Identification of Plk1 type II inhibitors by structure-based virtual screening

Protein kinases are targets for drug development. Dysregulation of kinase activity leads to various diseases, e.g. cancer, inflammation, diabetes. Human polo-like kinase 1 (Plk1), a serine/threonine kinase, is a cancer-relevant gene and a potential drug target which attracts increasing attention in the field of cancer therapy. Plk1 is a key player in mitosis and modulates entry into mitosis and the spindle checkpoint at the meta-/anaphase transition. Plk1 overexpression is observed in various human tumors, and it is a negative prognostic factor for cancer patients. The same catalytical mechanism and the same co-substrate (ATP) lead to the problem of inhibitor selectivity. A strategy to solve this problem is represented by targeting the inactive conformation of kinases. Kinases undergo conformational changes between active and inactive conformation and thus an additional hydrophobic pocket is created in the inactive conformation where the surrounding amino acids are less conserved. A "homology model" of the inactive conformation of Plk1 was constructed, as the crystal structure in its inactive conformation is unknown. A crystal structure of Aurora A kinase served as template structure. With this homology model a receptor-based pharmacophore search was performed using SYBYL7.3 software. The raw hits were filtered using physico-chemical properties. The resulting hits were docked using Gold3.2 software, and 13 candidates for biological testing were manually selected. Three compounds of the 13 tested exhibit anti-proliferative effects in HeLa cancer cells. The most potent inhibitor, SBE13, was further tested in various other cancer cell lines of different origins and displayed EC50 values between 12 microM and 39 microM. Cancer cells incubated with SBE13 showed induction of apoptosis, detected by PARP (Poly-Adenosyl-Ribose-Polymerase) cleavage, caspase 9 activation and DAPI staining of apoptotic nuclei

The Role of Fog in the Hydrological Functioning of Tropical Island Ecosystems

Fog is a critical water source in many tropical ecosystems, especially those that are semi-arid, or seasonally dry. Patterns of fog water input to these ecosystems are poorly understood, and currently limited by a lack of in-situ data spanning both space and time. Large gaps exist in our understanding of the spatiotemporal variability and mechanisms driving fog water deposition, and how fog travels through tropical systems. Given the significance of fog to semi-arid ecosystems across the globe, I use stable isotopes, remote sensing and plant physiological analyses to examine the role of fog in the semi-arid ecosystems of San Cristóbal Island, Galápagos and Ascension Island, UK by utilizing data from four field campaigns. I first create a ground-based optical fog detection scheme to trace fine-temporal scale mechanisms driving fog formation, evolution and dissipation across varying hydroclimatic zones. I then establish the isotopic signature of fog and other environmental waters to assess the overall contribution of fog to different microclimatic zones and under different hydroclimatic regimes. And finally, I trace fog through the Galápagos ecosystem, specifically examining how native versus invasive flora utilize fog under varying hydroclimatic conditions. In this research, I create a simple model to predict degree-of-fogginess with commonly measured meteorological variables, and I show how different fog formation mechanisms can be taking place in tandem over a concentrated spatial scale. I also demonstrate that fog is a common phenomenon on San Cristóbal Island, especially during the dry season, and that fog is consistently enriched compared to co-collected rainfall. Finally, I utilize the isotopic signature of fog and other environmental waters to trace water sources through the Galápagos ecosystem, suggesting that invasive guava’s water use strategy (including fog water utilization) plays a key role in its competitive capacity versus co-occurring native plants. Through this island lens, I address the critical disconnect between the hydrological and ecological role that fog plays in tropical island ecosystems. Taken together, these findings will be critical in projecting future water resource availability across many seasonally arid ecosystems, especially those that may become drier under future regimes of climate change.Doctor of Philosoph

Veganism

This flyer examines the ecological effect of the vegan diet and compares it against omnivorous and vegetarian diets

Investigation of the Role of Tensile Forces in Cellular Response to Chemotherapeutic Agents

