Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB.

Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Polγ, and its modulation by cognate and noncognate SSBs. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ∼55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ∼30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγ strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ∼25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ∼3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.

Detection of biosurfactants in Bacillus species: genes and products identification.

AIM: To screen environmental Bacillus strains for detection of genes encoding the enzymes involved in biosurfactant synthesis and to evaluate their products e.g. surfactin, iturin and fengycin. MATERIALS AND RESULTS: The taxonomic identification of isolated from the environment Bacillus strains was performed by Microgene ID Bacillus panel and GEN III Biolog system. The polymerase chain reaction (PCR) strategy for screening of genes in Bacillus strains was set up. Liquid chromatography-mass spectrometry (LC-MS/MS) method was used for the identification of lipopeptides (LPs). All studied strains exhibited the presence of srfAA gene and produced surfactin mostly as four homologues (C13 to C16). Moreover, in 2 strains (KP7, T'-1) simultaneous co-production of 3 biosurfactants: surfactin, iturin and fengycin was observed. Additionally, it was found out that isolate identified as Bacillus subtilis ssp. subtilis (KP7), beside LPs co-production, synthesizes surfactin with the efficiency much higher than other studied strains (40·2 mg l(-1) ) and with the yield ranging from 0·8 to 8·3 mg l(-1) . CONCLUSION: We showed that the combined methodology based on PCR and LC-MS/MS technique is an optimal tool for the detection of genes encoding enzymes involved in biosurfactant synthesis as well as their products, e.g. surfactin, iturin and fengycin. This approach improves the screening and the identification of environmental Bacillus co-producing biosurfactants-stimulating and facilitating the development of this area of science. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work will help to improve screening of biosurfactant producers. Discovery of novel biosurfactants and biosurfactants co-production ability has shed light on their new application fields and for the understanding of their interactions and properties.

Validation of daily microincrement deposition in otoliths of juvenile and adult Peruvian anchovy Engraulis ringens.

Wild adult specimens of the Peruvian anchovy Engraulis ringens were captured and reared to validate the daily periodicity of otolith microincrement formation. The postcapture stress generated spontaneous spawning, making it possible to conduct a rearing trial on larvae first in an artificial nutrient-enriched system (ANES) for 52 days followed by an artificial feeding regime in a culture tank until day 115 post-hatch. Microincrements of the sagittal otoliths of sacrificed juveniles [mean ± S.D. total length (LT ) = 5·13 ± 0·37 cm, range 5-6 cm; c.v. = 7·5%] showed very distinct light and dark zones. The slope of the relationship between the total number of increments after the hatch check and days elapsed after hatching was not significantly different from 1. The transfer from ANES to the artificial feeding regime induced a mark in the sagittal otoliths. The number of microincrements after this induced mark coincided with the number of days elapsed after the transfer date. In parallel experiments, adult E. ringens (mean ± S.D. LT = 14·92 ± 0·55 cm, range 13-16 cm) were exposed to one of two fluorescent marking immersion treatments with either alizarin red S (ARS; 25 mg l(-1) per 6 h) or oxytetracycline hydrochloride (OTC; 200 mg l(-1) per 10 h). The microincrements between fluorescent bands were distinct, ranging from 0·89 to 2·75 µm (mean ± S.D. =1·43 ± 0·28 µm; c.v. = 32%) and from 0·71 to 2·89 µm (1·53 ± 0·27 µm; c.v. = 35%) for ARS and OTC, respectively. The relationship between the number of microincrements between marks and the number of elapsed days for ARS and OCT treatments indicated that there was a significant correspondence between the number of increases observed and the number of days. Hence, daily microincrements of otoliths of E. ringens are likely to be formed in juveniles and adults under natural conditions.

DNA replication machineries: Structural insights from crystallography and electron microscopy.

Since the discovery of DNA as the genetic material, scientists have been investigating how the information contained in this biological polymer is transmitted from generation to generation. X-ray crystallography, and more recently, cryo-electron microscopy techniques have been instrumental in providing essential information about the structure, functions and interactions of the DNA and the protein machinery (replisome) responsible for its replication. In this chapter, we highlight several works that describe the structure and structure-function relationships of the core components of the prokaryotic and eukaryotic replisomes. We also discuss the most recent studies on the structural organization of full replisomes.

Minor ampullate silks from Nephila and Argiope spiders: tensile properties and microstructural characterization.

The mechanical behavior and microstructure of minor ampullate gland silk (miS) of two orb-web spinning species, Argiope trifasciata and Nephila inaurata, were extensively characterized, enabling detailed comparison with other silks. The similarities and differences exhibited by miS when compared with the intensively studied major ampullate gland silk (MAS) and silkworm (Bombyx mori) silk offer a genuine opportunity for testing some of the hypotheses proposed to correlate microstructure and tensile properties in silk. In this work, we show that miSs of different species show similar properties, even when fibers spun by spiders that diverged over 100 million years are compared. The tensile properties of miS are comparable to those of MAS when tested in air, significantly in terms of work to fracture, but differ considerably when tested in water. In particular, miS does not show a supercontraction effect and an associated ground state. In this regard, the behavior of miS in water is similar to that of B. mori silk, and it is shown that the initial elastic modulus of both fibers can be explained using a common model. Intriguingly, the microstructural parameters measured in miS are comparable to those of MAS and considerably different from those found in B. mori. This fact suggests that some critical microstructural information is still missing in our description of silks, and our results suggest that the hydrophilicity of the lateral groups or the large scale organization of the sequences might be routes worth exploring.

An assessment of the genotoxic effects of landfill leachates using bacterial and plant tests.

