Faculty Bibliography

Introduction: Dimethyl sulfoxide (DMSO), propylene glycol (PG) and miglyol (MG) are common organic solvents often used to dissolve neuro-pharmacological agents for in vivo assays in animal models of various pediatric brain disorders. Nonetheless, these compounds were reported to exhibit pharmacological and pathological effects on the central nervous system of their own. Here we report chronic effects of these solvents on behavior, motoric functions and brain morphology in young rats (P35-37), following neonatal exposure to one of the compounds (P6).
Method(s): Compounds were administered intraperitoneally (DMSO, 2 or 4 ml/kg; MG, 2 ml/kg) or per os (PG, 2.5 ml/kg) at concentrations considered safe and non-interfering with neuroscience research. Age/sex matched controls received phosphate-buffered saline (PBS). Animals were sacrificed following behavioral and locomotor assays (P40) and their brains were subjected to pathological analyses.
Result(s): Rats exposed to DMSO (n = 10; 4 ml/kg only), spent significantly more time in the open field center and traveled shorter distance (p < 0.05) vs. controls (n = 10). Rats exposed to DMSO and MG exhibited shorter social interactions vs. controls (p < 0.05). CatWalk gait analysis showed various disturbances (p < 0.05) in rats exposed to any of the three compounds (DMSO, 4 ml/kg only). Brain pathological analyses revealed increased expression of microglia (Iba-1+) and reactive astrocytes (GFAP+) in rats exposed to DMSO (p < 0.05).
Conclusion(s): Observed chronic behavioral, motoric and morphologic sequelae of neonatal exposure to DMSO, PG or MG at concentrations that are generally considered safe raise concerns about under-appreciated neuro-toxicity of these common organic solvents, which warrants further exploration in larger translational studies

The biological mediator hydrogen sulfide (H₂S) is produced by bacteria and has been shown to be cytoprotective against oxidative stress and to increase the sensitivity of various bacteria to a range of antibiotic drugs. Here we evaluated whether bacterial H₂S provides resistance against the immune response, using two bacterial species that are common sources of nosocomial infections, Escherichia coli and Staphylococcus aureus Elevations in H₂S increased the resistance of both species to immune-mediated killing. Clearance of infections with wild type and genetically H₂S-deficient E. coli and S. aureus was compared in vitro and in mouse models of abdominal sepsis and burn wound infection. Also, inhibitors of H₂S-producing enzymes were used to assess bacterial killing by leukocytes. We found that inhibition of bacterial H₂S production can increase susceptibility of both bacterial species to rapid killing by immune cells and can improve bacterial clearance after severe burn, an injury that increases susceptibility to opportunistic infections. These findings support the role of H₂S as a bacterial defense mechanism against the host response and implicate bacterial H₂S inhibition as a potential therapeutic intervention in the prevention or treatment of infections.

Isocitrate dehydrogenase (IDH) mutation have been reported to impose in gliomas a shortage of NADPH required to maintain a redox state and may rely on cysteine (Cys) availability for biosynthesis of glutathione (GSH) to ensure antioxidant levels. Cys may be replenished via extracellular intake or by de novo intracellular synthesis via transsulfuration (TS) pathway. The aim of this study was to investigate alterations of Cys metabolism in genetic variants of high-grade gliomas (HGG). Seventeen tumor samples from 15 adult patients (11 M / 4 F; average age 57 years, range 25 - 81 years), who underwent surgical resection for newly diagnosed or recurrent HGG were analyzed by HPLC. Levels of Cys, homocysteine and GSH were correlated with the genetic signature of HGG (wild-types vs. IDH1 mutation, PTEN deletion, EGFR amplification and MGMT methylation). Cys levels were significantly higher (2.1 fold increase; p=0.0038) in IDH1-mut (n=4) vs. IDH1-wt HGG (n=13), with comparable homocysteine and GSH levels. PTEN deletion and EGFR amplification did not significantly alter Cys metabolites with comparable levels of Cys, homocysteine and GSH detected in PTEN-del (n=7) and PTEN-intact (n=6) HGG, as well as in EGFR-amp (n=7) and EGFR-non amp (n=9) HGG. Significantly higher Cys levels (3.2 fold increase; p=0.0186) were also found in MGMT methylated (n=4) vs. non-methylated (n=3) HGG, with comparable levels of homocysteine and GSH. Increased Cys levels detected in IDH1-mut and MGMT methylated HGG support the hypothesis that these tumors may preferentially use the TS pathway for GSH synthesis. These findings are consistent with our report of increased TS pathway enzyme cystathionine B-synthase (CBS) in HGG, but concurrent increased intake of Cys cannot be excluded. Our results suggest utilizing Cys metabolites as potential markers and/or therapeutic targets in some genetic variants of HGG, a hypothesis that should be further explored in larger translational trials

