Surficial geologic map of the Des Moines Lobe of Iowa, Phase 5: Polk County

https://ir.uiowa.edu/igs_ofm/1030/thumbnail.jp

The Traitor

The proteolytic activation of protein kinase Cδ (PKCδ) generates a catalytic fragment called PKCδ-CF, which induces cell death. However, the mechanisms underlying PKCδ-CF-mediated cell death are largely unknown. On the basis of an engineering leukemic cell line with inducible expression of PKCδ-CF, here we employ SILAC-based quantitative phosphoproteomics to systematically and dynamically investigate the overall phosphorylation events during cell death triggered by PKCδ-CF expression. Totally, 3000 phosphorylation sites were analyzed. Considering the fact that early responses to PKCδ-CF expression initiate cell death, we sought to identify pathways possibly related directly with PKCδ by further analyzing the data set of phosphorylation events that occur in the initiation stage of cell death. Interacting analysis of this data set indicates that PKCδ-CF triggers complicated networks to initiate cell death, and motif analysis and biochemistry verification reveal that several kinases in the downstream of PKCδ conduct these networks. By analysis of the specific sequence motif of kinase-substrate, we also find 59 candidate substrates of PKCδ from the up-regulated phosphopeptides, of which 12 were randomly selected for <i>in vitro</i> kinase assay and 9 were consequently verified as substrates of PKCδ. To our greatest understanding, this study provides the most systematic analysis of phosphorylation events initiated by the cleaved activated PKCδ, which would vastly extend the profound understanding of PKCδ-directed signal pathways in cell death. The MS data have been deposited to the ProteomeXchange with identifier PXD000225

Epigenetic and Post-Translational Mechanisms in Pain: MicroRNA and Phosphorylation

The molecular and neurobiological mechanisms underlying persistent pain are poorly understood. My dissertation research focused on three problems relevant to chronic pain to elucidate epigenetic and post-translational mechanisms. A key issue leading to inadequate pain control is the development of opioid analgesic tolerance. I studied the role of microRNA as an epigenetic regulator of opioid tolerance by targeting the µ opioid receptor (MOR). Employing bioinformatics, a let-7 binding site in MOR 3’UTR was identified, which was experimentally confirmed as a direct target of let-7. Morphine significantly upregulated let-7 expression in human neuroblastoma SH-SY5Y cells and in a mouse model of opioid tolerance. A LNA-let-7 inhibitor decreased brain let-7 levels and partially attenuated opioid antinociceptive tolerance in mice. Mechanistically, let-7 functioned as a mediator translocating and sequestering MOR mRNA to P-bodies, leading to translation repression during opioid tolerance development. Paclitaxel, an effective and frequently used chemotherapeutic agent, produces neuropathic pain as an adverse effect. I found paclitaxel in the low nanomolar range potently triggers intracellular Ca2+ transients, PKA & PKC activation, and substance P release from primary afferent sensory neurons. A specific cellular signaling pathway involving PKA/AKAP150/PKC(ε, βII, & δ) was identified in mediating pain neurotransmitter release and, ultimately, neuropathic pain induced by paclitaxel. Moreover, patients with advanced breast cancer suffer from severe and long-lasting pain, which is extremely difficult to treat. Employing invasive breast cancer MDA-MB-231 cells, I developed a novel cellular model to study nociceptive signaling within the cancer microenvironment. Neuroimmune factors secreted from tumor cells were recognized as contributors to sensory neuron activation. Tumor-induced nociceptor hyperexcitability correlates with the behavioral manifestations of pain seen in vivo, thus validating the model and shedding light on the mechanisms of breast cancer pain. Taken together, these findings provide a better understanding on the specific mechanisms of cancer-related pain and opioid tolerance, which may facilitate the design and development of novel pharmacological treatments for pain relief

