrotic mosaic virus (CYNMV; genus Macluravirus). Phylogenetic analysis and comparison of the encoded amino acid sequences with those of other viruses demonstrated that the identified virus shows minimal sequence similarity to known viruses and should therefore be considered a member of a new genus in the family Potyviridae. The name bellflower veinal mottle virus (BVMoV) is proposed for this new virus. [ABSTRACT FROM AUTHOR]

Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on glutamine, inducing 'glutamine addiction'. Surprisingly, colon cancer cells that express high levels of MYC due to WNT pathway mutations are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous MYC via the 3′- UTR of the MYC mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine-nucleotide levels. Glutamine deprivation causes a global reduction in promoter association of RNA polymerase II ( RNAPII) and slows transcriptional elongation. While activation of MYC restores binding of MYC and RNAPII function on most promoters, restoration of elongation is imperfect and activation of MYC in the absence of glutamine causes stalling of RNAPII on multiple genes, correlating with R-loop formation. Stalling of RNAPII and R-loop formation can cause DNA damage, arguing that the MYC 3′- UTR is critical for maintaining genome stability when ribonucleotide levels are low. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are cellular, short, non-coding ribonucleotides acting as endogenous posttranscriptional repressors following incorporation in the RNA-induced silencing complex. Despite being chemically and mechanistically very similar, miRNAs exert a multitude of different cellular effects by acting on mRNA species, whose gene-products partake in a wide array of processes. Here, the aim was to review the knowledge of miRNA expression and action in the islet of Langerhans. We have focused on: 1) physiological consequences of islet or beta cell specific inhibition of miRNA processing, 2) mechanisms regulating processing of miRNAs in islet cells, 3) presence and function of miRNAs in alpha versus beta cells – the two main cell types of islets, and 4) miRNA mediators of beta cell decompensation. It is clear that miRNAs regulate pancreatic islet development, maturation, and function in vivo . Moreover, processing of miRNAs appears to be altered by obesity, diabetes, and aging. A number of miRNAs (such as miR-7, miR-21, miR-29, miR-34a, miR-212/miR-132, miR-184, miR-200 and miR-375) are involved in mediating beta cell dysfunction and/or compensation induced by hyperglycemia, oxidative stress, cytotoxic cytokines, and in rodent models of fetal metabolic programming prediabetes and overt diabetes. Studies of human type 2 diabetic islets underline that these miRNA families could have important roles also in human type 2 diabetes. Furthermore, there is a genuine gap of knowledge regarding miRNA expression and function in pancreatic alpha cells. Progress in this area would be enhanced by improved in vitro alpha cell models and better tools for islet cell sorting. [ABSTRACT FROM AUTHOR]

BACKGROUND 5′-ribonucleotides including uridine 5′-monophosphate (UMP), adenosine 5′-monophosphate (AMP), cytidine 5′-monophosphate (CMP), and guanosine 5′-monophosphate (GMP) have been widely used as food additives. Ion exchange is a potential technique to separate 5′-ribonucleotides. A knowledge of uptake equilibrium on ion exchangers is important for the design and scale-up of ion exchange processes. The uptake equilibria of 5′-ribonucleotides and Na+ on the strong acid cation exchange resin NH-1 were studied experimentally and modelled in binary and multicomponent systems. The uptake mechanism and thermodynamics were also studied. RESULTS A modified Helfferich model was developed combining ion exchange of cations with adsorption of zwitter ions. The model can predict the uptake equilibria of 5′-ribonucleotides and Na+ successfully in multicomponent systems. The hydrophobicity of bases of 5′-ribonucleotides determined their relative affinities to the resin. The resin shows the strongest affinity for AMP among 5′-ribonucleotides. The uptakes of 5′-ribonucleotides cations is an endothermic and entropy-driven process. CONCLUSION The equilibrium model proposed is important for simulation and design of the dynamic separation process of 5′-ribonucleotides. UMP, GMP, and CMP can be separated based on their difference in isoelectric point. AMP and CMP with similar isoelectric point can be separated based on their difference in affinity to the resin. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

