Ribonucleoside-5'-diphosphates (NDPs) support RNA polymerase transcription, suggesting NDPs may have been substrates for primordial nucleic acid biosynthesis.

A better understanding of the structural basis for the preferences of RNA and DNA polymerases for nucleoside-5'-triphosphates (NTPs) could help define the catalytic mechanisms for nucleotidyl transfer during RNA and DNA synthesis and the origin of primordial nucleic acid biosynthesis. We show here that ribonucleoside-5'-diphosphates (NDPs) can be utilized as substrates by RNA polymerase (RNAP). We found that NDP incorporation is template-specific and that noncognate NDPs are not incorporated. Compared with the natural RNAP substrates, NTPs, the Km of RNAP for NDPs was increased ∼4-fold, whereas the Vmax was decreased ∼200-fold. These properties could be accounted for by molecular modeling of NTP/RNAP co-crystal structures. This finding suggested that the terminal phosphate residue in NTP (not present in NDP) is important for positioning the nucleotide for nucleolytic attack in the nucleotidyl transfer reaction. Strikingly, a mutational substitution of the active-center ßR1106 side chain involved in NTP positioning also strongly inhibited NDP-directed synthesis, even though this residue does not contact NDP. Substitutions in the structurally analogous side chain in RB69 DNA polymerase (Arg-482) and HIV reverse transcriptase (Lys-65) were previously observed to inhibit dNDP incorporation. The unexpected involvement of these residues suggests that they affect a step in catalysis common for nucleic acid polymerases. The substrate activity of NDPs with RNAP along with those reported for DNA polymerases reinforces the hypothesis that NDPs may have been used for nucleic acid biosynthesis by primordial enzymes, whose evolution then led to the use of the more complex triphosphate derivatives.

A comprehensive time-course metabolite profiling of the model cyanobacterium Synechocystis sp. PCC 6803 under diurnal light:dark cycles.

Cyanobacteria are a model photoautotroph and a chassis for the sustainable production of fuels and chemicals. Knowledge of photoautotrophic metabolism in the natural environment of day/night cycles is lacking, yet has implications for improved yield from plants, algae and cyanobacteria. Here, a thorough approach to characterizing diverse metabolites-including carbohydrates, lipids, amino acids, pigments, cofactors, nucleic acids and polysaccharides-in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) under sinusoidal diurnal light:dark cycles was developed and applied. A custom photobioreactor and multi-platform mass spectrometry workflow enabled metabolite profiling every 30-120 min across a 24-h diurnal sinusoidal LD ('sinLD') cycle peaking at 1600 µmol photons m-2 sec-1 . We report widespread oscillations across the sinLD cycle with 90%, 94% and 40% of the identified polar/semi-polar, non-polar and polymeric metabolites displaying statistically significant oscillations, respectively. Microbial growth displayed distinct lag, biomass accumulation and cell division phases of growth. During the lag phase, amino acids and nucleic acids accumulated to high levels per cell followed by decreased levels during the biomass accumulation phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates displayed sharp oscillations per cell at the day-to-night transition. Potential bottlenecks in central carbon metabolism are highlighted. Together, this report provides a comprehensive view of photosynthetic metabolite behavior with high temporal resolution, offering insight into the impact of growth synchronization to light cycles via circadian rhythms. Incorporation into computational modeling and metabolic engineering efforts promises to improve industrially relevant strain design.

An overview on anti-biofilm properties of quercetin against bacterial pathogens.

Bacterial biofilms are multicellular aggregates enclosed in a self-created biopolymer matrix. Biofilm-producing bacteria have become a great public health problem worldwide because biofilms enable these microorganisms to evade several clearance mechanisms produced by host and synthetic sources. Over the past years, different flavonoids including quercetin have engrossed considerable interest among researchers owing to their potential anti-biofilm properties. To our knowledge, there is no review regarding effects of quercetin towards bacterial biofilms, prompting us to summarize experimental evidence on its anti-biofilm properties. Quercetin inhibits biofilm development by a diverse array of bacterial pathogens such as Enterococcus faecalis, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Pseudomonas aeruginosa. Prevention of bacterial adhesion, suppression of quorum-sensing pathways, disruption or alteration of plasma membrane, inhibition of efflux pumps, and blocking nucleic acid synthesis have been documented as major anti-biofilm mechanisms of quercetin. Overall, anti-biofilm activity of quercetin can open up new horizons in a wide range of biomedical areas, from food industry to medicine.

Salvianolic acid B regulates gene expression and promotes cell viability in chondrocytes.

