Dr. Todd Emrick
Professor, Department of Polymer Science & Engineering
A series of polymer−drug conjugates based on 2-methacryloyloxyethyl phosphorylcholine (MPC) was prepared with the glioblastoma drug temozolomide (TMZ) as pendent groups. Random and block copolymers were synthesized by reversible addition−fragmentation chain-transfer (RAFT) polymerization using a TMZ-containing methacrylate monomer. The solution properties of the polyMPC− TMZ copolymers were investigated by dynamic light scattering and transmission electron microscopy, revealing well-defined nanostructures from the block copolymers. Conjugation of TMZ to polyMPC enhanced drug stability, with decomposition half-life values ranging from 2- to 19-times longer than that of free TMZ. The cytotoxicity of polyMPC−TMZ was evaluated in both chemosensitive (U87MG) and chemoresistant (T98G) glioblastoma cell lines. Furthermore, the polyMPC−TMZ platform was expanded considerably by the preparation of redox-sensitive polyMPC−TMZ copolymers utilizing disulfides as the polymer-to-drug linker.
Read more about Dr. Todd Emrick.
Dr. Alejandro Heuck
Associate Professor, Biochemistry and Molecular Biology
Many Gram-negative bacterial pathogens use a type III secretion system to infect eukaryotic cells. The injection of bacterial toxins or protein effectors via this system is accomplished through a plasma membrane channel formed by two bacterial proteins, termed translocators, whose assembly and membraneinsertion mechanisms are currently unclear. Here, using purified proteins we demonstrate that the translocators PopB and PopD in Pseudomonas aeruginosa assemble heterodimers in membranes, leading to stably inserted hetero-complexes. Using site-directed fluorescence labeling with an environment-sensitive probe, we found that hydrophobic segments inPopDanchor the translocator to the membrane, but without adopting a typical transmembrane orientation. A fluorescence dual-quenching assay revealed that the presence of PopB changes the conformation adopted by PopD segments in membranes. Furthermore, analysis of PopD’s interaction with human cell membranes revealed that PopD adopts a distinctive conformation when PopB is present. An N-terminal region of PopD is only exposed to the host cytosol when PopB is present. We conclude that PopB assists with the proper insertion of PopD in cell membranes, required for the formation of a functional translocon and host infection.
Read more about Dr. Alejandro Heuck.
Dr. Craig Martin
Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase or one of its close family members. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyze in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confirm that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex, a process that favorably competes with promoter driven synthesis under high yield reaction conditions. While certain priming modes can be favored, the process is heterogeneous, both in initial priming and in the extent of priming, and already extended products can rebind for further extension, in a distributive process. Furthermore, addition of one or a few nucleotides, previously termed ‘nontemplated addition,’ also occurs via templated primer extension. At last, this work demonstrates the utility of RNA-Seq as a tool for in vitro mechanistic studies, providing information far beyond that provided by traditional gel electrophoresis.
Read more about Dr. Craig Martin.
Dr. David Julian McClements
Distinguished Professor, Food Science
In this study, nanoemulsion-based delivery systems fabricated using three different methods were compared with three commercially available curcumin supplements. Powdered curcumin was dispersed into the oil-in-water nanoemulsions using three methods: the conventional oil-loading method, the heat-driven method, and the pH-driven method. The conventional method involved dissolving powdered curcumin in the oil phase (60 °C, 2 h) and then forming a nanoemulsion. The heat-driven method involved forming a nanoemulsion and then adding powdered curcumin and incubating at an elevated temperature (100 °C, 15 min). The pH-driven method involved dissolving curcumin in an alkaline solution (pH 12.5) and then adding this solution to an acidified nanoemulsion (pH 6.0). The three commercial curcumin products were capsules or tablets purchased from an online supplier: Nature Made, Full Spectrum, and CurcuWin. Initially, the encapsulation efficiency of the curcumin in the three nanoemulsions was determined and decreased in the following order: pH-driven (93%) > heat-driven (76%) > conventional (56%) method. The different curcumin formulations were then subjected to a simulated gastrointestinal tract (GIT) model consisting of mouth, stomach, and small intestine phases. All three nanoemulsions had fairly similar curcumin bioaccessibility values (74−79%) but the absolute amount of curcumin in the mixed micelle phase was highest for the pH-driven method. A comparison of these nanoemulsions and commercial products indicated that the curcumin concentration in the mixed micelles decreased in the following order: CurcuWin ≈ pH-driven method > heat-driven method > conventional method ≫ Full spectrum > Nature Made. This study provides valuable information about the impact of the delivery system type on curcumin bioavailability. It suggests that encapsulating curcumin within small lipid particles may be advantageous for improving its absorption form the GIT.
