Synthetic Lethal Targeting of Superoxide Dismutase 1 Selectively Kills RAD54B-Deficient Colorectal Cancer Cells

Synthetic lethality is a rational approach to identify candidate drug targets for selective killing of cancer cells harboring somatic mutations that cause chromosome instability (CIN). To identify a set of the most highly connected synthetic lethal partner genes in yeast for subsequent testing in mammalian cells, we used the entire set of 692 yeast CIN genes to query the genome-wide synthetic lethal datasets. Hierarchical clustering revealed a highly connected set of synthetic lethal partners of yeast genes whose human orthologs are somatically mutated in colorectal cancer. Testing of a small matrix of synthetic lethal gene pairs in mammalian cells suggested that members of a pathway that remove reactive oxygen species that cause DNA damage would be excellent candidates for further testing. We show that the synthetic lethal interaction between budding yeast rad54 and sod1 is conserved within a human colorectal cancer context. Specifically, we demonstrate RAD54B-deficient cells are selectively killed relative to controls via siRNA-based silencing and chemical inhibition and further demonstrate that this interaction is conserved in an unrelated cell type. We further show that the DNA double strand breaks, resulting from increased reactive oxygen species following SOD1 inhibition, persist within the RAD54B-deficient cells and result in apoptosis. Collectively, these data identify SOD1 as a novel candidate cancer drug target and suggest that SOD1 inhibition may have broad-spectrum applicability in a variety of tumor types exhibiting RAD54B deficiencies.     

The influence of exogenous carbohydrate provision and pre-exercise alkalosis on the heat shock protein response to prolonged interval cycling

The aim of this study was to observe the intracellular heat shock protein 72 (HSP72) and heme oxygenase-1 (HSP32) response to prolonged interval cycling following the ingestion of carbohydrates (CHO) and sodium bicarbonate (NaHCO₃). Six recreationally active males (mean ± SD; age 23.2 ± 2.9 years, height 179.5 ± 5.5 cm, body mass 76.5 ± 6.8 kg, and peak power output 315 ± 36 W) volunteered to complete a 90 min interval cycling exercise on four occasions. The trials were completed in a random and blinded manner following ingestion of either: placebo and an artificial sweetener (P–P), NaHCO₃ and sweetener (B–P), placebo and CHO (P–CHO), and NaHCO₃ and CHO (B–CHO). Both HSP72 and HSP32 were significantly increased in monocytes and lymphocytes from 45 min post-exercise (p ≤ 0.039), with strong relationships between both cell types (HSP72, r = 0.83; HSP32, r = 0.89). Exogenous CHO had no influence on either HSP72 or HSP32, but the ingestion of NaHCO₃ significantly attenuated HSP32 in monocytes and lymphocytes (p ≤ 0.042). In conclusion, the intracellular stress protein response to 90 min interval exercise is closely related in monocytes and lymphocytes, and HSP32 appears to be attenuated with a pre-exercise alkalosis.     

Influence of a Polyphenol-Enriched Protein Powder on Exercise-Induced Inflammation and Oxidative Stress in Athletes: A Randomized Trial Using a Metabolomics Approach

Objectives

Polyphenol supplementation was tested as a countermeasure to inflammation and oxidative stress induced by 3-d intensified training.

Methods

Water soluble polyphenols from blueberry and green tea extracts were captured onto a polyphenol soy protein complex (PSPC). Subjects were recruited, and included 38 long-distance runners ages 19–45 years who regularly competed in road races. Runners successfully completing orientation and baseline testing (N = 35) were randomized to 40 g/d PSPC (N = 17) (2,136 mg/d gallic acid equivalents) or placebo (N = 18) for 17 d using double-blinded methods and a parallel group design, with a 3-d running period inserted at day 14 (2.5 h/d, 70% VO_2max). Blood samples were collected pre- and post-14 d supplementation, and immediately and 14 h after the third day of running in subjects completing all aspects of the study (N = 16 PSPC, N = 15 placebo), and analyzed using a metabolomics platform with GC-MS and LC-MS.

Results

Metabolites characteristic of gut bacteria metabolism of polyphenols were increased with PSPC and 3 d running (e.g., hippurate, 4-hydroxyhippurate, 4-methylcatechol sulfate, 1.8-, 1.9-, 2.5-fold, respectively, P<0.05), an effect which persisted for 14-h post-exercise. Fatty acid oxidation and ketogenesis were induced by exercise in both groups, with more ketones at 14-h post-exercise in PSPC (3-hydroxybutyrate, 1.8-fold, P<0.05). Established biomarkers for inflammation (CRP, cytokines) and oxidative stress (protein carbonyls) did not differ between groups.

Conclusions

PSPC supplementation over a 17-d period did not alter established biomarkers for inflammation and oxidative stress but was linked to an enhanced gut-derived phenolic signature and ketogenesis in runners during recovery from 3-d heavy exertion.

Trial Registration

ClinicalTrials.gov, U.S. National Institutes of Health, identifier: {"type":"clinical-trial","attrs":{"text":"NCT01775384","term_id":"NCT01775384"}}NCT01775384     

Fluorescence detection of intracellular cadmium with Leadmium Green

Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd²⁺) and lead (Pb²⁺). Leadmium Green has been used in various paradigms, such as tracking Cd²⁺ sequestration in plant cells, heavy metal export in protozoa, and Pb²⁺ absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd²⁺, Pb²⁺, and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer’s claims, Leadmium Green was sensitive to Cd²⁺ (K_D ~600 nM) and also Pb²⁺ (K_D ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca²⁺, Co²⁺, Mn²⁺ and Ni²⁺. Leadmium Green also responded to Zn²⁺ with a K_D of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd²⁺ or Zn²⁺ in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn²⁺ was much less than that produced by Cd²⁺. When tested in a model of oxidant-induced liberation of endogenous Zn²⁺, Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd²⁺, particularly in models where Cd²⁺ accumulates rapidly, and when concomitant fluctuations of intracellular Zn²⁺ are minimal.     

Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.