Disinfection of bacterial biofilms in pilot-scale cooling tower systems

The impact of continuous chlorination and periodic glutaraldehyde treatment on planktonic and biofilm microbial communities was evaluated in pilot-scale cooling towers operated continuously for 3 months. The system was operated at a flow rate of 10,080 l day(-1). Experiments were performed with a well-defined microbial consortium containing three heterotrophic bacteria: Pseudomonas aeruginosa, Klebsiella pneumoniae and Flavobacterium sp. The persistence of each species was monitored in the recirculating cooling water loop and in biofilms on steel and PVC coupons in the cooling tower basin. The observed bacterial colonization in cooling towers did not follow trends in growth rates observed under batch conditions and, instead, reflected differences in the ability of each organism to remain attached and form biofilms under the high-through flow conditions in cooling towers. Flavobacterium was the dominant organism in the community, while P. aeruginosa and K. pneumoniae did not attach well to either PVC or steel coupons in cooling towers and were not able to persist in biofilms. As a result, the much greater ability of Flavobacterium to adhere to surfaces protected it from disinfection, whereas P. aeruginosa and K. pneumoniae were subject to rapid disinfection in the planktonic state.     

Retinoic acid metabolism blocking agents (RAMBAs) for treatment of cancer and dermatological diseases

The naturally occurring retinoids and their synthetic analogs play a key role in differentiation, proliferation, and apoptosis, and their use/potential in oncology, dermatology and a variety of diseases are well documented. This review focuses on the role of all-trans-retinoic acid (ATRA), the principal endogenous metabolite of vitamin A (retinol) and its metabolism in oncology and dermatology. ATRA has been used successfully in differentiated therapy of acute promyelocytic leukemia, skin cancer, Kaposi's sarcoma, and cutaneous T-cell lymphoma, and also in the treatment of acne and psoriasis. However, its usefulness is limited by the rapid emergence of acquired ATRA resistance involving multifactoral mechanisms. A key mechanism of resistance involves ATRA-induced catabolism of ATRA. Thus, a novel strategy to overcome the limitation associated with exogenous ATRA therapy has been to modulate and/or increase the levels of endogenous ATRA by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzymes (particularly CYP26s) responsible for ATRA metabolism. These inhibitors are also referred to as retinoic acid metabolism blocking agents (RAMBAs). This review highlights development in the design, synthesis, and evaluation of RAMBAs. Major emphasis is given to liarozole, the most studied and only RAMBA in clinical use and also the new RAMBAs in development and with clinical potential.     

Potent anti-prostate cancer agents derived from a novel androgen receptor down-regulating agent

The search for novel androgen receptor (AR) down-regulating agents by catalyst HipHop pharmacophore modeling led to the discovery of some lead molecules. Unexpectedly, the effect of these leads on human prostate cancer LNCaP cell viability did not correlate with the ability of the compounds to cause down-regulation of AR protein expression. Through rational synthetic optimization of the lead compound (BTB01434), we have discovered a series of novel substituted diaryl molecules as potent anti-prostate cancer agents. Some compounds (1-6) were shown to be extremely potent inhibitors of LNCaP cell viability with GI(50) values in the nanomolar range (1.45-83 nM). The most potent compound (4-methylphenyl)[(4-methylphenyl)sulfonyl]amine (5) with a GI(50) value of 1.45 nM is 27,000 times more potent than our lead compound BTB01434 (GI(50)=39.8 microM). In addition, some of the compounds exhibited modest anti-androgenic activities and one was also a potent inhibitor (GI(50)=850 nM) of PC-3 (AR-null) cell growth. A clear structure-activity relationship (SAR) has been established for activity against LNCaP cells, where potent molecules possess two substituted/unsubstituted aromatic rings connected through a sulfonamide linker. These novel compounds are strong candidates for development for the treatment of hormone-sensitive and importantly hormone-refractory prostate cancers in humans.     

Identification of senescence-associated genes from daylily petals

The petals of daylily (Hemerocallis hybrid) have a genetically based program that leads to senescence and cell death ca. 24 h after the flower opens. In order to determine the components of this program, six cDNAs, whose levels increase during petal senescence, were isolated and sequenced and designated DSA3, 4, 5, 6, 12 and 15. All six DSAs are members of gene families and all but DSA5 and DSA6 have one to three other very similar genes. GenBank database homology searches indicate that DSA3 is most similar at the amino acid level to an in-chain fatty acid hydroxylase which is bound to cytochrome P450, DSA4 may be an aspartic proteinase, DSA5 is as yet unidentified, DSA6 is a putative S1-type nuclease, DSA12 is very similar to a cytochrome P450-containing allene oxide synthase, and DSA15 may be a fatty acid elongase. Except for DSA12, the genes are expressed at low levels in daylily roots. Levels of the DSA mRNAs in leaves are less than 4% of the maximum detected in petals, and there are no clear differences between younger and older leaves. With the exception of DSA4, accumulation of the DSA mRNAs is increased 3.2 to 43 times by a concentration of abscisic acid that causes premature senescence of the petals. The relationship of the putative DSA gene products to senescence and cell death of daylily petals is discussed.     

