In silico pathway reconstruction: Iron-sulfur cluster biogenesis in Saccharomyces cerevisiae

Background: Current advances in genomics, proteomics and other areas of molecular biology make the identification and reconstruction of novel pathways an emerging area of great interest. One such class of pathways is involved in the biogenesis of Iron-Sulfur Clusters (ISC). Results: Our goal is the development of a new approach based on the use and combination of mathematical, theoretical and computational methods to identify the topology of a target network. In this approach, mathematical models play a central role for the evaluation of the alternative network structures that arise from literature data-mining, phylogenetic profiling, structural methods, and human curation. As a test case, we reconstruct the topology of the reaction and regulatory network for the mitochondrial ISC biogenesis pathway in S. cerevisiae. Predictions regarding how proteins act in ISC biogenesis are validated by comparison with published experimental results. For example, the predicted role of Arh1 and Yah1 and some of the interactions we predict for Grx5 both matches experimental evidence. A putative role for frataxin in directly regulating mitochondrial iron import is discarded from our analysis, which agrees with also published experimental results. Additionally, we propose a number of experiments for testing other predictions and further improve the identification of the network structure. Conclusion: We propose and apply an iterative in silico procedure for predictive reconstruction of the network topology of metabolic pathways. The procedure combines structural bioinformatics tools and mathematical modeling techniques that allow the reconstruction of biochemical networks. Using the Iron Sulfur cluster biogenesis in S. cerevisiae as a test case we indicate how this procedure can be used to analyze and validate the network model against experimental results. Critical evaluation of the obtained results through this procedure allows devising new wet lab experiments to confirm its predictions or provide alternative explanations for further improving the models.

Corrigendum : Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis

Absorption, transport and storage of iron are tightly regulated, as expected for an element, which is both essential and potentially toxic. Iron deficiency is the leading cause of anaemia, and it also compromises immune function and cognitive development. Iron overload damages the liver and other organs in hereditary hemochromatosis, and in thalassaemia patients with both transfusion and non-transfusionrelated iron accumulation. Excess iron has harmful effects in chronic liver diseases caused by excessive alcohol, obesity or viruses. There is evidence for involvement of iron in neurodegenerative diseases and in Type 2 diabetes. Variation in transferrin saturation, a biomarker of iron status, has been associated with mortality in patients with diabetes and in the general population13. All these associations between iron and either clinical disease or pathological processes make it important to understand the causes of variation in iron status. Importantly, information on genetic causes of variation can be used in Mendelian randomization studies to test whether variation in iron status is a cause or consequence of disease. We have used biomarkers of iron status (serum iron, transferrin, transferrin saturation and ferritin), which are commonly used clinically and readily measurable in thousands of individuals, and carried out a meta-analysis of human genomewide association study (GWAS) data from 11 discovery and eight replication cohorts. Our aims were to identify additional loci affecting markers of iron status in the general population and to relate the significant loci to information on gene expression to identify relevant genes. We also made an initial assessment of whether any such loci affect iron status in HFE C282Y homozygotes, who are at genetic risk of HFE-related iron overload (hereditary hemochromatosis type 1, OMIM #235200)

Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression ofSSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.

Modelling of iron-filled magneto-active polymers with a dispersed chain-like microstructure

Magneto-active polymers are a class of smart materials commonly manufactured by mixing micron-sized iron particles in a rubber-like matrix. When cured in the presence of an externally applied magnetic field, the iron particles arrange themselves into chain-like structures that lend an overall anisotropy to the material. It has been observed through electron micrographs and X-ray tomographs that these chains are not always perfect in structure, and may have dispersion due to the conditions present during manufacturing or some undesirable material properties. We model the response of these materials to coupled magneto-mechanical loading in this paper using a probability based structure tensor that accounts for this imperfect anisotropy. The response of the matrix material is decoupled from the chain phase, though still being connected through kinematic constraints. The latter is based on the definition of a 'chain deformation gradient' and a 'chain magnetic field'. We conclude with numerical examples that demonstrate the effect of chain dispersion on the response of the material to magnetoelastic loading.

Enzymatic technology to improve oil extraction from Caryocar brasiliense camb. (Pequi) Pulp.

