Nutrigenomics of hepatic steatosis in a feline model: effect of monosodium glutamate, fructose, and Trans-fat feeding

Nonalcoholic fatty liver disease begins with a relatively benign hepatic steatosis, often associated with increased adiposity, but may progress to a more severe nonalcoholic steatohepatitis with inflammation. A subset of these patients develops progressive fibrosis and ultimately cirrhosis. Various dietary components have been shown to contribute to the development of liver disease, including fat, sugars, and neonatal treatment with high doses of monosodium glutamate (MSG). However, rodent models of progressive disease have been disappointing, and alternative animal models of diet-induced liver disease would be desirable, particularly if they contribute to our knowledge of changes in gene expression as a result of dietary manipulation. The domestic cat has previously been shown to be an appropriate model for examining metabolic changes–associated human diseases such as diabetes. Our aim was therefore to compare changes in hepatic gene expression induced by dietary MSG, with that of a diet containing Trans-fat and high fructose corn syrup (HFCS), using a feline model. MSG treatment increased adiposity and promoted hepatic steatosis compared to control (P < 0.05). Exposure to Trans-fat and HFCS promoted hepatic fibrosis and markers of liver dysfunction. Affymetrix microarray analysis of hepatic gene expression showed that dietary MSG promoted the expression of genes involved in cholesterol and steroid metabolism. Conversely, Trans-fat and HFCS feeding promoted the expression of genes involved in lipolysis, glycolysis, liver damage/regeneration, and fibrosis. Our feline model examining gene–diet interactions (nutrigenomics) demonstrates how dietary MSG, Trans-fat, and HFCS may contribute to the development of hepatic steatosis.     

A rapid,cost-effective procedure for the extraction of microbial DNA from soil

Here we describe a DNA extraction method that is based on a simple, rapid polyvinylpolypyrrolidone–calcium chloride precipitation to release microorganisms from the soil combined with lysozyme–proteinase–SDS lysis of the microbial community. The extracted DNA is of high quality and allows direct detection of specific genes by the polymerase chain reaction (PCR) as well as cloning of indigenous microbial DNA. This method facilitates the extraction of 36 500-mg soil samples simultaneously in a 2-h period by one person. The procedure is safe, inexpensive, and does not require specialized equipment or generate hazardous wastes.     

Genetic Heterogeneity in Bacillus sporothermodurans as Demonstrated by Ribotyping and Repetitive Extragenic Palindromic-PCR Fingerprinting

Thirty-eight strains of Bacillus sporothermodurans isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the PvuII and EcoRI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that B. sporothermodurans is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of B. sporothermodurans that can be found in UHT-treated or sterilized milk products.     

Parallel medicinal chemistry approaches to selective HDAC1/HDAC2 inhibitor (SHI-1:2) optimization

The successful application of both solid and solution phase library synthesis, combined with tight integration into the medicinal chemistry effort, resulted in the efficient optimization of a novel structural series of selective HDAC1/HDAC2 inhibitors by the MRL-Boston Parallel Medicinal Chemistry group. An initial lead from a small parallel library was found to be potent and selective in biochemical assays. Advanced compounds were the culmination of iterative library design and possess excellent biochemical and cellular potency, as well as acceptable PK and efficacy in animal models.     

A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8⁺ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8⁺ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8⁺ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies.     

Contextualization of drug-mediator relations using evidence networks

Background

Genomic analysis of drug response can provide unique insights into therapies that can be used to match the “right drug to the right patient.” However, the process of discovering such therapeutic insights using genomic data is not straightforward and represents an area of active investigation. EDDY (Evaluation of Differential DependencY), a statistical test to detect differential statistical dependencies, is one method that leverages genomic data to identify differential genetic dependencies. EDDY has been used in conjunction with the Cancer Therapeutics Response Portal (CTRP), a dataset with drug-response measurements for more than 400 small molecules, and RNAseq data of cell lines in the Cancer Cell Line Encyclopedia (CCLE) to find potential drug-mediator pairs. Mediators were identified as genes that showed significant change in genetic statistical dependencies within annotated pathways between drug sensitive and drug non-sensitive cell lines, and the results are presented as a public web-portal (EDDY-CTRP). However, the interpretability of drug-mediator pairs currently hinders further exploration of these potentially valuable results.

