Genomics: more than genes

The fundamentals of Real-time Polymerase Chain Reaction,Automated capillary electrophoresis -Sanger sequencing and Fragmentanalysis- and "Next-generation" sequencing are reviewed. An overview ofapplications is presented using our own examples carried out in our facility.

Postmortem angiography after vascular perfusion with diesel oil and a lipophilic contrast agent.

OBJECTIVE: The objective of our study was to establish optimal perfusion conditions for high-resolution postmortem angiography that would permit dynamic visualization of the arterial and venous systems. MATERIALS AND METHODS: Cadavers of two dogs and one cat were perfused with diesel oil through a peristaltic pump. The lipophilic contrast agent Lipiodol Ultra Fluide was then injected, and angiography was performed. The efficiency of perfusion was evaluated in the chick chorioallantoic membrane. RESULTS: Vessels could be seen up to the level of the smaller supplying and draining vessels. Hence, both the arterial and the venous sides of the vascular system could be distinguished. The chorioallantoic membrane assay revealed that diesel oil enters microvessels up to 50 microm in diameter and that it does not penetrate the capillary network. CONCLUSION: After establishing a postmortem circulation by diesel oil perfusion, angiography can be performed by injection of Lipiodol Ultra Fluide. The resolution of the images obtained up to 3 days after death is comparable to that achieved in clinical angiography.

Simple holographic polarogram

A new technique capable of obtaining quantitative values of the rotation angle of the polarization vector by using holography is presented. This is a two-stage holographic process; during the recording stage a hologram of the object of interest is obtained. The reference beam is composed of two beams that form a small angle between them and keep their polarization states at right angles to each other. In the reconstruction stage of the hologram, two images from the hologram are obtained along two different angles. As a result of the interference between these two images, a set of parallel fringes is formed at the image plane. The fringe contrast on the reconstruction is related to the angle of the polarization vector of the light at each position on the image plane. Measurements of the rotation of the polarization angle of a fraction of a degree were obtained. The main application of this technique is in the study of transient phenomena, where single-shot measurements are the only means of obtaining reliable data.

A simple gas chromatography method for the analysis of monoethanolamine in air

A simple method determining airborne monoethanolamine has been developed. Monoethanolamine determination has traditionally been difficult due to analytical separation problems. Even in recent sophisticated methods, this difficulty remains as the major issue often resulting in time-consuming sample preparations. Impregnated glass fiber filters were used for sampling. Desorption of monoethanolamine was followed by capillary GC analysis and nitrogen phosphorous selective detection. Separation was achieved using a specific column for monoethanolamines (35% diphenyl and 65% dimethyl polysiloxane). The internal standard was quinoline. Derivatization steps were not needed. The calibration range was 0.5-80 μg/mL with a good correlation (R(2) = 0.996). Averaged overall precisions and accuracies were 4.8% and -7.8% for intraday (n = 30), and 10.5% and -5.9% for interday (n = 72). Mean recovery from spiked filters was 92.8% for the intraday variation, and 94.1% for the interday variation. Monoethanolamine on stored spiked filters was stable for at least 4 weeks at 5°C. This newly developed method was used among professional cleaners and air concentrations (n = 4) were 0.42 and 0.17 mg/m(3) for personal and 0.23 and 0.43 mg/m(3) for stationary measurements. The monoethanolamine air concentration method described here was simple, sensitive, and convenient both in terms of sampling and analytical analysis.

Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4.

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

Scoring system for renal pathology in Fabry disease: report of the international study group of fabry nephropathy (ISGFN).

BACKGROUND: In Fabry nephropathy, alpha-galactosidase deficiency leads to accumulation of glycosphingolipids in all kidney cell types, proteinuria and progressive loss of kidney function. METHODS: An international working group of nephrologists from 11 Fabry centres identified adult Fabry patients, and pathologists scored histologic changes on renal biopsies. A standardized scoring system was developed with a modified Delphi technique assessing 59 Fabry nephropathy cases. Each case was scored independently of clinical information by at least three pathologists with an average final score reported. RESULTS: We assessed 35 males (mean age 36.4 years) and 24 females (43.9 years) who mostly had clinically mild Fabry nephropathy. The average serum creatinine was 1.3 mg/dl (114.9 micromol/l); estimated glomerular filtration rate was 81.7 ml/min/1.73 m(2) and urine protein to creatinine ratio was 1.08 g/g (122.0 mg/mmol). Males had greater podocyte vacuolization on light microscopy (mean score) and glycosphingolipid inclusions on semi-thin sections than females. Males also had significantly more proximal tubule, peritubular capillary and vascular intimal inclusions. Arteriolar hyalinosis was similar, but females had significantly more arterial hyalinosis. Chronic kidney disease stage correlated with arterial and glomerular sclerosis scores. Significant changes, including segmental and global sclerosis, and interstitial fibrosis were seen even in patients with stage 1-2 chronic kidney disease with minimal proteinuria. CONCLUSIONS: The development of a standardized scoring system of both disease-specific lesions, i.e. lipid deposition related, and general lesions of progression, i.e. fibrosis and sclerosis, showed a spectrum of histologic appearances even in early clinical stage of Fabry nephropathy. These findings support the role of kidney biopsy in the baseline evaluation of Fabry nephropathy, even with mild clinical disease. The scoring system will be useful for longitudinal assessment of prognosis and responses to therapy for Fabry nephropathy.