The present study aims to compare yield and quality of pequi pulp oil when applying two distinct processes: in the first, pulp drying in a tray dryer at 60ºC was combined with enzymatic treatment and pressing to oil extraction; in the second, a simple process was carried out by combining sun-drying pulp and pressing. In this study, raw pequi fruits were collected in Mato Grosso State, Brazil. The fruits were autoclaved at 121ºC and stored under refrigeration. An enzymatic extract with pectinase and CMCase activities was used for hydrolysis of pequi pulp, prior to oil extraction. The oil extractions were carried out by hydraulic pressing, with or without enzymatic incubation. The oil content in the pequi pulp (45% w/w) and the physicochemical characteristic of the oil was determined according to standard analytical methods. Free fatty acids, peroxide values, iodine and saponification indices were respectively 1.46 mgKOH/g, 2.98 meq/kg, 49.13 and 189.40. The acidity and peroxide values were lower than the obtained values in commercial oil samples, respectively 2.48 mgKOH/g and 5.22 meq/kg. Aqueous extraction has presented lower efficiency and higher oxidation of unsaturated fatty acids. On the other hand, pequi pulp pressing at room temperature has produced better quality oil. However its efficiency is still smaller than the combined enzymatic treatment and pressing process. This combined process promotes cellular wall hydrolysis and pulp viscosity reduction, contributing to at least 20% of oil yield increase by pressing.

Physicochemical characterization of nebulized superparamagnetic iron oxide nanoparticles (SPIONs)

Abstract Background: Aerosol-mediated delivery of nano-based therapeutics to the lung has emerged as a promising alternative for treatment and prevention of lung diseases. Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted significant attention for such applications due to their biocompatibility and magnetic properties. However, information is lacking about the characteristics of nebulized SPIONs for use as a therapeutic aerosol. To address this need, we conducted a physicochemical characterization of nebulized Rienso, a SPION-based formulation for intravenous treatment of anemia. Methods: Four different concentrations of SPION suspensions were nebulized with a one-jet nebulizer. Particle size was measured in suspension by transmission electron microscopy (TEM), photon correlation spectroscopy (PCS), and nanoparticle tracking analysis (NTA), and in the aerosol by a scanning mobility particle sizer (SMPS). Results: The average particle size in suspension as measured by TEM, PCS, and NTA was 9±2 nm, 27±7 nm, and 56±10 nm, respectively. The particle size in suspension remained the same before and after the nebulization process. However, after aerosol collection in an impinger, the suspended particle size increased to 159±46 nm as measured by NTA. The aerosol particle concentration increased linearly with increasing suspension concentration, and the aerodynamic diameter remained relatively stable at around 75 nm as measured by SMPS. Conclusions: We demonstrated that the total number and particle size in the aerosol were modulated as a function of the initial concentration in the nebulizer. The data obtained mark the first known independent characterization of nebulized Rienso and, as such, provide critical information on the behavior of Rienso nanoparticles in an aerosol. The data obtained in this study add new knowledge to the existing body of literature on potential applications of SPION suspensions as inhaled aerosol therapeutics.

Evolution and cellular function of monothiol glutaredoxins: involvement in iron-sulphur cluster assembly

A number of bacterial species, mostly proteobacteria, possess monothiol glutaredoxins homologous to the Saccharomyces cerevisiae mitochondrial protein Grx5, which is involved in iron–sulphur cluster synthesis. Phylogenetic profiling is used to predict that bacterial monothiol glutaredoxins also participate in the iron–sulphur cluster (ISC) assembly machinery, because their phylogenetic profiles are similar to the profiles of the bacterial homologues of yeast ISC proteins. High evolutionary cooccurrence is observed between the Grx5 homologues and the homologues of the Yah1 ferredoxin, the scaffold proteins Isa1 and Isa2, the frataxin protein Yfh1 and the Nfu1 protein. This suggests that a specific functional interaction exists between these ISC machinery proteins. Physical interaction analyses using low-definition protein docking predict the formation of strong and specific complexes between Grx5 and several components of the yeast ISC machinery. Two-hybrid analysis has confirmed the in vivo interaction between Grx5 and Isa1. Sequence comparison techniques and cladistics indicate that the other two monothiol glutaredoxins of S. cerevisiae, Grx3 and Grx4, have evolved from the fusion of a thioredoxin gene with a monothiol glutaredoxin gene early in the eukaryotic lineage, leading to differential functional specialization. While bacteria do not contain these chimaeric glutaredoxins, in many eukaryotic species Grx5 and Grx3/4-type monothiol glutaredoxins coexist in the cell.

