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.

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.

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.

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.

Ictiosis ligada al x tratada con tazarotene tópico / X-linked ichthyosis treated with tazarotene topic

La Ictiosis ligada al cromosoma X (ILX), es una genodermatosis poco frecuente que afecta a varones, se manifiesta a partir del período neonatal y se caracteriza por: xerodermia, hiperqueratosis, descamación y en algunos casos, manifestaciones extracutáneas. La delección total o parcial de la enzima sulfatasa esteroidea, es la causa de las manifestaciones clínicas. Presentamos el caso clínico, de un paciente con manifestaciones clínicas y hallazgos en la anatomía patológica, compatibles con esta entidad tratado con tazarotene (gel).

X-linked ichthyosis (XLI) is a rare genodermatosis that affects men and manifests from the neonatal period and is characterized by xeroderma, hyperkeratosis, desquamation and in some cases, extracutaneous manifestations. The disease is of chronic evolution and undergoes partial improvement in the summer season. The total or partial deletion of the steroid sulphatase enzyme is the cause of the clinical manifestations. We present the case of a patient with clinical manifestations and findings in the pathological anatomy compatible with this entity, as well as ITS treatment with topical tazarotene 0.1% (gel) Weekly controls were performed and clinical benefit of lesion-free skin was observed up to 6 weeks after treatment discontinuation, followed by the progressive appearance of brownish scales that became thick and adherent with the passage of time.

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.

Persistence and variation in microstructural design during the evolution of spider silk.

The extraordinary mechanical performance of spider dragline silk is explained by its highly ordered microstructure and results from the sequences of its constituent proteins. This optimized microstructural organization simultaneously achieves high tensile strength and strain at breaking by taking advantage of weak molecular interactions. However, elucidating how the original design evolved over the 400 million year history of spider silk, and identifying the basic relationships between microstructural details and performance have proven difficult tasks. Here we show that the analysis of maximum supercontracted single spider silk fibers using X ray diffraction shows a complex picture of silk evolution where some key microstructural features are conserved phylogenetically while others show substantial variation even among closely related species. This new understanding helps elucidate which microstructural features need to be copied in order to produce the next generation of biomimetic silk fibers.

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.

Simple measurement of the apparent viscosity of a cell from only one picture: Application to cardiac stem cells.

Mechanical deformability of cells is a key property that influences their ability to migrate and their contribution to tissue development and regeneration. We analyze here the possibility of characterizing the overall deformability of cells by their apparent viscosity, using a simplified method to estimate that parameter. The proposed method simplifies the quantitative analysis of micropipette-aspiration experiments. We have studied by this procedure the overall apparent viscosity of cardiac stem cells, which are considered a promising tool to regenerate damaged cardiac tissue. Comparison with the apparent viscosity of low-viscosity cells such as immune-system cells suggests that treatments to reduce the viscosity of these cells could enhance their ability to repair damaged cardiac tissue.

GHEP-ISFG collaborative exercise on mixture profiles of autosomal STRs (GHEP-MIX01, GHEP-MIX02 and GHEP-MIX03): results and evaluation.

One of the main objectives of the Spanish and Portuguese-Speaking Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the area of forensic genetics. Due to this fact, GHEP-ISFG holds different working commissions that are set up to develop activities in scientific aspects of general interest. One of them, the Mixture Commission of GHEP-ISFG, has organized annually, since 2009, a collaborative exercise on analysis and interpretation of autosomal short tandem repeat (STR) mixture profiles. Until now, three exercises have been organized (GHEP-MIX01, GHEP-MIX02 and GHEP-MIX03), with 32, 24 and 17 participant laboratories respectively. The exercise aims to give a general vision by addressing, through the proposal of mock cases, aspects related to the edition of mixture profiles and the statistical treatment. The main conclusions obtained from these exercises may be summarized as follows. Firstly, the data show an increased tendency of the laboratories toward validation of DNA mixture profiles analysis following international recommendations (ISO/IEC 17025:2005). Secondly, the majority of discrepancies are mainly encountered in stutters positions (53.4%, 96.0% and 74.9%, respectively for the three editions). On the other hand, the results submitted reveal the importance of performing duplicate analysis by using different kits in order to reduce errors as much as possible. Regarding the statistical aspect (GHEP-MIX02 and 03), all participants employed the likelihood ratio (LR) parameter to evaluate the statistical compatibility and the formulas employed were quite similar. When the hypotheses to evaluate the LR value were locked by the coordinators (GHEP-MIX02) the results revealed a minor number of discrepancies that were mainly due to clerical reasons. However, the GHEP-MIX03 exercise allowed the participants to freely come up with their own hypotheses to calculate the LR value. In this situation the laboratories reported several options to explain the mock cases proposed and therefore significant differences between the final LR values were obtained. Complete information concerning the background of the criminal case is a critical aspect in order to select the adequate hypotheses to calculate the LR value. Although this should be a task for the judicial court to decide, it is important for the expert to account for the different possibilities and scenarios, and also offer this expertise to the judge. In addition, continuing education in the analysis and interpretation of mixture DNA profiles may also be a priority for the vast majority of forensic laboratories.

[Comparison between topical anaesthesia with cocaine versus lidocaine plus adrenaline for outpatient laser dacryocystorhinostomy]. / Comparación entre la anestesia tópica con cocaína y lidocaína con adrenalina en la dacriocistorrinostomía con láser.