In eukaryotes, numerous genetic factors contribute to the lifespan including metabolic enzymes, signal transducers, and transcription factors. As previously reported, the forkhead-like transcription factor ( FHL1 ) gene was required for yeast replicative lifespan and cell proliferation. To determine how Fhl1p regulates the lifespan, we performed a DNA microarray analysis of a heterozygous diploid strain deleted for FHL1 . We discovered numerous Fhl1p-target genes, which were then screened for lifespan-regulating activity. We identified the ribonucleotide reductase (RNR) 1 gene ( RNR1 ) as a regulator of replicative lifespan. RNR1 encodes a large subunit of the RNR complex, which consists of two large (Rnr1p/Rnr3p) and two small (Rnr2p/Rnr4p) subunits. Heterozygous deletion of FHL1 reduced transcription of RNR1 and RNR3 , but not RNR2 and RNR4 . Chromatin immunoprecipitation showed that Fhl1p binds to the promoter regions of RNR1 and RNR3 . Cells harboring an RNR1 deletion or an rnr1 - C428A mutation, which abolishes RNR catalytic activity, exhibited a short lifespan. In contrast, cells with a deletion of the other RNR genes had a normal lifespan. Overexpression of RNR1 , but not RNR3 , restored the lifespan of the heterozygous FHL1 mutant to the wild-type (WT) level. The Δ fhl1 / FHL1 mutant conferred a decrease in dNTP levels and an increase in hydroxyurea (HU) sensitivity. These findings reveal that Fhl1p regulates RNR1 gene transcription to maintain dNTP levels, thus modulating longevity by protection against replication stress. [ABSTRACT FROM AUTHOR]

Heritable gene expression arises readily in a simple non-genetic system employing known small-RNA biochemistry. Pooled cross-templating ribonucleotides show varied chemical competence on which selection acts, even calculating only minimal effects. Evolution can be quick-computed progress toward encoded gene expression can require only days or weeks for two millimolar, partly activated complementary 5′ ribonucleotides. After only one product selection cycle, early templating can become prevailing pool behavior. Subsequently, a selected templated product is efficiently amplified as a pool ages, frequently accumulated in the same order of concentration as incoming nucleotides. Pools spontaneously favor templating because sporadic nucleotide accumulations increase it-and selection increases templating in pools of all ages. Nonetheless, templated chemical competence appears most easily in young pools. Pool history is critical-pools can perish from periodic hazards (like tides), or alternatively, from hazards roughly constant in time (like rainfall). Selection is greatly enhanced in constant hazard pools-more effective if pools have varied ages. Stronger selection is disproportionately more effective. Selected evolutionary change has an uncomplicated molecular basis-progress from chemical product synthesis to templated, proto-genetic inheritance exploits identity between templating and entropic catalysis. Though discovered by computation, selection of an elevated product of template catalysis is plausible, independent of any chemical or mathematical assumption. Selected chemical variation before genetics (chance utility) therefore inaugurates inheritance, even when hindered by unstable, dilute nucleotides, erratically supplied in undependable quantities. Remarkably, such uncontrolled conditions are not necessarily hostile, but can instead accelerate appearance of primordial gene-like behavior. [ABSTRACT FROM AUTHOR]

Cancer and genomic instability are highly impacted by the deoxyribonucleic acid (DNA) replication program. Inaccuracies in DNA replication lead to the increased acquisition of mutations and structural variations. These inaccuracies mainly stem from loss of DNA fidelity due to replication stress or due to aberrations in the temporal organization of the replication process. Here we review the mechanisms and impact of these major sources of error to the replication program. [ABSTRACT FROM AUTHOR]

The heterodimeric T1R1 + T1R3 receptor is considered critical for normal signaling of L-glutamate and 5'-ribonucleotides in the oral cavity. However, some taste-guided responsiveness remains in mice lacking one subunit of the receptor, suggesting that other receptors are sufficient to support some behaviors. Here, mice lacking both receptor subunits (KO) and wild-type (WT, both n = 13) mice were tested in a battery of behavioral tests. Mice were trained and tested in gustometers with a concentration series of Maltrin-580, a maltodextrin, in a brief-access test (10-s trials) as a positive control. Similar tests followed with monosodium glutamate (MSG) with and without the ribonucleotide inosine 5'-monophosphate (IMP), but always in the presence of the epithelial sodium channel blocker amiloride (A). Brief-access tests were repeated following short-term (30-min) and long-term (48-h) exposures to MSG + A + IMP and were also conducted with sodium gluconate replacing MSG. Finally, progressive ratio tests were conducted with Maltrin-580 or MSG + A + IMP, to assess appetitive behavior while minimizing satiation. Overall, MSG generated little concentration-dependent responding in either food-restricted WT or KO mice, even in combination with IMP. However, KO mice licked less to the amino acid stimuli, a measure of consummatory behavior in the brief-access tests. In contrast, both groups initiated a similar number of trials and had a similar breakpoint in the progressive ratio task, both measures of appetitive (approach) behavior. Collectively, these results suggest that while the T1R1 + T1R3 receptor is necessary for consummatory responding to MSG (+IMP), other receptors are sufficient to maintain appetitive responding to this "umami" stimulus complex in food-restricted mice. [ABSTRACT FROM AUTHOR]

Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as Thermococcus rely only upon RNaseH2 to initiate the pathway. Because Thermococcus DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ensure genome integrity. Here, we show that ribonucleotides are incorporated by the hyperthermophilic archaeon Thermococcus kodakarensis both in vitro and in vivo and a robust ribonucleotide excision repair pathway is critical to keeping incorporation levels low in wild-type cells. Using pre-steady-state and steady-state kinetics experiments, we also show that archaeal RNaseH2 rapidly cleaves at embedded ribonucleotides (200-450 s-1), but exhibits an ~1,000-fold slower turnover rate (0.06-0.17 s-1), suggesting a potential role for RNaseH2 in protecting or marking nicked sites for further processing. We found that following RNaseH2 cleavage, the combined activities of polymerase B (PolB), flap endonuclease (Fen1), and DNA ligase are required to complete ribonucleotide processing. PolB formed a ribonucleotide-containing flap by strand displacement synthesis that was cleaved by Fen1, and DNA ligase sealed the nick for complete repair. Our study reveals conservation of the overall mechanism of ribonucleotide excision repair across domains of life. The lack of redundancies in ribonucleotide repair in archaea perhaps suggests a more ancestral form of ribonucleotide excision repair compared with the eukaryotic pathway. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae RNase H2 resolves RNA-DNA hybrids formed during transcription and it incises DNA at single ribonucleotides incorporated during nuclear DNA replication. To distinguish between the roles of these two activities in maintenance of genome stability, here we investigate the phenotypes of a mutant of yeast RNase H2 ( rnh201-RED ; ribonucleotide excision defective) that retains activity on RNA-DNA hybrids but is unable to cleave single ribonucleotides that are stably incorporated into the genome. The rnh201-RED mutant was expressed in wild type yeast or in a strain that also encodes a mutant allele of DNA polymerase ε ( pol2-M644G ) that enhances ribonucleotide incorporation during DNA replication. Similar to a strain that completely lacks RNase H2 ( rnh201 Δ), the pol2-M644G rnh201-RED strain exhibits replication stress and checkpoint activation. Moreover, like its null mutant counterpart, the double mutant pol2-M644G rnh201-RED strain and the single mutant rnh201-RED strain delete 2–5 base pairs in repetitive sequences at a high rate that is topoisomerase 1-dependent. The results highlight an important role for RNase H2 in maintaining genome integrity by removing single ribonucleotides incorporated during DNA replication. [ABSTRACT FROM AUTHOR]

Non-coding RNAs such as microRNAs (miRNAs) are very tiny ribonucleotides having an essential role in gene regulation at both post-transcriptional and translational levels. They are very conserved and expressed in worms, flies, plants, and mammals in a sequence-specific manner. Furthermore, it is now possible to clone miRNAs using the new genome editing tool CRISPR/cas9, which shows benefit in control of untargeted effect. In this special issue, we tried to cover researches associated with functional roles of miRNAs accross model and complex organisms. [ABSTRACT FROM AUTHOR]