Epäpuhtaudet sinkin uutossa

Työssä tutkittiin metalli- ja anioniepäpuhtauksien myötäuuttautumista sinkin mukana di(2-etyyliheksyyli)fosforihappoalla (D2EHPA). Laboratoriokokeissa selvitettiin pH:n vaikutusta metalliepäpuhtauksien uuttautumiseen pH-alueella -O, l... 3 sekä pesujen vaikutusta sinkillä ladatun orgaanisen faasin metallipitoisuuksiin. Kokeita tehtiin sekä synteettisillä että autenttisilla prosessiliuoksilla. Anionikokeissa tutkittiin raudan ja sinkin vaikutusta kloridin ja fluoridin uuttautumiseen. Synteettisillä liuoksilla tehdyissä kokeissa tutkittiin kadmiumin, koboltin, nikkelin, kuparin sekä antimonin uuttautumista sinkkisulfaattiliuoksesta. Kokeissa havaittiin D2EHPA:n uuttavan sinkkiä selvästi em. metalleja paremmin. Sinkki uuttautui sulfaattiliuoksesta lähes täydellisesti tasapaino-pH:n ollessa yli 2,3. Sinkin jälkeen muista metalleista uuttautui eniten kadmium ja järjestyksessä sitten kupari, koboltti ja nikkeli. Epäpuhtausmetallien myötäuuttautumista lisääntyi uuton tasapaino-pH:n kasvaessa ja väheni sinkkilatauksen kasvaessa. Kahdella peräkkäisellä pesuaskeleella, joissa ensimmäinen pesuliuos sisälsi 5 g/L rikkihappoa ja toinen sekä 15 g/L rikkihappoa että 5 g/L sinkkiä saatiin kaikkien epäpuhtausmetallien pitoisuudet jäämään alle 3 mg/L. Antimonin uuttokokeissa huomattiin antimonin uuttautuvan täydellisesti D2EHPA:lla pH:sta riippumatta pH-alueella0...3. Prosessiliuoksilla tehdyissä kokeissa todettiin D2EHPA:n pystyvän tehokkaasti erottamaan sinkin magnesiumista ßzn,Mg ˜107 ja kadmiumista ßzn,cd ˜106. Havaittiin myös, että mitä suurempi sinkki- ja rautalataus orgaanisessa faasissa on sitä vähemmän magnesiumia ja kadmiumia uuttautuu. Ensimmäisessä pesussa saatiin sekä kadmiumin että magnesiumin pitoisuudet putoamaan keskimäärin 0,1 mg/L eli 30 alkuperäisestä pitoisuudesta. Toisella pesuaskeleella ei enää ollut vaikutusta kadmiumin ja magnesiumin pitoisuuksiin orgaanisessa faasissa. Kokeissa havaittiin myös, että D2EHPA:n latausasteen ylittäessä 0,7 alkaa sinkki-D2EHPA-kompleksit polymeroitua ja faasit eivät enää selkeytyneet helposti. Anionikokeissa huomattiin, ettei D2EHPA uuttanut kloridi tai fluoridi sinkin tai raudan mukana. D2EHPA:n havaittiin myös itsessään sisältävän hieman kloridia.

Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking.

The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.

Evaluation of the indication for surgical extraction of third molars according to the oral surgeon and the primary care dentist. Experience in the Master of Oral Surgery and Implantology at Barcelona University Dental School

Introduction: Third molar extraction is the most frequent procedure in oral surgery. The present study evaluates the indication of third molar extraction as established by the primary care dentist (PCD) and the oral surgeon, and compares the justification for extraction with the principal reason for patient consultation. Patients and method: A descriptive study was made of 319 patients subjected to surgical removal of a third molar in the context of the Master of Oral Surgery and Implantology (Barcelona University Dental School, Barcelona, Spain) between July 2004 and March 2005. The following parameters were evaluated: sex, age, molar, type of impaction, position according to the classifications of Pell and Gregory and of Winter, and the reasons justifying extraction. Results: The lower third molars were the most commonly extracted molars (73.7%). A total of 69.6% of the teeth were covered by soft tissues only. Fifty-six percent of the lower molars corresponded to Pell and Gregory Class IIB, while 42.1% were in the vertical position. The most common reason for patient reference to our Service of Oral Surgery on the part of the PCD was prophylactic removal (51.0% versus 46.1% in the case of the oral surgeon). Discussion and conclusions. Our results show prophylaxis to be the principal indication of third molar extraction, followed by orthodontic reasons. Regarding third molars with associated clinical symptoms or signs, infectious disease-including pericoronitis- was the pathology most often observed by the oral surgeon, followed by caries. This order of frequency was seen to invert in the case of third molars referred for extraction by the PCD. A vertical position predominated among the third molars with associated pathology

