The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans

FKBP5 encodes FK506-binding protein 5, a glucocorticoid receptor (GR)-binding protein implicated in various psychiatric disorders and alcohol withdrawal severity. The purpose of this study is to characterize alcohol preference and related phenotypes in Fkbp5 knockout (KO) mice and to examine the role of FKBP5 in human alcohol consumption. The following experiments were performed to characterize Fkpb5 KO mice. (1) Fkbp5 KO and wild-type (WT) EtOH consumption was tested using a two-bottle choice paradigm; (2) The EtOH elimination rate was measured after intraperitoneal (IP) injection of 2.0 g/kg EtOH; (3) Blood alcohol concentration (BAC) was measured after 3 h limited access of alcohol; (4) Brain region expression of Fkbp5 was identified using LacZ staining; (5) Baseline corticosterone (CORT) was assessed. Additionally, two SNPs, rs1360780 (C/T) and rs3800373 (T/G), were selected to study the association of FKBP5 with alcohol consumption in humans. Participants were college students (n = 1162) from 21–26 years of age with Chinese, Korean or Caucasian ethnicity. The results, compared to WT mice, for KO mice exhibited an increase in alcohol consumption that was not due to differences in taste sensitivity or alcohol metabolism. Higher BAC was found in KO mice after 3 h of EtOH access. Fkbp5 was highly expressed in brain regions involved in the regulation of the stress response, such as the hippocampus, amygdala, dorsal raphe and locus coeruleus. Both genotypes exhibited similar basal levels of plasma corticosterone (CORT). Finally, single nucleotide polymorphisms (SNPs) in FKBP5 were found to be associated with alcohol drinking in humans. These results suggest that the association between FKBP5 and alcohol consumption is conserved in both mice and humans.     

Capture‐Tag‐Release: A Strategy for Small Molecule Labeling of Native Enzymes

A strategy for labeling native enzymes in a manner that preserves their activity is reported: capture–tag–release (CTR). Key to this approach is the small molecule CTR probe that contains an enzyme inhibitor, benzophenone crosslinker, and aryl phosphine ester. After UV‐derived capture of the enzyme, addition of an azide‐containing tag triggers a Staudinger ligation that labels the enzyme. A further consequence of the Staudinger ligation is fragmentation of the CTR probe, thus releasing the inhibitor and restoring enzymatic activity. As a proof‐of‐principle, the CTR strategy was applied to the hydrolase β‐galactosidase. The enzyme was efficiently labeled with biotin, and the kinetic data for the biotinylated enzyme were comparable to those for unlabeled β‐galactosidase. The CTR probe exhibits excellent targeting specificity, as it selectively labeled β‐galactosidase in a complex protein mixture.     

Mechanisms of Enhanced Catalysis in Enzyme–DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme–DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9‐ and 2.4‐fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4‐aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems.     

Thermal conductivity of the ionic liquid [HMIm][Tf2N] with compressed carbon dioxide

The liquid thermal conductivity of the ionic liquid (IL), 1-hexyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)amide ([HMIm][Tf₂N]), saturated with compressed vapor and supercritical carbon dioxide was measured over three isotherms (298.15, 323.15, and 348.15 K) and pressures up to approximately 20 MPa using a transient hot-wire technique. Pure [HMIm][Tf₂N] thermal conductivity was also measured over a temperature range of 293.15–353.15 K at ambient pressure and with hydrostatic pressure to approximately 20 MPa. Literature vapor–liquid equilibrium data were used to predict the liquid CO₂ composition at the conditions investigated. Initially, the liquid thermal conductivity slightly decreased with pressure/composition of CO₂ followed by a gradual increase that is mainly attributed to hydrostatic pressure effects. Simple composition-based mixing rules for mixture properties are not qualitatively nor quantitatively accurate. These data could be used to engineer heat transfer equipment required for a variety of proposed IL applications in CO₂ capture, absorption refrigeration, biphasic CO₂/IL reaction platforms, etc.     

Biophysical Modulation of Mesenchymal Stem Cell Differentiation in the Context of Skeletal Repair

A prominent feature of the skeleton is its ability to remodel in response to biophysical stimuli and to repair under varied biophysical conditions. This allows the skeleton considerable adaptation to meet its physiological roles of stability and movement. Skeletal cells and their mesenchymal precursors exist in a native environment rich with biophysical signals, and they sense and respond to those signals to meet organismal demands of the skeleton. While mechanical strain is the most recognized of the skeletal biophysical stimuli, signaling phenomena also include fluid flow, hydrostatic pressure, shear stress, and ion-movement-related electrokinetic phenomena including, prominently, streaming potentials. Because of the complex interactions of these electromechanical signals, it is difficult to isolate the significance of each. The application of external electrical and electromagnetic fields allows an exploration of the effects of these stimuli on cell differentiation and extra-cellular matrix formation in the absence of mechanical strain. This review takes a distinctly translational approach to mechanistic and preclinical studies of differentiation and skeletal lineage commitment of mesenchymal cells under biophysical stimulation. In vitro studies facilitate the examination of isolated cellular responses while in vivo studies permit the observation of cell differentiation and extracellular matrix synthesis.     

Phospholipid Cyclosporine Prodrugs Targeted at Inflammatory Bowel Disease (IBD) Treatment: Design,Synthesis, and in Vitro Validation

Novel phospholipid (PL)-cyclosporine conjugates were prepared and studied as potential prodrugs for inflammatory bowel disease (IBD). Our approach relies on phospholipase A₂ (PLA₂), which is overexpressed in the inflamed intestinal tissues, as the prodrug activator to potentially release cyclosporine at the site of inflammation. PL-cyclosporine prodrug conjugates with methylene linkers of various lengths between the sn-2 position of the PL and cyclosporine were synthesized and evaluated for in vitro activation. Surprisingly, despite previous work indicating that conjugates with six methylene linkers between the lipid and drug would suffer rapid enzymatic hydrolysis, with cyclosporine this was not observed. However, compounds with longer linkers (n=10, 12 methylene units) display complete release of the drug by PLA₂-catalyzed hydrolysis, thus demonstrating the importance and profound impact of structural fine-tuning. This study represents a proof-of-concept for our hypothesis and a first step towards a truly targeted IBD treatment with cyclosporine that could be administered throughout the GI tract.     

Development of Undergraduate Students’ Professional Skills

The development of engineering students’ professional skills has gained considerable national attention from Accreditation Board for Engineering and Technology, the National Academy of Engineering, ASCE, and other constituents. There is little debate that these professional skills are necessary. Engineering programs have tried many approaches to develop these skills in the undergraduate programs. Colorado State University (CSU) has developed a new approach modeled on the type of professional development that occurs in the professional environment. This new Professional Learning Institute (PLI) provides students with a broad array of workshops, presentations, and experiential opportunities addressing the areas of cross cultural communication and teamwork, innovation, leadership, ethics, and public service. This program introduces students to the concept of professional development through required extracurricular activities, includes minimum requirements along with requirements to earn certificates in specialty areas for motivated students. The majority of offerings in the PLI are presented by leaders from the engineering profession who have teamed with CSU to provide high quality programs for our students.