Trichloroethylene biodegradation by mesophilic and psychrophilic ammonia oxidizers and methanotrophs in groundwater microcosms.

This study investigated the efficiency of methane and ammonium for stimulating trichloroethylene (TCE) biodegradation in groundwater microcosms (flasks and batch exchange columns) at a psychrophilic temperature (12 degrees C) typical of shallow aquifers in the northern United States or a mesophilic temperature (24 degrees C) representative of most laboratory experiments. After 140 days, TCE biodegradation rates by ammonia oxidizers and methanotrophs in mesophilic flask microcosms were similar (8 to 10 nmol day-1), but [14C]TCE mineralization (biodegradation to 14CO2) by ammonia oxidizers was significantly greater than that by methanotrophs (63 versus 53%). Under psychrophilic conditions, [14C]TCE mineralization in flask systems by ammonia oxidizers and methanotrophs was reduced to 12 and 5%, respectively. In mesophilic batch exchange columns, average TCE biodegradation rates for methanotrophs (900 nmol liter-1 day-1) were not significantly different from those of ammonia oxidizers (775 nmol liter-1 day-1). Psychrophilic TCE biodegradation rates in the columns were similar with both biostimulants and averaged 145 nmol liter-1 day-1. Methanotroph biostimulation was most adversely affected by low temperatures. At 12 degrees C, the biodegradation efficiencies (TCE degradation normalized to microbial activity) of methanotrophs and ammonia oxidizers decreased by factors of 2.6 and 1.6, respectively, relative to their biodegradation efficiencies at 24 degrees C. Collectively, these experiments demonstrated that in situ bioremediation of TCE is feasible at the psychrophilic temperatures common in surficial aquifers in the northern United States and that for such applications biostimulation of ammonia oxidizers could be more effective than has been previously reported.     

Induction of aquaporin-1 mRNA following cardiopulmonary bypass and reperfusion.

BACKGROUND: Cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA) are important components of congenital cardiac surgery. Ischemia/reperfusion injury and inflammatory cascade activation result in endothelial damage and vascular leak which are clinically manifested as pulmonary edema and low cardiac output postoperatively. Newborns are particularly susceptible. Subtraction cloning is a useful method of isolating induced genes and can be applied to CPB/HCA. MATERIALS AND METHODS: We used a newborn lamb model replicating infant CPB with HCA to obtain tissues during various periods of reperfusion. We utilized subtraction cloning to identify mRNA induced in lung following CPB/HCA and reperfusion. Ribonuclease protection was used to quantify mRNA levels. RESULTS: We isolated a cDNA encoding ovine aquaporin-1 in a subtracted cDNA screen comparing control lung with lung exposed to CPB/HCA and reperfusion. Aquaporin-1 mRNA levels increased 3-fold in lung (p = .006) exposed to CPB/HCA and 6 hr of reperfusion. No induction was observed immediately following bypass or after 3 hr of reperfusion. We found no significant induction of aquaporin-1 mRNA following bypass, arrest, and reperfusion in other tissues surveyed, including ventricle, atrium, skeletal muscle, kidney, brain, and liver. CONCLUSIONS: Our finding that aquaporin-1 mRNA is reproducibly induced in lung following CPB/HCA with 6 hr of reperfusion suggests an important role for the water channel in the setting of pulmonary edema. Induction of Aquaporin-1 is late compared with other inflammatory mediators (ICAM-1, E-selectin, IL-8). Further studies are needed to determine if aquaporin-1 contributes to the disease process or if it is part of the recovery phase.     

Behaviour of DNA,protein and acid hydrolases in response to thyroxine in isolated tail tips ofXenopus-larvae