Cells carrying deletions of genes encoding H-class ribonucleases display elevated rates of chromosome instability. The role of these enzymes is to remove RNA-DNA associations including persistent mRNA-DNA hybrids (R-loops) formed during transcription, and ribonucleotides incorporated into DNA during replication. RNases H1 and H2 can degrade the RNA component of R-loops, but only RNase H2 can initiate accurate ribonucleotide excision repair (RER). In order to examine the specific contributions of these activities to chromosome stability, we measured rates of loss-of-heterozygosity (LOH) in diploid Saccharomyces cerevisiae yeast strains carrying the rnh201-RED separation-of-function allele, encoding a version of RNase H2 that is RE R- d efective, but partly retains its other activity. The LOH rate in rnh201-RED was intermediate between RNH201 and rnh201Δ . In strains carrying a mutant version of DNA polymerase ε ( pol2-M644G ) that incorporates more ribonucleotides than normal, the LOH rate in rnh201-RED was as high as the rate measured in rnh201Δ . Topoisomerase 1 cleavage at sites of ribonucleotide incorporation has been recently shown to produce DNA double strand breaks. Accordingly, in both the POL2 and pol2-M644G backgrounds, the LOH elevation in rnh201-RED was suppressed by top1Δ . In contrast, in strains that incorporate fewer ribonucleotides ( pol2-M644L ) the LOH rate in rnh201-RED was low and independent of topoisomerase 1. These results suggest that both R-loop removal and RER contribute substantially to chromosome stability, and that their relative contributions may be variable across different regions of the genome. In this scenario, a prominent contribution of R-loop removal may be expected at highly transcribed regions, whereas RER may play a greater role at hotspots of ribonucleotide incorporation. [ABSTRACT FROM AUTHOR]

Background: Vasohibin 2 (VASH2) has previously been identified as an agiogenenic factor and a cancer related protein. Here we investigated the association of VASH2 expression and chemoresistance in pancreatic cancer. Methods: Immunohistochemical staining for VASH2 was performed on 102 human pancreatic cancer samples. Pancreatic cancer cell line models exhibiting overexpression or knockdown of VASH2 were generated. Gene expression analyses were carried out to determine genes differentially regulated by VASH2. Putative transcription factors that are downstream mediators of gene expression regulated by VASH2 were queried bioinformatically. Dual-luciferase reporter assays and ChIP assays were performed to confirm transactivation of target genes following VASH2 overexpression or knockdown. Results: VASH2 protein expression was higher in human pancreatic cancer than in paired adjacent tissues and elevated VASH2 levels were associated with gemcitabine chemoresistance. In cell line models of pancreatic cancer, VASH2 expression induced gemcitabine chemoresistance in vitro and in vivo. It was discovered that expression of ribonucleotide reductase regulatory subunit M2 (RRM2) is regulated by VASH2; immunohistochemical analysis demonstrated a positive association of VASH2 expression and RRM2 expression in human pancreatic cancer tissues. Bioinformatics analyses revealed that induction of the Jun proto-oncogene (JUN) by VASH2 is responsible for upregulation of RRM2 expression; this JUN-dependent regulation of RRM2 by VASH2 was confirmed by chromatin immunoprecipitation and dual luciferase reporter assays, which demonstrated that JUN directly binds with the RRM2 promoter to activate transcription. Conclusions: These data suggest that VASH2 reduces the chemosensitivity to gemcitabine in pancreatic cancer cells via JUN-dependent transactivation of RRM2. [ABSTRACT FROM AUTHOR]

Despite the apparent clinical benefits of high-dose cytarabine (Ara-C) over lower dose Ara-C in acute myeloid leukemia (AML) therapy, the mechanism behind high-dose Ara-C therapy remains uncertain. In this study, a LC-MS-based method was carried out to investigate the metabolic alteration of ribonucleotide and deoxyribonucleotide in human promyelocytic leukemia cells (HL-60) after treatment with Ara-C to reveal its antitumor mechanism. The metabolic results revealed that four nucleotides (ATP, ADP, CDP, and dCTP) could be used as potential biomarkers indicating the benefit of high-dose Ara-C over lower dose Ara-C treatment. Combining metabolic perturbation and cell cycle analysis, we conjectured that, apart from the acknowledged mechanism of Ara-C on tumor inhibition, high-dose Ara-C could present a specific action pathway. It was suggested that the pronounced rise in AMP/ATP ratio induced by high-dose Ara-C can trigger AMP-activated protein kinase (AMPK) and subsequently Forkhead Box, class O (FoxO), to promote cell cycle arrest. Moreover, the significant decrease in CDP pool induced by high-dose Ara-C might further accelerate the reduction of dCTP, which then aggravates DNA synthesis disturbance. As a result, all of these alterations led to heightened tumor inhibition. This study provides new insight in the investigation of potential mechanisms in the clinical benefits of high-dose Ara-C in therapy for AML. [ABSTRACT FROM AUTHOR]