Delivery of nitric oxide for analysis of the function of cytochrome c'

On delivery of nitric oxide (NO) to protein samples (e.g., cytochrome c'), for spectroscopic experiments it is important to avoid exposure to oxygen and to remove contaminants from the NO gas. We describe a number of techniques for steady-state UV/Vis spectrophotometry and pre-steady-state stopped-flow spectrophotometry analysis of cytochrome c'.

A FimH inhibitor prevents acute bladder infection and treats chronic cystitis caused by multidrug-resistant uropathogenic Escherichia coli ST131

Background. Escherichia coli O25b:H4-ST131 represents a predominant clone of multidrug-resistant uropathogens currently circulating worldwide in hospitals and the community. Urinary tract infections (UTIs) caused by E. coli ST131 are typically associated with limited treatment options and are often recurrent. Methods. Using established mouse models of acute and chronic UTI, we mapped the pathogenic trajectory of the reference E. coli ST131 UTI isolate, strain EC958. Results. We demonstrated that E. coli EC958 can invade bladder epithelial cells and form intracellular bacterial communities early during acute UTI. Moreover, E. coli EC958 persisted in the bladder and established chronic UTI. Prophylactic antibiotic administration failed to prevent E. coli EC958–mediated UTI. However, 1 oral dose of a small-molecular-weight compound that inhibits FimH, the type 1 fimbriae adhesin, significantly reduced bacterial colonization of the bladder and prevented acute UTI. Treatment of chronically infected mice with the same FimH inhibitor lowered their bladder bacterial burden by >1000-fold. Conclusions. In this study, we provide novel insight into the pathogenic mechanisms used by the globally disseminated E. coli ST131 clone during acute and chronic UTI and establish the potential of FimH inhibitors as an alternative treatment against multidrug-resistant E. coli.

Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA

We report that tumor cells devoid of their mitochondrial genome (mtDNA) show delayed tumor growth and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory supercomplexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest a novel pathophysiological process for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.

Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.

School connectedness: Student voices examine power and subjectivity

Ideally, school would be a place where all students felt that they belonged. However, the reality is that many students feel as though they do not belong to their school community. Alienated or disaffected students are an endemic problem in schools in Australia, affecting the whole school community, as well as life chances for the students themselves after school. The crux of this matter, we believe, are the tensions between the desire to connect to the school community, and the frustration experienced by some students as a result of their subjectification by the school system. Perhaps students that we tend to identify as alienated or disaffected in their schools may be resisting the accepted negotiations of power that underpin the school system.

A Chlamydia trachomatis strain with a chemically generated amino acid substitution (P370L) in the cthtrA gene shows reduced elementary body production

Background Chlamydia (C.) trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Genetic approaches to investigate C. trachomatis have been only recently developed due to the organism’s intracellular developmental cycle. HtrA is a critical stress response serine protease and chaperone for many bacteria and in C. trachomatis has been previously shown to be important for heat stress and the replicative phase of development using a chemical inhibitor of the CtHtrA activity. In this study, chemically-induced SNVs in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterized. Methods SNVs were initially biochemically characterized in vitro using recombinant protein techniques to confirm a functional impact on proteolysis. The C. trachomatis strains containing the SNVs with marked reductions in proteolysis were investigated in cell culture to identify phenotypes that could be linked to CtHtrA function. Results The strain harboring the SNV with the most marked impact on proteolysis (cthtrAP370L) was detected to have a significant reduction in the production of infectious elementary bodies. Conclusions This provides genetic evidence that CtHtrA is critical for the C. trachomatis developmental cycle.

An analysis of ethical issues in using wastewater analysis to monitor illicit drug use

Aims To discuss ethical issues that may arise in using WWA to monitor illicit drug use in the general population and in entertainment precincts, prisons, schools and work-places. Method Review current applications of WWA and identify ethical and social issues that may be raised with current and projected future uses of this method. Results Wastewater analysis (WWA) of drug residues is a promising method of monitoring illicit drug use that may overcome some limitations of other monitoring methods. When used for monitoring purposes in large populations, WWA does not raise major ethical concerns because individuals are not identified and the prospects of harming residents of catchment areas are remote. When WWA is used in smaller catchment areas (entertainment venues, prisons, schools or work-places) their results could, possibly, indirectly affect the occupants adversely. Researchers will need to take care in reporting their results to reduce media misreporting. Fears about possible use of WWA for mass individual surveillance by drug law enforcement officials are unlikely to be realized, but will need to be addressed because they may affect public support adversely for this type of research. Conclusions Using wastewater analysis to monitor illicit drug use in large populations does not raise major ethical concerns, but researchers need to minimize possible adverse consequences in studying smaller populations, such as workers, prisoners and students.

