Transport behavior of the pharmaceutical compounds carbamazepine, sulfamethoxazole, gemfibrozil, ibuprofen, and naproxen, and the lifestyle drug caffeine, in saturated laboratory columns.

Despite the large number of pharmaceutically active compounds found in natural environments little is known about their transport behavior in groundwater, which is complicated by their wide range of physical and chemical properties. The transport behavior of five widely used and often detected pharmaceutical compounds and one lifestyle drug has therefore been investigated, using a set of three column experiments. The investigated compounds were the anticonvulsant carbamazepine, the lifestyle drug caffeine, the antibiotic sulfamethoxazole, the lipid regulator gemfibrozil, and the nonsteroidal anti-inflammatories ibuprofen and naproxen. The columns were filled with three different types of sand. The substrates consisted of artificially prepared iron-coated sand, artificially prepared organic carbon sand (with 5% leaf compost), and natural aquifer sand from Long Point, Ontario (Canada). The experiments were conducted simultaneously under the same hydraulic conditions and with the same input solution of about 1µg·L-1 of each compound. The transport behavior of the organic compounds differed significantly between both the different columns and the different compounds. A strong correlation was observed between the retardation factors for carbamazepine, gemfibrozil, and ibuprofen and the organic carbon content of the substrate. While the retardation increased with increasing organic carbon content, no direct relationship was observed between the organic carbon content and the removal of these compounds. In contrast, the retardation factors for sulfamethoxazole and naproxen showed no correlation with the organic carbon content but these compounds were significantly removed in the presence of organic matter. The influence of the Fe3+ surfaces in the iron-coated sand was less significant than expected, with all compounds except for sulfamethoxazole having retardation factors <1.8. Caffeine was so strongly removed during transport through those substrates containing organic carbon that no reliable retardation factor could be determined.

Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks.

Topoisomerase II is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating transient double-strand DNA breaks. While the enzyme cannot perform its essential cellular functions without cleaving DNA, this scission activity is inherently dangerous to chromosomal integrity. In fact, etoposide and other clinically important anticancer drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. Cells rely heavily on recombination to repair double-strand DNA breaks, but the specific pathways used to repair topoisomerase II-generated DNA damage have not been defined. Therefore, Saccharomyces cerevisiae was used as a model system to delineate the recombination pathways that repair DNA breaks generated by topoisomerase II. Yeast cells that expressed wild-type or a drug-hypersensitive mutant topoisomerase II or overexpressed the wild-type enzyme were examined. Based on cytotoxicity and recombination induced by etoposide in different repair-deficient genetic backgrounds, double-strand DNA breaks generated by topoisomerase II appear to be repaired primarily by the single-strand invasion pathway of homologous recombination. Non-homologous end joining also was triggered by etoposide treatment, but this pathway was considerably less active than single-strand invasion and did not contribute significantly to cell survival in S.cerevisiae.

Screening and optimization of chemically defined media and feeds with integrated and statistical approaches.

The majority of therapeutic proteins are expressed in mammalian cells, predominantly in Chinese Hamster Ovary cells. While cell culture media and feed supplements are crucial to protein productivity, medium optimization can be labor intensive and time-consuming. In this chapter, we describe some basic concepts in medium development and introduce a rational and rapid workflow to screen and optimize media and feeds. The major goal of medium screening is to select a base formulation as the foundation for further optimization, but ironically, the most conventional screening method may actually rule out ideal chemically defined medium candidates. Appropriate cell adaptation is the key to identifying an optimal base medium, particularly when cells were originally cultured in serum-free medium containing recombinant proteins and/or undefined hydrolysates. The efficient workflow described herein integrates the optimization of both medium and feed simultaneously using a Design-of-Experiment (DOE) approach. The feasibility of the workflow is then demonstrated with a case study, in which chemically defined medium and feed were optimized in a single fed-batch study using a high-throughput microbioreactor system (SimCell™), which resulted in improving protein titers three- to sixfold.

Acesulfame-K and pharmaceuticals as co-tracers of municipal wastewater in a receiving river.