Social network extraction and analysis based on multimodal dyadic interaction

Social interactions are a very important component in people"s lives. Social network analysis has become a common technique used to model and quantify the properties of social interactions. In this paper, we propose an integrated framework to explore the characteristics of a social network extracted from multimodal dyadic interactions. For our study, we used a set of videos belonging to New York Times" Blogging Heads opinion blog. The Social Network is represented as an oriented graph, whose directed links are determined by the Influence Model. The links" weights are a measure of the"influence" a person has over the other. The states of the Influence Model encode automatically extracted audio/visual features from our videos using state-of-the art algorithms. Our results are reported in terms of accuracy of audio/visual data fusion for speaker segmentation and centrality measures used to characterize the extracted social network.

More than 10 years of experience with immediate sequential bilateral cataract extraction (ISBCE) - a retrospective study

Background: To evaluate the safety of immediate sequential bilateral cataract extraction (ISBCE) with respect to indications, visual outcomes, complications, benefits and disadvantages. Methods: This is a retrospective review of all ISBCEs performed at Kantonsspital Winterthur, Switzerland, between April 2000 and September 2013. The case notes of 500 eyes of 250 patients were reviewed. Of these 500 eyes, 472 (94.4%) had a straight forward phacoemulsification with posterior chamber intraocular lens implantation; 21 (4.2%) had a planned extracapsular cataract extraction; 4 (0.8%) had an intracapsular cataract extraction and 3 (0.6%) had a combined phacoemulsification with trabeculectomy. Results: Over 66% of eyes achieved improved visual acuity (at least 3 Snellen lines) following ISBCE. Median preoperative best corrected visual acuity (BCVA) was 0.5 LogMAR; the interquartile range was [0.4, 1] LogMAR. At one week control the median BCVA was 0.3 LogMAR, IQR [0.1, 0.5] LogMAR. At one month the median BCVA was 0.15 LogMAR, IQR [0.05, 0.3] (p < 0.01). There were no sight-threatening intraoperative or postoperative complications observed. Conclusions: ISBCE is an effective and safe option with high degree of patient satisfaction. The relative benefits of ISBCE should be balanced against the theoretically enhanced risks.

Characterization of volatile organic gunshot residues in fired handgun cartridges by headspace sorptive extraction

In forensic investigation of firearm-related cases, determination of the residual amount of volatile compounds remaining inside a cartridge could be useful in estimating the time since its discharge. Published approaches are based on following the decrease of selected target compounds as a function of time by using solid phase micro-extraction (SPME). Naphthalene, as well as an unidentified decomposition product of nitrocellulose (referred to as "TEA2"), are usually employed for this purpose. However, reliability can be brought into question given their high volatility and the low reproducibility of their extracted quantities. In order to identify alternatives and therefore develop improved dating methods, an extensive study on the composition and variability of volatile residues in nine different types of cartridges was carried out. Analysis was performed using headspace sorptive extraction (HSSE), which is a more exhaustive technique compared to SPME. 166 compounds were identified (several of which for the first time), and it was observed that the final compositional characteristics of each residue were strongly dependent on its source. Variability of single identified compounds within and between different types of cartridge, as well as their evolution over time, was also studied. Many explosion products containing up to 4 aromatic rings were found to be globally present in high proportions amongst residues. 27 of them (excluding naphthalene) also presented detectable decreases during the first 24 h. Therefore, they could be used as complementary target analytes in future dating methods.

COMPARISON OF RNA EXTRACTION METHODS FOR Passiflora edulis SIMS LEAVES

ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), TRIzol® Reagent (Invitrogen) and TRIzol® Reagent (Invitrogen)/ice -commercial products specifically designed to extract RNA, and to determine which method is the most effective for extracting RNA from the leaves of passion fruit plants. In contrast to the RNA extracted using the other 2 methods, the RNA extracted using TRIzol® Reagent (Invitrogen) did not have acceptable A260/A280 and A260/A230 ratios and did not have ideal concentrations. Agarose gel electrophoresis showed a strong DNA band for all of the Concert™ method extractions but not for the TRIzol® and TRIzol®/ice methods. The TRIzol® method resulted in smears during electrophoresis. Due to its low levels of DNA contamination, ideal A260/A280 and A260/A230 ratios and superior sample integrity, RNA from the TRIzol®/ice method was used for reverse transcription-polymerase chain reaction (RT-PCR), and the resulting amplicons were highly similar. We conclude that TRIzol®/ice is the preferred method for RNA extraction for P. edulis leaves.