Surréanimation liquidienne pré et intrahospitatière des patients brûlés: fréquente et néfaste [Pre- and intra-hospital over-resuscitation in burns: frequent and deleterious].

Major burns are characterized by an initial capillary leak which requires fluid resuscitation for hemodynamic stabilisation. While under-resuscitation was the major cause of death until the 80ies, over-resuscitation has become an important source of complications: abdominal compartment syndrome, escharotomies, impaired gas exchange and prolonged mechanical ventilation and hospital stay. The fluid creep started in the 90ies with an increasing proportion of the first 24 hours' fluid delivery above the 4 ml/kg/% BSA Parkland prediction. The first alerts were published under the form of case reports of increased mortality due to abdominal compartment syndrome and respiratory failure. The paper analyses the causes of this fluid creep, and the ways to prevent it, which includes rationing prehospital fluid delivery, avoiding early colloids and permissive hypovolemia.

Geochemical evolution of active porphyry copper tailings impoundments : from alkaline deposition towards acidification

J. Smuda: Geochemical evolution of active porphyry copper tailings impoundments Thesis abstract Mine waste is the largest volume of materials handled in the world. The oxidation of sulfidic mine waste may result in the release of acid mine drainage (AMD) rich in heavy metals and arsenic to the environment, one of the major problems the mining industry is facing today. To control and reduce this environmental impact, it is crucial to identify the main geochemical and hydrological processes influencing contaminant liberation, transport, and retention. This thesis presents the results of a geochemical, mineralogical and stable isotope study (δ2H, δ18O, δ34S) from two active porphyry copper tailings impoundments in Mediterranean (Carén tailings impoundment, El Teniente mine, Central Chile) and hyper-arid climate (Talabre tailings impoundment, Chuquicamata, Northern Chile) from the deposition in alkaline environment (pH 10.5) towards acidification after several years of exposure. The major hydrological results were the identification of vertical contaminant and water transport in the uppermost, not water-saturated zone, triggered by capillary rise due to evaporation, and infiltration downwards due to new tailings deposition, and of horizontal transport in the groundwater zone. At the surface of the sedimented tailings, evaporation of pore water led to the precipitation of Na-Ca-Mg sulfates (e.g., gypsum, tenorite), in hyper-arid climate also halite. At the Carén tailings impoundment, renewed deposition in a 4-week interval inhibited a pH decrease below neutral values and the formation of an efflorescent salt crust. At the Talabre tailings impoundment, deposition breaks of several years resulted in the formation of acidic oxidation zones in the timeframe of less than 4 years. This process enabled the transport of liberated Cu, Zn, and Fe via capillary rise to the surface, where these metals precipitated as heavy-metal sulfates (e.g., devilline, krohnkite) and chlorides (eriochalcite, atacamite). Renewed depositing may dissolve efflorescent salts and transport liberated elements towards the groundwater zone. This zone was found to be highly dynamic due to infiltration and mixing with water from different sources, like groundwater, catchment water, and infiltration from superficial waters. There, Cu was found to be partially mobile due to complexation with Cl (in Cl-rich groundwater, Talabre) and dissolved organic matter (in zones with infiltration of catchment water rich in dissolved organic matter, Carén). A laboratory study on the isotopic fractionation of sulfur and oxygen of sulfate in different minerals groups (water-soluble sulfates, low- and high-crystalline Fe(III) oxyhydroxides) contributed to the use of stable isotopes as tracer of geochemical and transport processes for environmental studies. The results highlight that a detailed geochemical, stable isotope and mineralogical study permits the identification of contamination processes and pathways already during the deposition of mine tailings. This knowledge allows the early planning of adequate actions to reduce and control the environmental impact during tailings deposition and after the closing of the impoundment. J. Smuda: Geochemical evolution of active porphyry copper tailings impoundments Résumé de these Les déchets miniers constituent les plus grands volumes de matériel gérés dans le monde. L'oxydation des déchets miniers sulfuriques peut conduire à la libération de drainages