Research using in vitro cell cultures are frequently conducted under static growth conditions. Cells growing in vivo, however, grow in a highly dynamic and interactive environment, where they receive cues in the form of mechanical stimuli from their surroundings. Lung cells, for example, are continually exposed in vivo to a cyclic tensile stretch during normal inhalation and exhalation. The absence of a mechanically representative environment could have important implications for research and development, particularly in the context of drug discovery. We hypothesize that tensile (mechanical) forces applied to two non-small cell lung cancer cell lines, bronchoalveolar H358 cells and alveolar A549 cells, play an important role in determining cellular response to chemotherapeutic agents. In order to investigate changes resulting from exposure to tensile stretch, we first looked at changes in proliferation and expression of a few cellular markers associated with epithelial-mesenchymal transition (EMT). Next, we looked at changes in cell cycle distribution and expression of a few cell-cycle checkpoint proteins. Finally, we studied the effect of a tensile force on the efficacy of three chemotherapeutic agents. We found that a tensile force significantly reduces cellular proliferation and causes significant shifts in cell cycle distribution. Mechanically active culture environments led to decreased efficacy of cisplatin and increased efficacy of Zactima. These results indicate that a mechanically active culture environment does impact cell survival and protein expression, and has important implications in the context of the discovery and screening of new antitumor drug therapies

DNA microarray-based characterization and antimicrobial resistance phenotypes of clinical MRSA strains from animal hosts

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe infections in humans and animals worldwide. Studies elucidating the population structure, staphylococcal cassette chromosome mec types, resistance phenotypes, and virulence gene profiles of animal-associated MRSA are needed to understand spread and transmission. Objectives: The objective of this study was to determine 1) clonal complexes and spa types, 2) resistance phenotypes, and 3) virulence/resistance gene profiles of MRSA isolated from animals in Switzerland. Methods: We analyzed 31 presumptive MRSA isolates collected from clinical infections in horses, dogs, cattle, sheep, and pigs, which had tested positive in the Staphaurex Latex Agglutination Test. The isolates were characterized by spa typing and DNA microarray profiling. In addition, we performed antimicrobial susceptibility testing using the VITEK 2 Compact system. Results: Characterization of the 31 presumptive MRSA isolates revealed 3 methicillin-resistant Staphylococcus pseudintermedius isolates, which were able to grow on MRSA2 Brilliance agar. Of the 28 MRSA isolates, the majority was assigned to CC398 (86%), but CC8 (11%) and CC1 (4%) were also detected. The predominant spa type was t011 (n = 23), followed by t009 (n = 2), t034 (n = 1), t008 (n = 1), and t127 (n = 1). Conclusions: The results of this study extend the current body of knowledge on the population structure, resistance phenotypes, and virulence and resistance gene profiles of MRSA from livestock and companion animal

A novel multiplex qPCR targeting 23S rDNA for diagnosis of swine dysentery and porcine intestinal spirochaetosis

Figure S1. Consensus sequence alignment of the target DNA region within 23S ribosomal DNA. Primers (Brachy primer for. and Brachy primer rev.) on the target DNA are marked in grey. The probe for B. hyodysenteriae (Probe_hyo) is highlighted in yellow, the probe for B. pilosicoli (Probe_pilo) in purple, and the probe for the B. intermedia/B. innocens/B. murdochii (probe inter) in green. Differences in single residues are marked in red. (PDF 112 kb

The bactericidal effect of two photoactivated chromophore for keratitis-corneal crosslinking protocols (standard vs. accelerated) on bacterial isolates associated with infectious keratitis in companion animals