Effect of Haemoproteus belopolskyi (Haemosporida: Haemoproteidae) on body mass of the blackcap Sylvia atricapilla

The effect of initial Haemoproteus belopolskyi infection on the weight of its natural host, the blackcap Sylvia atricapilla, was investigated. Fourteen blackcap nestlings were taken at the age of 4-5 days and raised by hand in the laboratory. They were free of blood parasites. Seven 20- to 21-day-old nestlings were infected experimentally by inoculation in their pectoral muscle with approximately 45 sporozoites, which had developed in the experimentally infected biting midge Culicoides impunctatus. Seven nestlings were used as negative controls. Parasitemia developed in 6 inoculated nestlings, with a prepatent period of 11-12 days. No infections were detected in the controls during this study. The weight of experimentally infected and control birds was measured 2 days before parasitemia became patent and for a 45-day period after patency. Blood smears were prepared from all birds on the days when they were weighed. When compared with controls, there was a significant weight loss of experimentally infected blackcaps during 6 days after the decline of parasitemia at 10-16 days of patency, indicating a short-term influence of the infection on the birds' body mass. Clinical symptoms of the infection were not recorded. All birds from both groups survived until the end of the experiment.     

A novel planar flow cell for studies of biofilm heterogeneity and flow-biofilm interactions

Biofilms are microbial communities growing on surfaces, and are ubiquitous in nature, in bioreactors, and in human infection. Coupling between physical, chemical, and biological processes is known to regulate the development of biofilms; however, current experimental systems do not provide sufficient control of environmental conditions to enable detailed investigations of these complex interactions. We developed a novel planar flow cell that supports biofilm growth under complex two-dimensional fluid flow conditions. This device provides precise control of flow conditions and can be used to create well-defined physical and chemical gradients that significantly affect biofilm heterogeneity. Moreover, the top and bottom of the flow chamber are transparent, so biofilm growth and flow conditions are fully observable using non-invasive confocal microscopy and high-resolution video imaging. To demonstrate the capability of the device, we observed the growth of Pseudomonas aeruginosa biofilms under imposed flow gradients. We found a positive relationship between patterns of fluid velocity and biofilm biomass due to faster microbial growth under conditions of greater local nutrient influx, but this relationship eventually reversed because high hydrodynamic shear leads to the detachment of cells from the surface. These results reveal that flow gradients play a critical role in the development of biofilm communities. By providing new capability for observing biofilm growth, solute and particle transport, and net chemical transformations under user-specified environmental gradients, this new planar flow cell system has broad utility for studies of environmental biotechnology and basic biofilm microbiology, as well as applications in bioreactor design, environmental engineering, biogeochemistry, geomicrobiology, and biomedical research.     

Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

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Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain.

New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost-effectiveness of the process. Spider venom is one of many potential sources of novel insect-specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin-1 from the wolf spider were produced in high throughput using an automated integrated plasmid-based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin-1 open reading frames (ORF) in a novel small ubiquitin-like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so-called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin alpha-helix to interact with insect cell membranes or to increase its pore-forming ability, leading to cell lysis. The toxin peptides have potential as value-added coproducts to increase the cost-effectiveness of fuel ethanol bioproduction. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanism of stimulation of plus-strand synthesis by an RNA replication enhancer in a tombusvirus

Replication of RNA viruses is regulated by cis-acting RNA elements, including promoters, replication silencers, and replication enhancers (REN). To dissect the function of an REN element involved in plus-strand RNA synthesis, we developed an in vitro trans-replication assay for tombusviruses, which are small plus-strand RNA viruses. In this assay, two RNA strands were tethered together via short complementary regions with the REN present in the nontemplate RNA, whereas the promoter was located in the template RNA. We found that the template activity of the tombusvirus replicase preparation was stimulated in trans by the REN, suggesting that the REN is a functional enhancer when located in the vicinity of the promoter. In addition, this study revealed that the REN has dual function during RNA synthesis. (i) It binds to the viral replicase. (ii) It interacts with the core plus-strand initiation promoter via a long-distance RNA-RNA interaction, which leads to stimulation of initiation of plus-strand RNA synthesis by the replicase in vitro. We also observed that this RNA-RNA interaction increased the in vivo accumulation and competitiveness of defective interfering RNA, a model template. We propose that REN is important for asymmetrical viral RNA replication that leads to more abundant plus-strand RNA progeny than the minus-strand intermediate, a hallmark of replication of plus-strand RNA viruses.     

Effects of itaconic acid on neuronal viability and brain mitochondrial functions

Recent studies have identified that under stimulation by bacterial lipopolysaccharide mammalian macrophages produce itaconic acid. Yet, it is unknown whether itaconate has any effect on viability of brain cells. Here we used extracellularly added itaconate to investigate its effects on viability of cerebellar granule cells (CGC) in cultures and respiratory functions of these cells and isolated brain mitochondria. We found that 3–5 mM itaconate had no effect on the viability of neurons, but 10 mM itaconate was toxic and induced neuronal apoptosis. Removal of itaconate after 24 h incubation resulted in further decrease in viability and number of neurons. Respiration of intact neurons was not affected by itaconate, but permeabilized cells as well as isolated brain mitochondria demonstrated decreased rates of respiration in the presence of itaconate. Using isolated adult rat brain mitochondria we found that itaconate decreased mitochondrial phosphorylating respiration, mitochondrial calcium retention capacity, production of reactive oxygen species with Complex I and Complex II substrates as well as inhibition of Complex I, Complex IV and ATP synthase. In conclusion, the results suggest that itaconic acid at millimolar concentrations affects mitochondrial functions and viability of neurons.