Summary Analysis of factors influencing survival of tail tips ofXenopus larvae in vitro has shown that prevention of infection by antibiotic pretreatment of donor tadpoles and amputated tips is most critical. In tail tips exceeding not more than 1/3 of the body length, maintenance of N-content and regenerative capacity are superior in Niu-Twitty and Steinberg's saline than in Holtfreter's solution, all media being supplemented by 0.04 % sulfathiazole. Addition of nutrient ingredients to saline (glucose, Parker's medium 199, serum protein) does not improve viability of tail explants.In isolated tail tips 2.5×10^–7M L-thyroxine (T₄) induces involution in vitro, resulting in losses of about 85% in DNA and 70% of protein respectively after 12 days of treatment. A significant decrease in DNA occurs after three days, and for protein after 6 days of hormone treatment, when tail fins are almost fully resorbed.Statistical analysis of the regression curves for decrease in DNA and protein, produced by different concentrations of T₄, indicates the presence of an upper threshold (2.5×10^–7M) and a lower threshold (2.5×10^–8M) of sensitivity to hormone. Intermediate concentrations affect the latency time required for onset of DNA and protein loss, lengthening it at lower concentration.In tail tips exposed to 2.5×10^–7M T₄ a concurrent rise in activity of cathepsins, DNase and acid phosphatase has been demonstrated. The specific activities of these acid hydrolases are significantly higher in T₄-treated tails after four days of hormone administration, this response proceeding detectable loss in protein by two days. Extent and duration in the rise of activity are characteristic for each enzyme, the increase in both specific and total activities being highest for cathepsins, intermediate for DNase and lowest for acid phosphatase.

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Zusammenfassung Die Untersuchung der für das Überleben von isolierten Schwanzspitzen vonXenopus-Larven wesentlichen Kulturbedingungen hat ergeben, daß die Verhütung von Infektionen durch Vorbehandlung der Kaulquappen und der amputierten Schwanzspitzen für den Erfolg entscheidend ist. Schwanzspitzen, deren Länge 1/3 der Körperlänge der Kaulquappen nicht überschreitet, zeigen einen geringeren Verlust an N-haltigen Stoffen und ein besseres Regenerationsvermögen in Niu-Twitty- und Steinberglösung gegenüber der Lösung nach Holtfreter, obwohl alle Medien 0,04% Sulfathiazol enthalten. Zusatz von Nährstoffen (Glucose, Parker's Medium 199, Serumprotein) ergeben keine Verbesserung der Überlebensfähigkeit.In isolierten Schwanzspitzen bewirken 2,5×10^–7M L-Thyroxin (T₄) die Rückbildung in vitro, wobei im Verlaufe von 12 Tagen der DNS-Gehalt um 85% und derjenige an Protein um 70% sinkt. Der DNS-Verlust ist bereits nach 3 Tagen signifikant, während eine signifikante Abnahme an Protein erst nach 6 Tagen Hormonbehandlung nachgewiesen werden kann, wenn die Flossensäume bereits resorbiert sind.Aufgrund der statistischen Analyse der für den DNS- und Proteinverlust in Abhängigkeit von verschiedenen T₄-Konzentrationen ermittelten Regressionsgeraden ergeben sich folgende Aussagen: 2,5×10^–7 M repräsentiert die obere und 2,5×10^–8 M T₄ die untere Empfindlichkeitsschwelle. Innerhalb dieses Bereiches zeigt die Latenzzeit für den Beginn der DNS -und Proteinabnahme, nicht aber die Geschwindigkeit des Involutionsprozesses eine Konzentrationsabhängigkeit, indem die Latenzzeit mit fallender T₄-Konzentration zunimmt.Unter der Einwirkung von 2,5×10^–7 M T₄ nimmt in den Schwanzspitzen die Aktivität von Kathepsinen, DNase und saurer Phosphatase gleichzeitig zu. Die spezifische Aktivität dieser sauren Hydrolasen ist bereits nach 4 Tagen Hormonbehandlung signifikant erhöht, während ein signifikanter Proteinverlust erst am 6. Tag der Behandlung nachgewiesen werden kann. Ausmaß und Dauer der Aktivitätszunahme sind für jedes Enzym charakteristisch; die Zunahme der spezifischen und Gesamtaktivität ist am größten bei den Kathepsinen, etwas geringer bei der DNase und am niedrigsten bei der sauren Phosphatase.

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These studies were carried out within the scope of a project supported by the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung under the direction of Prof. B. Weber.

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The author would like to acknowledge the advice and encouragement of Prof. R. Weber in all phases of these studies, and the assistance of Prof. S. Rosin with the statistical techniques. Further, the technical assistance of Miss A. Rohner, Miss B. Fahrer, Mr. J. Zbären, and Mr. T. Wyler is gratefully acknowledged.     