New chemical inhibitors of protein-protein interactions are needed to propel advances in molecular pharmacology. Peptoids are peptidomimetic oligomers with the capability to inhibit protein-protein interactions by mimicking protein secondary structure motifs. Here we report the in silico design of a macrocycle primarily composed of peptoid subunits that targets the β-catenin:TCF interaction. The β-catenin:TCF interaction plays a critical role in the Wnt signaling pathway which is over-activated in multiple cancers, including prostate cancer. Using the Rosetta suite of protein design algorithms, we evaluate how different macrocycle structures can bind a pocket on β-catenin that associates with TCF. The in silico designed macrocycles are screened in vitro using luciferase reporters to identify promising compounds. The most active macrocycle inhibits both Wnt and AR-signaling in prostate cancer cell lines, and markedly diminishes their proliferation. In vivo potential is demonstrated through a zebrafish model, in which Wnt signaling is potently inhibited.

Glycogen is synthesized and stored to maintain postprandial blood glucose homeostasis and to ensure an uninterrupted energy supply between meals. Although the regulation of glycogen turnover has been well studied, the effects of glycogen on aging and disease development have been largely unexplored. In Caenorhabditis elegans fed a high sugar diet, glycogen potentiates resistance to oxidants, but paradoxically, shortens lifespan. Depletion of glycogen by oxidants or inhibition of glycogen synthesis extends the lifespan of worms by an AMPK-dependent mechanism. Thus, glycogen is not merely an inert storage molecule, but also an active regulator of energy balance and aging. Its depletion by oxidants may be beneficial in the treatment of hyperglycemia and glycogen-related diseases.

Fast photochemical oxidation of proteins (FPOP) may be used to characterize changes in protein structure by measuring differences in the apparent rate of peptide oxidation by hydroxyl radicals. The variability between replicates is high for some peptides and limits the statistical power of the technique, even using modern methods controlling variability in radical dose and quenching. Currently, the root cause of this variability has not been systematically explored, and it is unknown if the major source(s) of variability are structural heterogeneity in samples, remaining irreproducibility in FPOP oxidation, or errors in LC-MS quantification of oxidation. In this work, we demonstrate that coefficient of variation of FPOP measurements varies widely at low peptide signal intensity, but stabilizes to ≈ 0.13 at higher peptide signal intensity. We dramatically reduced FPOP variability by increasing the total sample loaded onto the LC column, indicating that the major source of variability in FPOP measurements is the difficulties in quantifying oxidation at low peptide signal intensities. This simple method greatly increases the sensitivity of FPOP structural comparisons, an important step in applying the technique to study subtle conformational changes and protein-ligand interactions. Graphical Abstract ᅟ.

Unlike transcription initiation and termination, which have easily discernable signals such as promoters and terminators, elongation is regulated through a dynamic network involving RNA/DNA pause signals and states- rather than sequence-specific protein interactions. A report by Nedialkov et al. (in press) provides experimental evidence for sequence-specific recruitment of elongation factor RfaH to transcribing RNA polymerase (RNAP) and outlines the mechanism of gene expression regulation by restraint ("locking") of the DNA non-template strand. According to this model, the elongation complex pauses at the so called "operon polarity sequence" (found in some long bacterial operons coding for virulence genes), when the usually flexible non-template DNA strand adopts a distinct hairpin-loop conformation on the surface of transcribing RNAP. Sequence-specific binding of RfaH to this DNA segment facilitates conversion of RfaH from its inactive closed to its active open conformation. The interaction network formed between RfaH, non-template DNA, and RNAP locks DNA in a conformation that renders the elongation complex resistant to pausing and termination. The effects of such locking on transcript elongation can be mimicked by restraint of the non-template strand due to its shortening. This work advances our understanding of regulation of transcript elongation and has important implications for the action of general transcription factors, such as NusG, which lack apparent sequence-specificity, as well as for the mechanisms of other processes linked to transcription such as transcription-coupled DNA repair.

Molecular chaperone HSP70 (HSPA1A) has therapeutic potential in conformational neurological diseases. Here we evaluate the neuroprotective function of the chaperone in a rat model of Parkinson's disease (PD). We show that the knock-down of HSP70 (HSPA1A) in dopaminergic neurons of the Substantia nigra causes an almost 2-fold increase in neuronal death and multiple motor disturbances in animals. Conversely, pharmacological activation of HSF1 transcription factor and enhanced expression of inducible HSP70 with the echinochrome derivative, U-133, reverses the process of neurodegeneration, as evidenced by а increase in the number of tyrosine hydroxylase-containing neurons, and prevents the motor disturbances that are typical of the clinical stage of the disease. The neuroprotective effect caused by the elevation of HSP70 in nigral neurons is due to the ability of the chaperone to prevent α-synuclein aggregation and microglia activation. Our findings support the therapeutic relevance of HSP70 induction for the prevention and/or deceleration of PD-like neurodegeneration.