miniers acides (DMA) riches en métaux et arsenic dans l'environnement, ce qui est l'un des principaux problèmes de l'industrie minière aujourd'hui. Pour contrôler et réduire ces impacts sur l'environnement, il est crucial d'identifier les principaux processus géochimiques et hydrologiques influençant la libération, le transport et la rétention des contaminants. Cette thèse présente les résultats d'une étude géochimique, minéralogique et des isotopes stables (δ2H, δ18O, δ34S) sur des déchets miniers de 2 sites de dépôt actifs en climat méditerranéen (Dépôt de déchets de Carén, mine de El Teniente, Centre du Chili) et en climat hyper-aride (Dépôt de déchets de Talabre, mine de Chuquicamata, Nord du Chili). L'objectif était d'étudier l'évolution des déchets de la déposition en milieu alcalin (pH = 10.5) vers l'acidification après plusieurs années d'exposition. Le principal résultat hydrologique a été l'identification de 2 types de transport : un transport vertical de l'eau et des contaminants dans la zone non saturée en surface, induit par la montée capillaire due à l'évaporation et par l'infiltration subséquente de la déposition de sédiments frais ; et un transport horizontal dans la zone des eaux souterraines. À la surface des déchets, l'évaporation de l'eau interstitielle conduit à la précipitation de sulfates de Na-Ca-Mg (ex. gypse, ténorite) et halite en climat hyper-aride. Dans le site de Carén, une nouvelle déposition de déchets frais à 4 semaines intervalle a empêché la baise du pH en deçà des valeurs neutres et la formation d'une croûte de sels efflorescentes en surface. Dans le site de Talabre, les fentes de dessiccation des dépôts ont entraîné la formation d'une zone d'oxydation à pH acide en moins de 4 ans. Ce processus a permis la libération et le transport par capillarité de Cu, Zn, Fe vers la surface, où ces éléments précipitent sous forme de sulfates de métaux lourds (ex., dévilline, krohnkite) de chlorures (ex. ériochalcite, atacamite). Une nouvelle déposition de sédiments frais pourrait dissoudre ces sels et les transporter vers la zone des eaux souterraines. Cette dernière zone était très dynamique en raison du mélange d'eaux provenant de différentes sources, comme les eaux souterraines, l'eau de captage et l'infiltration des eaux superficielles. Egalement dans cette zone, le cuivre était partiellement mobile à cause de la formation de complexe avec le chlore (dans les zone riche en Cl, Talabre) et avec la matière organique dissoute (dans les zones où s'infiltre l'eau de captage riche en matière organique, Carén). Une étude en laboratoire sur le fractionnement des isotopes stables de sulfure et d'oxygène des sulfates dans différents groupes de minéraux (sulfates hydrosolubles, sulfures de oxy-hydroxyde de Fe(III) faiblement ou fortement cristallins) a permis d'apporter une contribution à leur utilisation comme traceurs dans l'étude des processus géochimiques et de transport lors d'études environnementales. Les résultats montrent qu'une étude détaillée de la géochimie, des isotopes stables et de la minéralogie permet d'identifier les processus et les voies de contamination déjà pendant la période de dépôt des déchets miniers. Cette connaissance permet de planifier, dès le début de l'exploitation, des mesures adéquates pour réduire et contrôler l'impact sur l'environnement pendant la période de dépôts de déchets miniers et après la fermeture du site.

Épidémiologie et physiopathologie de la rétinopathie du prématuré [Epidemiology and pathophysiology of retinopathy of prematurity].

Retinopathy of prematurity (ROP) is a major cause of visual impairment in premature infants. It is characterized by an arrest in normal retinal vascular development associated with microvascular degeneration, followed by an abnormal hypoxiainduced neovascularization. Recent studies point out that ROP is a multifactorial disease, implicating both oxygen-dependent and oxygen-independent mechanisms. Oxygen-dependent factors leading to microvascular degeneration include generation of reactive oxygen species and suppression of specific oxygen-regulated vascular survival factors, such as vascular endothelial growth factor (VEGF) and erythropoietin. The other major mechanism for the initial capillary loss is oxygen-independent and implicates a deficit in growth factor IGF-1/IGFBP3. The proliferative, second phase of ROP is triggered by increases in vascular growth factors concentrations, in an attempt to compensate for the hypoxic retina. Novel signaling pathways for vascular repair, implicating both metabolite signaling and inflammatory lipids signaling, represent new therapeutic avenues for ROP.