Articular chondrocytes reside in lacunae distributed in cartilage responsible for the remodelling of the tissue with limited ability of damage repairing. The in vitro expanded chondrocytes enhanced by factors/agents to obtain large numbers of cells with strengthened phenotype are essential for successful repair of cartilage lesions by clinical cell implantation therapies. Because the salvianolic acid B (Sal B), a major hydrophilic therapeutic agent isolated from Salvia miltiorrhiza, has been widely used to treat diseases and able to stimulate activity of cells, this study examines the effects of Sal B on passaged chondrocytes. Chondrocytes were treated with various concentrations of Sal B in monolayer culture, their morphological properties and changes, and mitochondrial membrane potential were analysed using microscopic analyses, including cellular biochemical staining and confocal laser scanning microscopy. The proteins were quantified by BCA and Western blotting, and the transcription of genes was detected by qRT-PCR. The passaged chondrocytes treated with Sal B showed strengthened cellular synthesis and stabilized mitochondrial membrane potential with upregulated expression of the marker genes for chondrocyte phenotype, Col2-α1, Acan and Sox9, the key Wnt signalling molecule ß-catenin and paracrine cytokine Cytl-1. The treatments using CYTL-1 protein significantly increased expression of Col2-α1 and Acan with no effect on Sox9, indicating the paracrine cytokine acts on chondrocytes independent of SOX9. Sal B has ultimately promoted cell growth and enhanced chondrocyte phenotype. The chondrocytes treated with pharmaceutical agent and cytokine in the formulated medium for generating large number of differentiated chondrocytes would facilitate the cell-based therapies for cartilage repair.

Isothermal Amplification of Nucleic Acids.

Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

Approximate analytic solution of a simple system of kinetic equations describing the enzymatic synthesis of nucleic acids.

Macroscopic kinetic models describing the process of polymerase chain reaction (PCR) are currently solved only by numerical methods, which hampers the development of effective software algorithms for processing the results of the reaction. This paper considers the application of the homotopy perturbation method for obtaining approximate analytical solution of the simplest system of enzymatic kinetic equations describing the synthesis of nucleic acid molecules during PCR. The resulting approximate analytic solution with high accuracy reproduces the results of a numerical solution of the system in a wide range of ratios of enzyme and substrate concentrations both for the case of a large excess of the substrate over the enzyme and vice versa.

Alternative solution of the simplest model of enzymatic synthesis of nucleic acids by homotopy perturbation method.

Development of methods for obtaining approximate analytical solutions of nonlinear differential equations and their systems is a rapidly developing field of mathematical physics. Earlier, an approximate solution of the simplest system of kinetic enzymatic equations for calculating dynamics of complementary strands of nucleic acids was obtained. In this study, we consider an alternative approach to selecting the basic linear approximation of the used method, which makes it possible to obtain more accurate analytical solutions of the set problem.

Profiling of the effects of antifungal agents on yeast cells based on morphometric analysis.

The incidence of fungal infection and evolution of multidrug resistance have increased the need for new antifungal agents. To gain further insight into the development of antifungal drugs, the phenotypic profiles of currently available antifungal agents of three classes-ergosterol, cell wall and nucleic acid biosynthesis inhibitors-were investigated using yeast morphology as a chemogenomic signature. The comparison of drug-induced morphological changes with the deletion of 4718 non-essential genes not only confirmed the mode of action of the drugs but also revealed an unexpected connection among ergosterol, vacuolar proton-transporting V-type ATPase and cell-wall-targeting drugs. To improve, simplify and accelerate drug development, we developed a systematic classifier that sorts a newly discovered compound into a class with a similar mode of action without any mutant information. Using well-characterized agents as target unknown compounds, this method successfully categorized these compounds into their respective classes. Based on our data, we suggest that morphological profiling can be used to develop novel antifungal drugs.

Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.

Doing it in reverse: 3'-to-5' polymerization by the Thg1 superfamily.

The tRNA(His) guanylyltransferase (Thg1) family of enzymes comprises members from all three domains of life (Eucarya, Bacteria, Archaea). Although the initial activity associated with Thg1 enzymes was a single 3'-to-5' nucleotide addition reaction that specifies tRNA(His) identity in eukaryotes, the discovery of a generalized base pair-dependent 3'-to-5' polymerase reaction greatly expanded the scope of Thg1 family-catalyzed reactions to include tRNA repair and editing activities in bacteria, archaea, and organelles. While the identification of the 3'-to-5' polymerase activity associated with Thg1 enzymes is relatively recent, the roots of this discovery and its likely physiological relevance were described ≈ 30 yr ago. Here we review recent advances toward understanding diverse Thg1 family enzyme functions and mechanisms. We also discuss possible evolutionary origins of Thg1 family-catalyzed 3'-to-5' addition activities and their implications for the currently observed phylogenetic distribution of Thg1-related enzymes in biology.