Two bacterial tests (the Ames test and the umu-test) and the Allium test were used to assess the genotoxicity of aqueous leachates from municipal solid waste landfill sites in Southern Poland. A comparison of the sensitivity of the applied tests was performed. None of the tested samples revealed genotoxic activity in the umu-test and thus did not appear to be sensitive enough for evaluations of leachates. Two out of 22 leachates were described as genotoxic in the Ames test and 3 out of 6 leachates in the Allium test. All of the analyzed leachates samples affected cell divisions. Two of the tests applied, the Allium and Ames test, revealed the high genotoxicity of leachate 4. Among the bioassays used in these studies, the Allium test proved to be more sensitive than bacterial tests for the investigation of leachate toxicity. The results suggest that the Allium cytogenetic bioassay is efficient and simple for genotoxicity studies of leachates. The potential correlations between the chemical characteristics and genotoxic effects is discussed. The biological effects of selected appeared to be related to chemical parameters. Leachates have a genotoxic potential and pose a risk to human health and the environment. A combination of biotests and chemical analyses is the best approach for the assessment of the risk or impact of leachates.

DNA replication machinery: Insights from in vitro single-molecule approaches.

The replisome is the multiprotein molecular machinery that replicates DNA. The replisome components work in precise coordination to unwind the double helix of the DNA and replicate the two strands simultaneously. The study of DNA replication using in vitro single-molecule approaches provides a novel quantitative understanding of the dynamics and mechanical principles that govern the operation of the replisome and its components. 'Classical' ensemble-averaging methods cannot obtain this information. Here we describe the main findings obtained with in vitro single-molecule methods on the performance of individual replisome components and reconstituted prokaryotic and eukaryotic replisomes. The emerging picture from these studies is that of stochastic, versatile and highly dynamic replisome machinery in which transient protein-protein and protein-DNA associations are responsible for robust DNA replication.

In vitro single-molecule manipulation studies of viral DNA replication.

Faithfull replication of genomic information relies on the coordinated activity of the multi-protein machinery known as the replisome. Several constituents of the replisome operate as molecular motors that couple thermal and chemical energy to a mechanical task. Over the last few decades, in vitro single-molecule manipulation techniques have been used to monitor and manipulate mechanically the activities of individual molecular motors involved in DNA replication with nanometer, millisecond, and picoNewton resolutions. These studies have uncovered the real-time kinetics of operation of these biological systems, the nature of their transient intermediates, and the processes by which they convert energy to work (mechano-chemistry), ultimately providing new insights into their inner workings of operation not accessible by ensemble assays. In this chapter, we describe two of the most widely used single-molecule manipulation techniques for the study of DNA replication, optical and magnetic tweezers, and their application in the study of the activities of proteins involved in viral DNA replication.

The effect of hydraulic retention time in onsite wastewater treatment and removal of pharmaceuticals, hormones and phenolic utility substances.

Micropollutants such as pharmaceuticals, hormones and phenolic utility chemicals in sewage water are considered to be an emerging problem because of increased use and observed adverse effects in the environment. The study provides knowledge on the removal efficiency of micropollutants with a range of physical and chemical properties in three commercially available onsite wastewater treatment facilities (OWTFs), tested on influent wastewater collected from 2500 person equivalents in Bildchen, Germany. A longer hydraulic retention time would in theory be expected to have a positive effect, and this study presents results for three different OWTFs in full-scale comparable tests under natural conditions. A range of 24 different pharmaceuticals, five phenols and three hormones were analyzed. Flow-proportional consecutive sampling was performed in order to determine the removal efficiency. Twenty-eight substances were detected in the effluent wastewater out of 32 substances included. Average effluent concentrations of Simvastatin, Estrone, Estradiol and Ethinylestradiol were above the indicative critical-effect concentration of pharmacological effect on fish in all facilities. Average effluent concentrations of both Diclofenac and Estradiol were higher than the Environmental Quality Standards applied in Sweden (190-240 times and 9-35 times respectively). The removal efficiency of micropollutants was high for substances with high logKow, which enhance the adsorption and removal with sludge. Low removal was observed for substances with low logKow and acidic characteristics, and for substances with stabilizing elements of the chemical structure. Facilities that use activated sludge processes removed hormones more efficiently than facilities using trickling filter treatment technique. Moreover, longer hydraulic retention time increased the removal of pharmaceuticals, hormones, turbidity and total nitrogen. Removal of Caffeine, Ibuprofen, Estrone, Naproxen and Estradiol, was strongly correlated to the sludge and particles removal. Thus, the efficiency of the tested OWTFs could be improved by adjusting the technical methods and increasing the hydraulic retention time.

[Mechanisms and management of hyperacusis (decreased sound tolerance)]. / Fisiopatología y tratamiento de la hiperacusia (hipersensibilidad al sonido).

Hyperacusis is a decreased sound tolerance. Prevalence of the disease is described in 9-15% of the population, but this percentage increases among the tinnitus patients. Pathophysiological mechanisms involve some disruptions in the amplification and regulation processes of the external hair cells or affect the central sound processing at the subcortical level. The role of the serotonin, also involved in other diseases related with hyperacusis (migraine, depression), can be crucial in this disorder. Other theories confirm the effect of the endorphins that activate the excitatory function of the glutamate, the auditory neurotransmitter, increasing its toxicity. The activation of the limbic and autonomic nervous systems produces the emotional reaction of the hyperacusis (anxiety, fear and depression). Proposed treatments are based on acoustic stimulation by a progressive introduction of sound (tinnitus retraining therapy TRT). Noise generators and hearing aids can be fitted in severe cases. The role of some drugs involved in the metabolism of the serotonin open new approaches for the management of hyperacusis.