Theory of Wetting-Induced Fluid Entrainment by Advancing Contact Lines on Dry Surfaces

We report on the onset of fluid entrainment when a contact line is forced to advance over a dry solid of arbitrary wettability. We show that entrainment occurs at a critical advancing speed beyond which the balance between capillary, viscous, and contact-line forces sustaining the shape of the interface is no longer satisfied. Wetting couples to the hydrodynamics by setting both the morphology of the interface at small scales and the viscous friction of the front. We find that the critical deformation that the interface can sustain is controlled by the friction at the contact line and the viscosity contrast between the displacing and displaced fluids, leading to a rich variety of wetting-entrainment regimes. We discuss the potential use of our theory to measure contact-line forces using atomic force microscopy and to study entrainment under microfluidic conditions exploiting colloid-polymer fluids of ultralow surface tension.

Concrete Pavement Mixture Design and Analysis (MDA): Evaluation of the Fresh and Hardened Properties of Concrete Mixtures Containing Permeability-Reducing Admixtures to Develop Standard Test Protocol, 2014

Concrete durability may be considered as the ability to maintain serviceability over the design life without significant deterioration, and is generally a direct function of the mixture permeability. Therefore, reducing permeability will improve the potential durability of a given mixture and, in turn, improve the serviceability and longevity of the structure. Given the importance of this property, engineers often look for methods that can decrease permeability. One approach is to add chemical compounds known as integral waterproofing admixtures or permeability-reducing admixtures, which help fill and block capillary pores in the paste. Currently, there are no standard approaches to evaluate the effectiveness of permeability-reducing admixtures or to compare different products in the US. A review of manufacturers’ data sheets shows that a wide range of test methods have been used, and rarely are the same tests used on more than one product. This study investigated the fresh and hardened properties of mixtures containing commercially available hydrophilic and hydrophobic types of permeability-reducing admixtures. The aim was to develop a standard test protocol that would help owners, engineers, and specifiers compare different products and to evaluate their effects on concrete mixtures that may be exposed to hydrostatic or non-hydrostatic pressure. In this experimental program, 11 concrete mixtures were prepared with a fixed water-to-cement ratio and cement content. One plain mixture was prepared as a reference, 5 mixtures were prepared using the recommended dosage of the different permeability-reducing admixtures, and 5 mixtures were prepared using double the recommended dosage. Slump, air content, setting time, compressive and flexural strength, shrinkage, and durability indicating tests including electrical resistivity, rapid chloride penetration, air permeability, permeable voids, and sorptivity tests were conducted at various ages. The data are presented and recommendations for a testing protocol are provided.

Determination and Evaluation of Alternate Methods for Managing and Controlling Highway-Related Dust, TR-449, 2002

Road dust is caused by wind entraining fine material from the roadway surface and the main source of Iowa road dust is attrition of carbonate rock used as aggregate. The mechanisms of dust suppression can be considered as two processes: increasing particle size of the surface fines by agglomeration and inhibiting degradation of the coarse material. Agglomeration may occur by capillary tension in the pore water, surfactants that increase bonding between clay particles, and cements that bind the mineral matter together. Hygroscopic dust suppressants such as calcium chloride have short durations of effectiveness because capillary tension is the primary agglomeration mechanism. Somewhat more permanent methods of agglomeration result from chemicals that cement smaller particles into a mat or larger particles. The cements include lignosulfonates, resins, and asphalt products. The duration of the cements depend on their solubility and the climate. The only dust palliative that decreases aggregate degradation is shredded shingles that act as cushions between aggregate particles. It is likely that synthetic polymers also provide some protection against coarse aggregate attrition. Calcium chloride and lignosulfonates are widely used in Iowa. Both palliatives have a useful duration of about 6 months. Calcium chloride is effective with surface soils of moderate fine content and plasticity whereas lignin works best with materials that have high fine content and high plasticity indices. Bentonite appears to be effective for up to two years and works well with surface materials having low fines and plasticity and works well with limestone aggregate. Selection of appropriate dust suppressants should be based on characterization of the road surface material. Estimation of dosage rates for potential palliatives can be based on data from this report, from technical reports, information from reliable vendors, or laboratory screening tests. The selection should include economic analysis of construction and maintenance costs. The effectiveness of the treatment should be evaluated by any of the field performance measuring techniques discussed in this report. Novel dust control agents that need research for potential application in Iowa include; acidulated soybean oil (soapstock), soybean oil, ground up asphalt shingles, and foamed asphalt. New laboratory evaluation protocols to screen additives for potential effectiveness and determine dosage are needed. A modification of ASTM D 560 to estimate the freeze-thaw and wet-dry durability of Portland cement stabilized soils would be a starting point for improved laboratory testing of dust palliatives.