Statistical physics of self-replication.

Self-replication is a capacity common to every species of living thing, and simple physical intuition dictates that such a process must invariably be fueled by the production of entropy. Here, we undertake to make this intuition rigorous and quantitative by deriving a lower bound for the amount of heat that is produced during a process of self-replication in a system coupled to a thermal bath. We find that the minimum value for the physically allowed rate of heat production is determined by the growth rate, internal entropy, and durability of the replicator, and we discuss the implications of this finding for bacterial cell division, as well as for the pre-biotic emergence of self-replicating nucleic acids.

Antimicrobials that affect the synthesis and conformation of nucleic acids.

Several antimicrobials act by inhibiting the synthesis of nucleic acids (rifamycins, sulfamides, diaminopyridines), modifying their conformation (quinolones, coumarins) or causing irreversible lesions (nitroimidazoles, nitrofurans). The resistance mechanisms are: a reduction in intracytoplasmic accumulation, modification of the target or the production of a new low-affinity target and, more rarely, enzyme inactivation. Although the mechanisms affecting the targets are specific to each family and can lead to high-level resistance, the reduced permeability of the membrane and the increased efflux are non-specific and result in low-level cross-resistance between several families. The genetic mediation is usually chromosomal for rifamycins and quinolones, although plasmid-mediated resistant genes have been observed. On the other hand, for sulfamides and trimethoprim, plasmid-borne genes are frequent. Resistance to nitroimidazoles and nitrofurans is still not widely understood.

[The influence of the androgens on the process of the experimental diabetes and the activity of the synthesis of the nucleic acids in the male rats liver].

The aim of this research is to study the dependence the gravity of diabetes course and the activity of the biosynthetical processes in the liver from the level of the sexual steroides in the male-rats blood on the model of the alloxan diabetes. The experiments were carried out on 80 male rats (weight 180-200g). The diabetes has been reproduced by the single injection of alloxan per 200mg/kg. The content of glucose, testosterone, estradiole and corticosterone have been determined in the blood of the animals. Activity of the syntheses of the nucleic acids in the liver has been researched by the radiometrical method on 14, 30, 45 and 90 days of the experiment. On all stages of the observation has been revealed the inverse relation between the concentration of the testosterone in the blood and the gravity of the experimental diabetes which was determined by the degree of hyperglycemya and the changes of the coefficient of sexual hormones ratio, the content of estradiol, corticosterone and activity of the syntheses of the nucleic acids in the tissue of the liver. The injection of the androgen during two week caused jump increasing the testosterone concentration, approaching the sexual steroids ratio to the norm, decreasing the glucose, estradiole, corticosterone content and also the reduction the nucleic acids syntheses activity in the liver. It was shown the positive influence of the androgens on development of the compensatory processes in the male rats, which course the decrease the experimental diabetes gravity.

Autophagy sustains glutamate and aspartate synthesis in Saccharomyces cerevisiae during nitrogen starvation.

Autophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer.

Effect of stalling after mismatches on the error catastrophe in nonenzymatic nucleic acid replication.

The frequency of errors during genome replication limits the amount of functionally important information that can be passed on from generation to generation. During the origin of life, mutation rates are thought to have been quite high, raising a classic chicken-and-egg paradox: could nonenzymatic replication propagate sequences accurately enough to allow for the emergence of heritable function? Here we show that the theoretical limit on genomic information content may increase substantially as a consequence of dramatically slowed polymerization after mismatches. As a result of postmismatch stalling, accurate copies of a template tend to be completed more rapidly than mutant copies and the accurate copies can therefore begin a second round of replication more quickly. To quantify this effect, we characterized an experimental model of nonenzymatic, template-directed nucleic acid polymerization. We found that most mismatches decrease the rate of primer extension by more than 2 orders of magnitude relative to a matched (Watson-Crick) control. A chemical replication system with this property would be able to propagate sequences long enough to have function. Our study suggests that the emergence of functional sequences during the origin of life would be possible even in the face of the high intrinsic error rates of chemical replication.

Antigenic modulation. Loss of TL antigen from cells exposed to TL antibody. Study of the phenomenon in vitro.

Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro. An ascites leukemia, phenotype TL.1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon. Over a wide range of TL antibody concentrations modulation at 37 degrees C was detectable within 10 min and was complete within approximately 1 hr. The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test. The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0 degrees C. Thus modulation apparently is an active cellular process. Antigens TL. 1,2, and 3 are all modulated by anti-TL.1,3 serum and by anti-TL.3 serum. This modulation affects all three TL components together, even when antibody to one or two of them is lacking. aAnti-TL.2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies. The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro. Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region. Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ --> TL- modulation and may require several cell divisions.

Revealing fosfomycin primary effect on Staphylococcus aureus transcriptome: modulation of cell envelope biosynthesis and phosphoenolpyruvate induced starvation.

BACKGROUND: Staphylococcus aureus is a highly adaptable human pathogen and there is a constant search for effective antibiotics. Fosfomycin is a potent irreversible inhibitor of MurA, an enolpyruvyl transferase that uses phosphoenolpyruvate as substrate. The goal of this study was to identify the pathways and processes primarily affected by fosfomycin at the genome-wide transcriptome level to aid development of new drugs. RESULTS: S. aureus ATCC 29213 cells were treated with sub-MIC concentrations of fosfomycin and harvested at 10, 20 and 40 minutes after treatment. S. aureus GeneChip statistical data analysis was complemented by gene set enrichment analysis. A visualization tool for mapping gene expression data into biological pathways was developed in order to identify the metabolic processes affected by fosfomycin. We have shown that the number of significantly differentially expressed genes in treated cultures increased with time and with increasing fosfomycin concentration. The target pathway - peptidoglycan biosynthesis - was upregulated following fosfomycin treatment. Modulation of transport processes, cofactor biosynthesis, energy metabolism and nucleic acid biosynthesis was also observed. CONCLUSIONS: Several pathways and genes downregulated by fosfomycin have been identified, in contrast to previously described cell wall active antibiotics, and was explained by starvation response induced by phosphoenolpyruvate accumulation. Transcriptomic profiling, in combination with meta-analysis, has been shown to be a valuable tool in determining bacterial response to a specific antibiotic.

Effects of pesticides and antibiotics on penaeid shrimp with special emphases on behavioral and biomarker responses.

The purpose of the present study is to provide information on the current state of knowledge regarding the effects of pesticides and antibiotics used in aquaculture on penaeid shrimp, one of the most common aquatic products for human consumption, with a special emphasis on the use of behavioral, physiological, and biochemical response. These include behavior; feeding rate changes; respiration rate, oxygen consumption, and osmoregulation alterations; nucleic acids, protein, and glycogen synthesis; cholinesterase activity inhibition; ATPase activity; and oxidative stress responses. This paper also deals with residues of antibiotics and pesticides in penaeid shrimp. Antibiotics and pesticides used in aquaculture may have adverse effects on treated animals and human consumers health if they are not correctly used. As a complement to the measurement of antibiotic and pesticide residues in tissues, the use of behavioral and biomarker responses can provide more relevant biological information on the potential adverse effects of antibiotics and pesticides on penaeid shrimp health.

[Seasonal variations of nucleic acid content in an experiment on non-pharmacological correction of the hepatic function].

This experiment was preformed on an animal model of CCl4 hepatitis using 320 Wistar rats. The animals underwent combined effect of saprogel and a magnetic field during different seasons. It was shown that non-medicamentous treatment of experimental CCl4 hepatitis had the most pronounced beneficial effect on the synthetic liver function in the winter time. An appreciable suppression of the hepatic function was documented in the spring.

Bacterial bug-out bags: outer membrane vesicles and their proteins and functions.

Among the major bacterial secretions, outer membrane vesicles (OMVs) are significant and highly functional. The proteins and other biomolecules identified within OMVs provide new insights into the possible functions of OMVs in bacteria. OMVs are rich in proteins, nucleic acids, toxins and virulence factors that play a critical role in bacteria-host interactions. In this review, we discuss some proteins with multifunctional features from bacterial OMVs and their role involving the mechanisms of bacterial survival and defence. Proteins with moonlighting activities in OMVs are discussed based on their functions in bacteria. OMVs harbour many other proteins that are important, such as proteins involved in virulence, defence, and competition. Overall, OMVs are a power-packed aid for bacteria, harbouring many defensive and moonlighting proteins and acting as a survival kit in case of an emergency or as a defence weapon. In summary, OMVs can be defined as bug-out bags for bacterial defence and, therefore, survival.