NanoMethViz: An R/Bioconductor package for visualizing long-read methylation data

A key benefit of long-read nanopore sequencing technology is the ability to detect modified DNA bases, such as 5-methylcytosine. The lack of R/Bioconductor tools for the effective visualization of nanopore methylation profiles between samples from different experimental groups led us to develop the NanoMethViz R package. Our software can handle methylation output generated from a range of different methylation callers and manages large datasets using a compressed data format. To fully explore the methylation patterns in a dataset, NanoMethViz allows plotting of data at various resolutions. At the sample-level, we use dimensionality reduction to look at the relationships between methylation profiles in an unsupervised way. We visualize methylation profiles of classes of features such as genes or CpG islands by scaling them to relative positions and aggregating their profiles. At the finest resolution, we visualize methylation patterns across individual reads along the genome using the spaghetti plot and heatmaps, allowing users to explore particular genes or genomic regions of interest. In summary, our software makes the handling of methylation signal more convenient, expands upon the visualization options for nanopore data and works seamlessly with existing methylation analysis tools available in the Bioconductor project. Our software is available at https://bioconductor.org/packages/NanoMethViz.     

Characterization of the bovine ampkγ1 gene

AMP-activated protein kinase (AMPK) represents the mammalian form of the core component of a kinase cascade that is conserved between fungi, plants, and animals. AMPK plays a major role in protecting mammalian cells from metabolic stress by switching off biosynthetic pathways that require ATP and switching on ATP-regenerating pathways. In this report, we describe the isolation and characterization of the gene for the noncatalytic bovine gamma1 subunit of AMPK. The bovine ampkgamma1 (PRKAG1) gene spans in excess of 14 kb and is located at BTA 5q21-q22. It consists of 12 exons ranging in size from 38 b to 166 b, interspersed with 11 introns that range between 97 b and 6753 b in length. The coding region of the bovine gene shares 93% and 90% nucleotide sequence similarity with its human and rat counterparts, and the bovine AMPKgamma1 protein is 98% and 95% identical to its human and rat homologs, respectively, in amino acid sequence. SNP discovery using a cattle DNA panel revealed a number of polymorphisms that may be useful for the evaluation of ampkgamma1 as a candidate gene for energy metabolism-related production traits.     

Systematics and Leaf Architecture of the Gunneraceae

Cladistic and phenetic analyses of leaf and other morphological characters ofGunnera strongly support monophyly of the genus, with the Saxifragaceae s.str. as the closest sister group. This morphologically based phylogeny provides a more coherent understanding of the evolutionary history ofGunnera than do recent phylogenetic hypotheses based on genetic data sets with Myrothamnaceae as the sister group. Simple, crenate, palinactinodromously veined leaves lacking freely ending veinlets and tricolpate, tectate-perforate pollen with a reticulate exine indicate a shared ancestry. Within the genusGunnera all six traditionally recognized subgenera are monophyletic, as supported by leaf architectural apomorphies. The monotypic subgenusOstenigunnera is the sister group to the other five subgenera, which can be divided into two principal lineages. One lineage includes the subgeneraMilligania andMisandra, characterized by a prostate stoloniferous habit with small, low-rank leaves and exclusively unisexual flowers, whereas the other lineage includes the subgeneraPerpensum, Pseudo Gunnera, andPanke, all of which possess at least some hermaphroditic flowers and larger, high-rank leaves. When the phylogeny of the subgenera is considered in light of biogeography and the fossil record, a number of cladogenetic events can be explained by continental vicariance in the Late Cretaceous. The AfricanPerpensum became distinct from the other large-leafed lineage with the separation of the African continent ca. 90 Ma. The two small-leafed lineages, the subgeneraMilligania andMisandra, split with the separation of New Zealand from Western Gondwana, about 80 Ma.Pseudo-Gunnera became isolated fromPanke prior to this time, whenPanke fossils occur in North America.Gunnera probably arose out of an early herbaceous radiation of tricolpate eudicots having close affinity to the basal Saxifragaceae, espethe genusChrysosplenium.     

HOPS proofreads the trans-SNARE complex for yeast vacuole fusion

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the K(m) value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Q(c)). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Q(a)) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.     

Differential mass spectrometry of rat plasma reveals proteins that are responsive to 17beta-estradiol and a selective estrogen receptor modulator PPT

Estrogens are a class of steroid hormones that interact with two related but distinct nuclear receptors, estrogen receptor (ER) alpha and beta. To identify potential ER biomarkers, we profiled the rat plasma glycoproteome after treatment with vehicle or 17beta-estradiol (E2) or an ERalpha-selective agonist PPT by differential mass spectrometry. Our comparative proteomic experiment identifies novel E2- and PPT-responsive proteins, such as serine protease inhibitor family members.