Methods

In this study, we address this challenge by constructing evidence networks built with protein and drug interactions from the STITCH and STRING interaction databases. STITCH and STRING are sister databases that catalog known and predicted drug-protein interactions and protein-protein interactions, respectively. Using these two databases, we have developed a method to construct evidence networks to “explain” the relation between a drug and a mediator. 

Results

We applied this approach to drug-mediator relations discovered in EDDY-CTRP analysis and identified evidence networks for ~70% of drug-mediator pairs where most mediators were not known direct targets for the drug. Constructed evidence networks enable researchers to contextualize the drug-mediator pair with current research and knowledge. Using evidence networks, we were able to improve the interpretability of the EDDY-CTRP results by linking the drugs and mediators with genes associated with both the drug and the mediator.

Conclusion

We anticipate that these evidence networks will help inform EDDY-CTRP results and enhance the generation of important insights to drug sensitivity that will lead to improved precision medicine applications.

     

Imaging mass spectrometry of intact proteins from alcohol-preserved tissue specimens: bypassing formalin fixation

Imaging mass spectrometry is becoming a key technology for the investigation of the molecular content of biological tissue sections in direct correlation with the underlying histology. Much of our work has been done with fresh-frozen tissue sections that has undergone minimal protein degradation between the time a tissue biopsy is sampled and the time it is snap-frozen so that no preserving or fixing agents need to be added to the frozen biopsy. However, in many sampling environments, immediate flash freezing may not be possible and so we have explored the use of ethanol-preserved, paraffin-embedded tissue specimens for proteomic analyses. Solvent-only preserved tissue specimens provide long-term preservation at room temperature, generation of high quality histological sections and little if any chemical alteration of the proteins. Using mouse organs, several key steps involved in the tissue dehydration process have been investigated to assess the potential of such preserved specimens for profiling and imaging mass spectrometry investigations.     

Pacing pattern and physiological responses to a 5-minute maximal exercise bout

The purpose of this study was to describe the pacing strategy of experienced cyclists in a 5-minute maximal exercise bout and to describe selected physiological responses associated with this effort. Six experienced and well-trained competitive cyclists (five males, one female) with a mean (+/-SD) age, height, and mass of 27.0 +/- 4.77 years, 174.7 +/- 8.57 cm, and 71.0 +/- 6.45 kg, performed a 5-minute maximal exercise bout in a laboratory on a racing cycle. Subjects were free to determine their work rate throughout. During exercise, data were collected for work rate, heart rate (HR), [latin capital V with dot above]O2, electromyography of the rectus femoris and vastus lateralis, oxygen saturation, and rating of perceived exertion. All six subjects selected a pacing strategy characterized by a surge in work rate in the first minute followed by a gradual decline until the last minute, when a sprint to the end occurred. Values for HR, respiratory exchange ratio, and blood lactate concentration (182.8 +/- 2.8 bpm, 1.08 +/- 0.07, and 15.5 +/- 2.1 mmol x L-1, respectively) indicated that [latin capital V with dot above]O2 (3.6 +/- 0.4 L x min-1) was close to or at maximum from minutes 2 to 5. Oxygen saturation dropped continuously across time, reaching <94% in the last minute, and rating of perceived exertion was 19.5 +/- 0.8. Electromyographic activity of the rectus femoris and vastus lateralis was not significantly related to work rate during the bout (p > 0.05). It is concluded that work rate or pace is uneven in an all-out, 5-minute exercise bout in experienced cyclists, yet the physiological responses are near maximal in minutes 2-5. Cyclists seem to pace themselves in a common pattern in short-term stochastic exercise bouts. The possible benefits of including some stochastic exercise in the training programs of athletes might be worthy of examination.     

SAR profiles of spirocyclic nicotinamide derived selective HDAC1/HDAC2 inhibitors (SHI-1:2)

A potent family of spirocyclic nicotinyl aminobenzamide selective HDAC1/HDAC2 inhibitors (SHI-1:2) is profiled. The incorporation of a biaryl zinc-binding motif into a nicotinyl scaffold resulted in enhanced potency and selectivity versus HDAC3, but also imparted hERG activity. It was discovered that increasing polar surface area about the spirocycle attenuates this liability. Compound 12 induced a 4-fold increase in acetylated histone H2B in an HCT-116 xenograft model study with acute exposure, and inhibited tumor growth in a 21-day efficacy study with qd dosing.