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ABSTRACT OF DISSERTATIONInvestigating the Effects of a Small-Molecule Allosteric Inhibitor of Core-Binding Factor Subunit Beta in Human OsteosarcomaOsteosarcoma (OS) is the most common primary malignant bone tumor in humans, however, there have been no new successful therapies that improve long-term survival in several decades. This indicates the need for novel therapeutics and targets for treating OS, particularly in reducing its metastatic potential which is the leading cause of osteosarcoma-related deaths. Core Binding Factor is a heterodimeric transcriptional complex, comprised of proteins runt-related transcription factor 2 (RUNX2) and core-binding factor subunit beta (CBFb), that promotes transcription of genes related to bone formation, osteoblast differentiation, and bone mineralization. RUNX2 protein expression is deregulated in OS tumors which contributes to chemoresistance, increased proliferation, and defective differentiation, suggesting it could be a therapeutic target in OS. This study utilizes small molecule allosteric inhibitor of CBFb, AI-14- 91, to disrupt the interaction of RUNX2 and CBFb, thereby reducing RUNX2 binding to target genes, to evaluate the CBFb and RUNX2 interaction as a pharmacological target in OS. We hypothesized that AI-14-91 will reduce the malignant phenotype of human OS cell lines, show favorable pharmacokinetic and pharmacodynamic qualities in vivo, and effectively reduce the development of cell line-derived xenografts (CDXs) in vivo.In vitro cell-based assays were performed with a panel of human OS cell lines treated with AI-14-91. RUNX2 knockout (KO) and CBFb KO cell lines were also generated from the parental U2OS wild-type (WT) by CRISPR/Cas9 editing, to compare the effects of AI-14-91 treatment versus the loss of each component of the Core Binding Factor complex. Concentration- and time-iidependent proliferation assays were performed with all cell lines, demonstrating AI-14-91 IC50 values ranging from approximately 13-25 μM across the panel. Clonogenic assays showed AI-14-91 treatment significantly reduced colony formation and colony size in most cell lines. AI-14-91 disrupted the cell cycle progression in some of the cell lines as well with a significant decrease of cell number in G0/G1 phase and an increase in S phase upon 48-hour exposure to 20 μM AI-14- 91. Induction of apoptosis by AI-14-91 was quantified with caspase-3/-7 activity fluorometric assay and western blotting with cleaved caspase-3 and cleaved PARP-1. Apoptotic and necrotic cell populations were evaluated in U2OS WT cells by annexin V/PI staining analyzed by flow cytometry. All of these showed no significant induction of apoptosis. Lastly, a transwell invasion and migration assay demonstrated that AI-14-91-treated LM7 and U2OS cells had reduced invasive ability. These anti-proliferative and -clonogenic effects as well as disruptions in cell cycle were seen in the U2OS WT and both KO cell lines, suggesting off-target effects.Batch 3’ Tag-Seq RNA-seq was performed on parental U2OS WT cells treated with 5 and 20 μM AI-14-91 for 6 and 48 hours, U2OS WT cells treated with DMSO vehicle control, and on untreated RUNX2 KO and CBFb KO cell lines. Differential gene expression (DGE), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA) were the analyses performed to evaluate pertinent transcriptional pathways altered by inhibitor treatment, and to compare the effects of inhibitor treatment with knockout of putative inhibitor targets. RNA-seq data plotted on multidimensional scaling plots showed that inhibitor treatment produced a unique transcriptional profile when compared to the CBFb KO cell lines or DMSO treated cells. The DGE analysis revealed that loss of CBFb and 6- hour treatment with AI-14-91 produced unique expression changes in U2OS WT cells and that loss of RUNX2 had a smaller effect on gene expression than loss of CBFb. In the AI-14-91 versusiiiDMSO comparison, KEGG pathway analysis showed enrichment in cancer pathways where RUNX2 is associated with metastasis to the bone. GO analysis highlighted terms and genes that would support the anti-tumor effects seen in the in vitro experiments such as pathways in regulation of cell migration and mitotic cell cycle. These are further supported by specific changes in expression of genes such as CDKN1C, BRINP1 and BMP4. These pathways that were altered support the anti-tumor properties of AI-14-91 and reduced metastatic potential seen in U2OS WT cells.Lastly, we determined concentrations and pharmacokinetic (PK) parameter values for both serum and lung tissue samples after a single intraperitoneal injection of 100 mg/kg AI-14-91 over a time course of 0-24 hours in nude mice. There were cell line growth-inhibiting concentrations of AI-14-91 found in the blood, exceeding the determined in vitro IC50 values (the highest being 23 μM). This effective concentration was seen in the blood for 1 hour post-injection. The measured concentration in the lung was not as high but reached 10 μM at its peak. Both peaks in concentration were seen with an early time of peak concentration (Tmax) of 15 and 30 min, respectively. The half-life of AI-14-91 in the serum was comparable to that of published literature and exceeded 10 hours in the lung. With a pulmonary metastatic CDX nude mouse model utilizing the LM7 cell line and live bioluminescent imaging, we were unable to demonstrate that a 6-day treatment period with AI-14-91 can reduce the rate of CDX growth. This study is still ongoing at this time and will continue to be monitored for CDX development. If successful future CDX development occurs, the efficacy of AI-14-91 would be evaluated by bioluminescent signal and metastatic burden in the lung along with survival curve analysis.Through these experiments, we have demonstrated that AI-14-91 has desirable anti-tumor properties in multiple OS cell lines, alters gene transcription that would support these properties,ivand has favorable pharmacokinetic parameters in vivo. The effects that AI-14-91 exerted in multiple human OS cell lines were also seen in U2OS cells without CBFb protein, the target of thesmall-molecule inhibitor. This suggested there are alternate mechanisms of action of this compound in OS, which began to be investigated here, but should be continued to be expanded upon. Additionally, this PK study could inform future studies that further characterize the ADMET properties of AI-14-91 and utilize this compound in pulmonary CDX models in mice. Overall, the small-molecule inhibitor of CBFb, AI-14-91, deserves further investigations that characterize its mechanism of action, anti-tumor properties, and efficacy in models of OS.v