OBJECTIVE: To evaluate the effectiveness of topical anaesthesia with cocaine versus lidocaine plus adrenaline for outpatient transcanalicular and endonasal dacryocystorhinostomy (TCLDCR) with diode laser under sedation. METHODS: A double blind randomised clinical trial was designed using topical anaesthesia for outpatient TCLDCR in the treatment of adult epiphora. A total of 92 patients were enrolled, and randomly allocated to be operated on under sedation and topical anaesthesia with cocaine 4% pledgets versus sedation and topical anaesthesia with lidocaine 2% plus 1/100.000 adrenaline pledgets. Main outcome measures were postoperative comfort, evaluated by a visual analogue scale, presence of secondary effects (blood pressure, heart rate), and resolution of epiphora, evaluated by Munk's scale and endoscopic control. RESULTS: Patients in both groups reported being comfortable during and immediately after TCLDCR. Visualization of the operative field was adequate, and surgery was successfully completed in all cases. Complications were more common in the cocaine group: Sixteen patients from the cocaine group had high blood pressures, versus 2 patients from the lidocaine group (RR=8). Mean blood loss was 6.09 ml in cocaine group, versus 2.05 ml in lidocaine group (RR=6). Both parameters were statistically significant (p=1,1×10(-9)). There were no cases of postoperative epistaxis requiring nasal packing or hospital admission in any group. Success rate was similar in the 2 groups (86.96% group 1 and 89.13% group 2), after 6 months of follow-up. CONCLUSIONS: The combination of topical lidocaine and adrenaline is more effective for outpatient transcanalicular and endonasal dacryocystorhinostomy than topical cocaine. Patient comfort was adequate in both groups, but high blood pressure and blood loss more common after cocaine.

[Endoscopic dacryocystorhinostomy: role of the ophthalmologist]. / Dacriocistorrinostomía endonasal: participación del oftalmólogo.

OBJECTIVE: To evaluate the results of endoscopic dacryocystorhinostomy (DCR) with or without support of the ophthalmologist. MATERIAL AND METHODS: A retrospective study of 100 cases of endoscopic DCR surgery conducted by an otolaryngologist between June 2008 and December 2009. Of the 100 cases, 50 were operated with surgical support of the ophthalmologist, who inserted Bowman probes in the upper and lower canaliculi, while in the other 50 cases it was the otolaryngologist who performed this, without support of the ophthalmologist. The evaluation of the results after 2 years included the subjective perception, the lacrimal patency after lacrimal syringing, and lacrimal functional test after modified Jones test. RESULTS: Of the 100 DCR reviewed, more than 50% required complementary treatment by the otolaryngologist, mainly septoplasty. As for the resolution of epiphora, without support of the ophthalmologist, 75% the patients reported an overall subjective improvement, but this reached 92% in the surgical group with support of the ophthalmologist, which was a statistically significant difference. CONCLUSIONS: Endoscopic DCR is effective in the treatment of epiphora, but its results improve when the ophthalmologist inserts the probes in the lacrimal canaliculi during the surgical procedure.

Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk.

Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ß-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the -GGX- and -GPG- motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract.

GHEP-ISFG proficiency test 2011: Paper challenge on evaluation of mitochondrial DNA results.

The GHEP-ISFG Working Group performed a collaborative exercise to monitor the current practice of mitochondrial (mt)DNA reporting. The participating laboratories were invited to evaluate a hypothetical case example and assess the statistical significance of a match between the haplotypes of a case (hair) sample and a suspect. A total of 31 forensic laboratories participated of which all but one used the EMPOP database. Nevertheless, we observed a tenfold range of reported LR values (32-333.4), which was mainly due to the selection of different reference datasets in EMPOP but also due to different applied formulae. The results suggest the need for more standardization as well as additional research to harmonize the reporting of mtDNA evidence.

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.

Supercontraction of dragline silk spun by lynx spiders (Oxyopidae).

Supercontraction is commonly considered as a functional adaptation of major ampullate gland (MA) silk to its role as the main structural material in orb-webs. However, the observation of supercontraction in the dragline silk of a lynx spider species, as it is shown in this work, offers a strong support to the hypothesis that the appearance of supercontraction preceded the advent of capture webs. Moreover, the absence of proline in the sequence of dragline silk spidroin in Oxyopidae and related spiders indicates that the presence of this amino acid may not be required for the existence of supercontraction. In this regard, the presence of particular subrepeats--in orb-web and non-orb-web building spiders--adds new clues for the understanding of supercontraction and associated effects.

Supramolecular organization of regenerated silkworm silk fibers.

The microstructures of N-methylmorpholine-N-oxide (NMMO) regenerated silk fibers have been characterized by atomic force microscopy from the micrometer to the nanometer scale and compared with those previously found from natural silks. Regenerated fibers show poor tensile properties and a brittle behavior, but their mechanical properties improve if subjected to post-spinning drawing. Consequently, it was hypothesized that post-spinning drawing would lead to a microstructure more similar to that of the natural material. Here we show that the microstructure of the samples not subjected to post-spinning drawing is composed of nanoglobules that differ from those found in natural silkworm silk both in size and orientation with respect to the macroscopic axis of the fiber. The microstructure of samples subjected to post-spinning drawing evolves in the sense of decreasing the size but increasing the orientation of the nanoglobules, but these effects are only observed in some regions of the fibers.

[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.