Background: Bacterial corneal infections are common and potentially blinding diseases in all species. As antibiotic resistance is a growing concern, alternative treatment methods are an important focus of research. Photoactivated chromophore for keratitis-corneal crosslinking (PACK-CXL) is a promising oxygen radical-mediated alternative to antibiotic treatment. The main goal of this study was to assess the anti-bactericidal efficacy on clinical bacterial isolates of the current standard and an accelerated PACK-CXL treatment protocol delivering the same energy dose (5.4 J/cm$^{2}$). Methods: Clinical bacterial isolates from 11 dogs, five horses, one cat and one guinea pig were cultured, brought into suspension with 0.1% riboflavin and subsequently irradiated. Irradiation was performed with a 365 nm UVA light source for 30 min at 3mW/cm$^{2}$ (standard protocol) or for 5 min at 18mW/cm$^{2}$ (accelerated protocol), respectively. After treatment, the samples were cultured and colony forming units (CFU’s) were counted and the weighted average mean of CFU’s per μl was calculated. Results were statistically compared between treated and control samples using a linear mixed effects model. Results: Both PACK-CXL protocols demonstrated a significant bactericidal effect on all tested isolates when compared to untreated controls. No efficacy difference between the two PACK-CXL protocols was observed. Conclusion: The accelerated PACK-CXL protocol can be recommended for empirical use in the treatment of bacterial corneal infections in veterinary patients while awaiting culture results. This will facilitate immediate treatment, the delivery of higher fluence PACK-CXL treatment within a reasonable time, and minimize the required anesthetic time or even obviate the need for general anesthesia

Lake Site Assessments: US EPA TIME-New England Lakes

TIME (Temporally Integrated Monitoring of Ecosystems) is a statistically selected population of lakes in New Eng- land and the Hudson Valley (31 lakes) and the Adirondacks (43 lakes) that were selected from the original 1991 EMAP-SW (Environmental Monitoring and Assessment Program–Surface Waters) population with acid neutralizing capacity less than 100 meq/L (Young & Stoddard 1996). Samples are taken annually, during a summer base-flow ‘index period’. This sampling strategy is used to reduce hydrologic impact on water chemistry and hence provide an assessment of trends in chemistry with the least number of samples (e.g., Stoddard et al. 2003). The EMAP program sampled these lakes and many others one or more times between 1991-1994. As part of EMAP, the lakes were characterized with respect to landscape features, hydrology, geology, and chemistry as well as biological studies (fish, breeding birds, zooplankton) and a paleo-limnological coring study to reconstruct pH and other variables. The program was discontinued, but some sampling of the lakes continued through other funding sources during the hiatus. In 1999, the TIME project officially began, with a goal of assessing the effectiveness of the Clean Air Act Amendments of 1990 at reducing acidification of surface waters (Stoddard et al. 2003, Kahl et al. 2004). As of this writing, the lakes have records spanning two decades or more

Susceptibility Provision Enhances Effective De-escalation (SPEED): utilizing rapid phenotypic susceptibility testing in Gram-negative bloodstream infections and its potential clinical impact

Abstract Objectives We evaluated the performance and time to result for pathogen identification (ID) and antimicrobial susceptibility testing (AST) of the Accelerate Pheno™ system (AXDX) compared with standard of care (SOC) methods. We also assessed the hypothetical improvement in antibiotic utilization if AXDX had been implemented. Methods Clinical samples from patients with monomicrobial Gram-negative bacteraemia were tested and compared between AXDX and the SOC methods of the VERIGENE® and Bruker MALDI Biotyper® systems for ID and the VITEK® 2 system for AST. Additionally, charts were reviewed to calculate theoretical times to antibiotic de-escalation, escalation and active and optimal therapy Results ID mean time was 21 h for MALDI-TOF MS, 4.4 h for VERIGENE® and 3.7 h for AXDX. AST mean time was 35 h for VITEK® 2 and 9.0 h for AXDX. For ID, positive percentage agreement was 95.9% and negative percentage agreement was 99.9%. For AST, essential agreement was 94.5% and categorical agreement was 93.5%. If AXDX results had been available to inform patient care, 25% of patients could have been put on active therapy sooner, while 78% of patients who had therapy optimized during hospitalization could have had therapy optimized sooner. Additionally, AXDX could have reduced time to de-escalation (16 versus 31 h) and escalation (19 versus 31 h) compared with SOC. Conclusions By providing fast and reliable ID and AST results, AXDX has the potential to improve antimicrobial utilization and enhance antimicrobial stewardship