Despite being the frontline therapy for type 2 diabetes, the mechanisms of action of the biguanide drug metformin are still being discovered. In particular, the detailed molecular interplays between the AMPK and the mTORC1 pathway in the hepatic benefits of metformin are still ill defined. Metformin-dependent activation of AMPK classically inhibits mTORC1 via TSC/RHEB, but several lines of evidence suggest additional mechanisms at play in metformin inhibition of mTORC1. Here we investigated the role of direct AMPK-mediated serine phosphorylation of RAPTOR in a new RaptorAA mouse model, in which AMPK phospho-serine sites Ser722 and Ser792 of RAPTOR were mutated to alanine. Metformin treatment of primary hepatocytes and intact murine liver requires AMPK regulation of both RAPTOR and TSC2 to fully inhibit mTORC1, and this regulation is critical for both the translational and transcriptional response to metformin. Transcriptionally, AMPK and mTORC1 were both important for regulation of anabolic metabolism and inflammatory programs triggered by metformin treatment. The hepatic transcriptional response in mice on high-fat diet treated with metformin was largely ablated by AMPK deficiency under the conditions examined, indicating the essential role of this kinase and its targets in metformin action in vivo.

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy, and the limited resolution of fluorescence microscopy makes it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution.

Here, we report the immobilization of Co-protoporphyrin IX (Co-PPIX) substituted cytochrome c (Co-cyt c) on Antimony-doped Tin Oxide (ATO) as a catalyst for photoelectrochemical oxidation of water. Under visible light irradiation (λ &gt, 450 nm), the ATO-Co-cyt c photoanode displays ~6-fold enhancement in photocurrent density relative to ATO-Co-PPIX at 0.25 V vs. RHE at pH 5.0. The light-induced water oxidation activity of the system was demonstrated by detecting evolved stoichiometric oxygen by gas chromatography, and incident photon to current efficiency was measured as 4.1% at 450 nm. The faradaic efficiency for the generated oxygen was 97%, with a 671 turnover number (TON) for oxygen. The current density had a slow decay over the course of 6 h of constant irradiation and applied potential, which exhibits the robustness of catalyst-ATO interaction.