Read more about Dr. David Julian McClements.
Dr. Margaret Riley
The feasibility of using colicins to create an antimicrobial lubricant to prevent extraluminal catheter contamination during urinary catheter insertion was assessed. Levels of resistance of uropathogenic Escherichia coli to antibiotics and colicins were compared. The results showed that antibiotics and colicins possess similar frequencies of resistance to a single drug, whereas colicins exhibit significantly lower levels of multidrug resistance (22%) than antibiotics (42%). Colicins and antibiotics showed complementary inhibitory activity, with each targeting different subsets of pathogenic isolates. The collateral impact of these two antimicrobials on genera that are members of the fecal/vaginal/urinary microbiome was assessed, with colicins showing significantly less collateral damage than antibiotics. Using a novel colicin, SR4, minimum inhibitory concentrations (MICs) for a panel of 30 uropathogenic isolates were determined and showed that SR4 achieved the same antimicrobial efficacy as gentamicin using 20-30% less drug. An SR4-impregnated catheter lubricant was created and its ability to prevent extraluminal urinary catheter contamination in vitro was demonstrated. These data indicate that a colicin-impregnated lubricant may provide a viable prophylactic option for preventing catheter-associated urinary tract infections.
Read more about Dr. Margaret Riley.
Dr. Sloan Siegrist
Assistant Professor, Microbiology
Rod-shaped mycobacteria expand from their poles, yet D-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or L,D-transpeptidation and in an L,D-transpeptidase mutant. Dipeptides complemented defects in D-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked L,D-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked D,D-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.
Read more about Dr. Sloan Siegrist.
Dr. Kim Tremblay
Associate Professor, Veterinary and Animal Sciences
During development, the endoderm initiates organ-restricted gene expression patterns in a spatiotemporally controlled manner. This process, termed induction, requires signals from adjacent mesodermal derivatives. Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) emanating from the cardiac mesoderm and the septum transversum mesenchyme (STM), respectively, are believed to be simultaneously and uniformly required to directly induce hepatic gene expression from the murine endoderm. Using small molecule inhibitors of BMP signals during liver bud induction in the developing mouse embryo, we found that BMP signaling was not uniformly required to induce hepatic gene expression. Although BMP inhibition caused an overall reduction in the number of induced hepatoblasts, the STM-bounded posterior liver bud demonstrated the most severe loss of the essential hepatic transcription factor, hepatocyte nuclear factor 4-a (HNF4a), whereas the sinus venosus–lined anterior liver bud was less affected. We found that the posterior liver bud progenitors were anteriorly displaced and aberrantly activated pancreatobiliary markers, including sex-determining region Ybox 9 (SOX9). Additionally, we found that ectopically expressed SOX9 inhibited HNF4a and that BMP was indirectly required for hepatoblast induction. Finally, because previous studies have demonstrated that FGF signals are essential for anterior but not posterior liver bud induction, we examined synchronous BMP and FGF inhibition and found this led to a nearly complete loss of hepatoblasts. Conclusion: BMP signaling is required to maintain the hepato-pancreatobiliary boundary, at least in part, by indirectly repressing SOX9 in the hepatic endoderm. BMP and FGF signals are each required for the induction of spatially complementary subsets of hepatoblasts. These results underscore the importance of studying early inductive processes in the whole embryo.
Read more about Dr. Kim Tremblay.