Wastewater treatment plant (WWTP) effluents are important sources of emerging contaminants at environmentally-relevant concentrations. In this study, water samples were collected from a river downstream of two WWTPs to identify practical tracers for tracking wastewater. The results of the study indicate elevated concentrations of Cl(-), nutrients (NH3-N and NO2(-)), the artificial sweetener acesulfame-K (ACE-K), and the pharmaceuticals carbamazepine (CBZ), caffeine (CAF), sulfamethoxazole (SMX), ibuprofen (IBU), gemfibrozil (GEM), and naproxen (NAP) in the river close to the WWTPs that decreased with distance downstream. A correlation analysis using the Spearman Rank method showed that ACE-K, CBZ, GEM, NAP, and Cl(-) were strongly correlated with each other over a 31 km stretch of the river in the study area. The strong correlations of these target compounds indicate that the artificial sweetener ACE-K and the pharmaceuticals CBZ, GEM, and NAP can potentially be used as co-tracers to track wastewater.

Reduced Rif2 and lack of Mec1 target short telomeres for elongation rather than double-strand break repair.

Telomerase in Saccharomyces cerevisiae binds and preferentially elongates short telomeres, and this process requires the checkpoint kinase Tel1. Here we show that the Mre11 complex bound preferentially to short telomeres, which could explain the preferential binding of Tel1 to these ends. Compared to wild-type length telomeres, short telomeres generated by incomplete replication had low levels of the telomerase inhibitory protein Rif2. Moreover, in the absence of Rif2, Tel1 bound equally well to short and wild-type length telomeres, suggesting that low Rif2 content marks short telomeres for preferential elongation. In congenic strains, a double-strand break bound at least 140 times as much Mec1 in the first cell cycle after breakage as did a short telomere in the same time frame. Binding of replication protein A was also much lower at short telomeres. The absence of Mec1 at short telomeres could explain why they do not trigger a checkpoint-mediated cell-cycle arrest.

ATM-like kinases and regulation of telomerase: lessons from yeast and mammals.

Telomeres, the essential structures at the ends of eukaryotic chromosomes, are composed of G-rich DNA and asociated proteins. These structures are crucial for the integrity of the genome, because they protect chromosome ends from degradation and distinguish natural ends from chromosomal breaks. The complete replication of telomeres requires a telomere-dedicated reverse transcriptase called telomerase. Paradoxically, proteins that promote the very activities against which telomeres protect, namely DNA repair, recombination and checkpoint activation, are integral to both telomeric chromatin and telomere elongation. This review focuses on recent findings that shed light on the roles of ATM-like kinases and other checkpoint and repair proteins in telomere maintenance, replication and checkpoint signaling.

Telomerase and Tel1p preferentially associate with short telomeres in S. cerevisiae.

In diverse organisms, telomerase preferentially elongates short telomeres. We generated a single short telomere in otherwise wild-type (WT) S. cerevisiae cells. The binding of the positive regulators Ku and Cdc13p was similar at short and WT-length telomeres. The negative regulators Rif1p and Rif2p were present at the short telomere, although Rif2p levels were reduced. Two telomerase holoenzyme components, Est1p and Est2p, were preferentially enriched at short telomeres in late S/G2 phase, the time of telomerase action. Tel1p, the yeast ATM-like checkpoint kinase, was highly enriched at short telomeres from early S through G2 phase and even into the next cell cycle. Nonetheless, induction of a single short telomere did not elicit a cell-cycle arrest. Tel1p binding was dependent on Xrs2p and required for preferential binding of telomerase to short telomeres. These data suggest that Tel1p targets telomerase to the DNA ends most in need of extension.

A flexible protein linker improves the function of epitope-tagged proteins in Saccharomyces cerevisiae.

Epitope tagging permits the detection of proteins when protein-specific antibodies are not available. However, the epitope tag can reduce the function of the tagged protein. Here we describe a cassette that can be used to introduce an eight amino acid flexible linker between multiple Myc epitopes and the open reading frame of a given gene. We show that inserting the linker improves the in vivo ability of the telomerase subunits Est2p and Est1p to maintain telomere length. The methods used here are generally applicable to improve the function of tagged proteins in both Saccharomyces cerevisiae and Schizosaccharomyces pombe.