The current trend in atmospheric carbon dioxide concentrations is causing increasing concerns for its environmental impacts, and spurring the developments of sustainable methods to reduce CO2 to usable molecules. We report the light-driven CO2 reduction in water in mild conditions by artificial protein catalysts based on cytochrome b562 and incorporating cobalt protoporphyrin IX as cofactor. Incorporation into the protein scaffolds enhances the intrinsic reactivity of the cobalt porphyrin toward proton reduction and CO generation. Mutations around the binding site modulate the activity of the enzyme, pointing to the possibility of further improving catalytic activity through rational design or directed evolution.

The current trend in atmospheric carbon dioxide concentrations is causing increasing concerns for its environmental impacts, and spurring the developments of sustainable methods to reduce CO2 to usable molecules. We report the light-driven CO2 reduction in water in mild conditions by artificial protein catalysts based on cytochrome b 562 and incorporating cobalt protoporphyrin IX as cofactor. Incorporation into the protein scaffolds enhances the intrinsic reactivity of the cobalt porphyrin toward proton reduction and CO generation. Mutations around the binding site modulate the activity of the enzyme, pointing to the possibility of further improving catalytic activity through rational design or directed evolution.

BackgroundNeurons are an integral component of the immune system that functions to coordinate responses to bacterial pathogens. Sensory nociceptive neurons that can detect bacterial pathogens are found throughout the body with dense innervation of the intestinal tract.MethodsIn this study, we assessed the role of these nerves in the coordination of host defenses to Citrobacter rodentium. Selective ablation of nociceptive neurons significantly increased bacterial burden 10 days postinfection and delayed pathogen clearance.ResultsBecause the sensory neuropeptide CGRP (calcitonin gene-related peptide) regulates host responses during infection of the skin, lung, and small intestine, we assessed the role of CGRP receptor signaling during C rodentium infection. Although CGRP receptor blockade reduced certain proinflammatory gene expression, bacterial burden and Il-22 expression was unaffected.ConclusionsOur data highlight that sensory nociceptive neurons exert a significant host protective role during C rodentium infection, independent of CGRP receptor signaling.

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy. We developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution. These probes reveal that ER-associated actin drives fission of multiple organelles including mitochondria, endosomes, lysosomes, peroxisomes, and the Golgi.

Arrays of one, two and four electron-transfer active [4Fe–4S] clusters were constructed on modular tetratricopeptide repeat protein scaffolds, with the number of clusters determined solely by the size of the scaffold. The constructs show reversible redox activity and transient charge stabilization necessary to facilitate charge transfer.

Incorporation of biotinylated aminopyridine cobalt complexes derived from the triazacyclononane scaffold into the streptavidin protein leads to formation of artificial metalloenzymes for water reduction to hydrogen. The synthesized artificial metalloenzymes have lower overpotential (at the half-peak up to 100 mV) and higher photocatalytic hydrogen evolution activity (up to 14- and 10-fold increase in TOF and TON, respectively, at pH 12.5) than the free biotinylated cobalt complexes. 1H-NMR, EPR and XAS highlight the presence of the metal complexes upon supramolecular attachment to the streptavidin. pHdependent catalytic studies and molecular dynamics (MD) simulations suggest that the increase in the catalytic activity could be induced by the protein residues positioned close to the metal centers. These findings illustrate the ability of the biotin−streptavidin technology to produce artificial metalloproteins for photo- and electrocatalytic hydrogen evolution reaction.

Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK−/− EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation.

Protein-based self-assembled nanostructures hold tremendous promise as smart materials. One strategy to control the assembly of individual protein modules takes advantage of the directionality and high affinity bonding afforded by metal chelation. Here, we describe the use of 2,2'-bipyridine units (Bpy) as side chains to template the assembly of large structures (MW approx. 35 000 Da) in a metal-dependent manner. The structures are trimers of independently folded 3-helix bundles, and are held together by 2 Me(Bpy)3 complexes. The assemblies are stable to thermal denaturation, and are more than 90% helical at 90°C. Circular dichroism spectroscopy shows that one of the 2 possible (Bpy)3 enantiomers is favored over the other. Because of the sequence pliability of the starting peptides, these constructs could find use to organize functional groups at controlled positions within a supramolecular assembly.

This experiment has the meaning for effective classification between Adenovirus serotype 2 and Human Herpes Virus-4 by discovering the Kernel Function which has low error rate using Support Vector Machine. Analyzing the differences would state the potential of developing medical remedy for Herpesvirus which has no vaccine of virus-specific therapy.