Authentication of vegetable oils by bulk and molecular carbon isotope analyses with emphasis on olive oil and pumpkin seed oil

The authenticity of vegetable oils consumed in Slovenia and Croatia was investigated by carbon isotope analysis of the individual fatty acids by the use of gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS), and through carbon isotope analysis of the bulk oil. The fatty acids from samples of olive, pumpkin, sunflower, maize, rape, soybean, and sesame oils were separated by alkaline hydrolysis and derivatized to methyl esters for chemical characterization by capillary gas chromatography/mass spectrometry (GC/MS) prior to isotopic analysis. Enrichment in heavy carbon isotope (C-13) of th, bulk oil and of the individual fatty acids are related to (1) a thermally induced degradation during processing (deodorization, steam washing, or bleaching), (2) hydrolytic rancidity (lipolysis) and oxidative rancidity of the vegetable oils during storage, and (3) the potential blend with refined oil or other vegetable oils. The impurity or admixture of different oils may be assessed from the delta C-13(16:0) VS. delta C-13(18:1) covariations. The fatty acid compositions of Slovenian and Croatian olive oils are compared with those from the most important Mediterranean producer countries (Spain, Italy, Greece, and France).

Carbonate Rock Pore Size Distribution Determination through Iowa Pore Index Testing, MLR-15-01, 2015

The Iowa Pore Index (IPI) measures the pore system of carbonate (limestone and dolomite) rocks using pressurized water to infiltrate the pore system. This technique provides quantitative results for the primary and capillary (secondary) pores in carbonate rocks. These results are used in conjunction with chemical and mineralogical test results to calculate a quality number, which is used as a predictor of aggregate performance in Portland cement concrete (PCC) leading to the durability classification of the aggregate. This study had two main objectives: to determine the effect different aggregate size has on IPI test results and to establish the precision of IPI test and test apparatus. It was found that smaller aggregate size fractions could be correlated to the standard 1/2”-3/4” size sample. Generally, a particle size decrease was accompanied by a slight decrease in IPI values. The IPI testing also showed fairly good agreement of the secondary pore index number between the 1/2”-3/4”and the 3/8”-1/2” fraction. The #4-3/8” showed a greater difference of the secondary number from the 1/2”-3/4” fraction. The precision of the IPI test was established as a standard deviation (Sr) of 2.85 (Primary) and 0.87 (Secondary) with a repeatability limit (%r) of 8.5% and 14.9% for the primary and secondary values, respectively.

Effects of erythropoietin on muscle O2 transport during exercise in patients with chronic renal failure.

Erythropoietin (rHuEPO) has proven to be effective in the treatment of anemia of chronic renal failure (CRF). Despite improving the quality of life, peak oxygen uptake after rHuEPO therapy is not improved as much as the increase in hemoglobin concentration ([Hb)] would predict. We hypothesized that this discrepancy is due to failure of O2 transport rates to rise in a manner proportional to [Hb]. To test this, eight patients with CRF undergoing regular hemodialysis were studied pre- and post-rHuEPO ([Hb] = 7.5 +/- 1.0 vs. 12.5 +/- 1.0 g x dl-1) using a standard incremental cycle exercise protocol. A group of 12 healthy sedentary subjects of similar age and anthropometric characteristics served as controls. Arterial and femoral venous blood gas data were obtained and coupled with simultaneous measurements of femoral venous blood flow (Qleg) by thermodilution to obtain O2 delivery and oxygen uptake (VO2). Despite a 68% increase in [Hb], peak VO2 increased by only 33%. This could be explained largely by reduced peak leg blood flow, limiting the gain in O2 delivery to 37%. At peak VO2, after rHuEPO, O2 supply limitation of maximal VO2 was found to occur, permitting the calculation of a value for muscle O2 conductance from capillary to mitochondria (DO2). While DO2 was slightly improved after rHuEPO, it was only 67% of that of sedentary control subjects. This kept maximal oxygen extraction at only 70%. Two important conclusions can be reached from this study. First, the increase in [Hb] produced by rHuEPO is accompanied by a significant reduction in peak blood flow to exercising muscle, which limits the gain in oxygen transport. Second, even after restoration of [Hb], O2 conductance from the muscle capillary to the mitochondria remains considerably below normal.