Preliminary study of the effects of carbon nanotubes on Cpu cooling

Managing heat produced by computer processors is an important issue today, especially when the size of processors is decreasing rapidly while the number of transistors in the processor is increasing rapidly. This poster describes a preliminary study of the process of adding carbon nanotubes (CNTs) to a standard silicon paste covering a CPU. Measurements were made in two rounds of tests to compare the rate of cool-down with and without CNTs present. The silicon paste acts as an interface between the CPU and the heat sink, increasing the heat transfer rate away from the CPU. To the silicon paste was added 0.05% by weight of CNTs. These were not aligned. A series of K-type thermocouples was used to measure the temperature as a function of time in the vicinity of the CPU, following its shut-off. An Omega data acquisition system was attached to the thermocouples. The CPU temperature was not measured directly because attachment of a thermocouple would have prevented its automatic shut-off A thermocouple in the paste containing the CNTs actually reached a higher temperature than the standard paste, an effect easily explained. But the rate of cooling with the CNTs was about 4.55% better.

Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

A quantitative survey of the benthic infauna of northern Monterey Bay, California: Annual report, Part 8, 1973

(PDF contains 241 pages)

Population response of triploid grass carp to declining levels of hydrilla in the Santee Cooper Reservoirs, South Carolina

Approximately 768,500 triploid grass carp ( Ctenopharyngodon idella Valenciennes) were stocked into the Santee Cooper reservoirs, South Carolina between 1989 and 1996 to control hydrilla ( Hydrilla verticillata (L.f.) Royle). Hydrilla coverage was reduced from a high of 17,272 ha during 1994 to a few ha by 1998. During 1997, 1998 and 1999, at least 98 triploid grass carp were collected yearly for population monitoring. Estimates of age, growth, and mortality, as well as population models, were used in the study to monitor triploid grass carp and predict population trends. Condition declined from that measured during a previous study in 1994. The annual mortality rate was estimated at 28% in 1997, 32% in 1998 and 39% in 1999; however, only the 1999 mortality rate was significantly different. Few (2 out of 98) of the triploid grass carp collected during 1999 were older than age 9. We expect increased mortality due to an aging population and sparse hydrilla coverage. During 1999, we estimated about 63,000 triploid grass carp system wide and project less than 3,000 fish by 2004, assuming no future stocking. management, population size Ctenopharyngodon idella, Hydrilla

A schematic analysis and discussion of species taken by otter trawl net by the research vessel Orion from July, 1965 to June 1966 in the Chesapeake Bay and major tributaries

From July 1965 to June 1964 the Natural Resources Institute's Research Vessel ORION took 16 minute tows with a forty (40) foot otter trawl net at 38 selected locations in Chesapeake Bay from the south of the Potomac River to Turkey Point at the head of the Bay and including some tributaries. Shallow and deep hauls were taken at most stations with depths ranging from 5 to 140 feet. A schematic summary of the 54 different species caught was compared with "Fishes of the Chesapeake Bay" by S. F. Hildebrand and W. C. Schroeder. Sixteen species including five not contained in the above references were selected for discussion. (PDF contains 21 pages)

Accelerating loss of seagrasses across the globe threatens coastal ecosystems

Coastal ecosystems and the services they provide are adversely affected by a wide variety of human activities. In particular, seagrass meadows are negatively affected by impacts accruing from the billion or more people who live within 50 km of them. Seagrass meadows provide important ecosystem services, including an estimated $1.9 trillion per year in the form of nutrient cycling; an order of magnitude enhancement of coral reef fish productivity; a habitat for thousands of fish, bird, and invertebrate species; and a major food source for endangered dugong, manatee, and green turtle. Although individual impacts from coastal development, degraded water quality, and climate change have been documented, there has been no quantitative global assessment of seagrass loss until now. Our comprehensive global assessment of 215 studies found that seagrasses have been disappearing at a rate of 110 square kilometers per year since 1980 and that 29% of the known areal extent has disappeared since seagrass areas were initially recorded in 1879. Furthermore, rates of decline have accelerated from a median of 0.9% per year before 1940 to 7% per year since 1990. Seagrass loss rates are comparable to those reported for mangroves, coral reefs, and tropical rainforests and place seagrass meadows among the most threatened ecosystems on earth.

Patterns of succession in benthic infaunal communities following dredging and dredged material disposal in Monterey Bay

This report is the final product of a two-year study conducted for the Office, Chief of Engineers, by the Moss Landing Marine Laboratories, Moss Landing, California, under Contract No. DACW39-74-C-OI51 with the Environmental Effects Laboratory (EEL), U. S. Army Engineer Waterways Experiment Station (WES), Yicksburg, Mississippi. (PDF contains 192 pages)

A Baseline Study of the Moss Landing/Elkhorn Slough Environment

In July, 1974 we began a baseline study of the Moss Landing-Elkhorn Slough marine environment for PG&E as mandated by the Coastal Commission. This report constitutes results of the first year's program. It is divided into three sections, oceanography, benthic invertebrate ecology, and fish and zooplankton ecology. (PDF contains 226 pages)