Let-7 micro RNAs and opioid tolerance

This chapter will focus on the role of microRNAs (miRs) in regulating the actions of opioid drugs through the opioid receptors. Opioids, such as morphine, are analgesics that are used for treating many forms of acute and chronic pain. However, their chronic use is limited by undesirable effects such as opioid tolerance. The mu opioid receptor (MOR) is the primary receptor responsible for opioids' analgesia and antinociceptive tolerance. The long 3'-untranslated region (3'-UTR) of MOR mRNA is of great interest since this region may contain elements for the post-transcriptional regulation of receptor expression, such as altering the stability of mRNA, influencing translational efficiency, and controlling mRNA transport. MicroRNAs are small non-coding RNA molecules that exert their functions through base-pairing with partially complementary sequences in the 3'-UTR of target mRNAs, resulting in decreased polypeptide formation from those mRNAs. Since the discovery of the first miR, lin-4 in Caenorhabditis elegans, hundreds of miRs have been identified from humans to viruses, which have provided a crucial and pervasive layer of post-transcriptional gene regulation. The nervous system is a rich source of miR expression, with a diversity of miR functions in fundamental neurobiological processes including neuronal development, plasticity, metabolism, and apoptosis. Recently, the let-7 family of miRs is found to be a critical regulator of MOR function in opioid tolerance. Let-7 is the first identified human miR. Its family members are highly conserved across species in sequence and function. In the review, we will present a brief review of the opioid receptors, their regulation, and opioid tolerance as well as an overview of miRs and a perspective how miRs may interact with MOR and serve as a regulator of opioid tolerance

Biomimetic Approach to the Catalytic Enantioselective Synthesis of Flavonoids

Herein is reported the direct asymmetric addition of phenol nucleophiles to benzopyrylium salts as a means to produce enantioenriched flavonoid-like compounds. This enantioselective C–C bond construction was achieved through a chiral anion phase-transfer strategy that mimics the proposed biosynthesis of this structurally diverse set of natural products. The utility of this methodology was demonstrated in enantioselective synthesis of a 2,8-dioxabicyclo[3.3.1]­nonane and a 2,4-diarylbenzopyran

Efficient Mo(VI)-Catalyzed Hydration of Nitrile with Acetaldoxime

<div><p></p><p>A method for the selective hydration of nitrile to amide by employing commercially available acetaldoxime and inexpensive oxometallate such as molybdate, vanadate, and tungstate in environmentally friendly water is described. Under this protocol, nitriles including aromatic nitriles, heterocyclic nitriles, and aliphatic nitriles were converted into the corresponding amides in good to excellent yields.</p> <p>[Supplementary materials are available for this article. Go to the publisher's online edition of <i>Synthetic Communications®</i> for the following free supplemental resource(s): Full experimental and spectral details.]</p> </div

Biomimetic Approach to the Catalytic Enantioselective Synthesis of Flavonoids

Herein is reported the direct asymmetric addition of phenol nucleophiles to benzopyrylium salts as a means to produce enantioenriched flavonoid-like compounds. This enantioselective C–C bond construction was achieved through a chiral anion phase-transfer strategy that mimics the proposed biosynthesis of this structurally diverse set of natural products. The utility of this methodology was demonstrated in enantioselective synthesis of a 2,8-dioxabicyclo[3.3.1]­nonane and a 2,4-diarylbenzopyran

Tandem Repeat Modification during Double-Strand Break Repair Induced by an Engineered TAL Effector Nuclease in Zebrafish Genome

<div><p>Tandem repeats (TRs) are abundant and widely distributed in eukaryotic genomes. TRs are thought to have various functions in gene transcription, DNA methylation, nucleosome position and chromatin organization. Variation of repeat units in the genome is observed in association with a number of diseases, such as Fragile X Syndrome, Huntington's disease and Friedreich's ataxia. However, the underlying mechanisms involved are poorly understood, largely owing to the technical limitations in modification of TRs at definite sites in the genome <i>in vivo</i>. Transcription activator-like effector nucleases (TALENs) are widely used in recent years in gene targeting for their specific binding to target sequences when engineered <i>in vitro</i>. Here, we show that the repair of a double-strand break (DSB) induced by TALENs adjacent to a TR can produce serial types of mutations in the TR region. Sequencing analysis revealed that there are three types of mutations induced by the DSB repair, including indels only within the TR region or within the flanking TALEN target region or simutaneously within both regions. Therefore, desired TR mutant types can be conveniently obtained by using engineered TALENs. These results demonstrate that TALENs can serve as a convenient tool for modifying TRs in the genome in studying the functions of TRs.</p></div

Speculative mechanisms involved in TALEN induced DSB repair.

<p>Blue lines represent the genome with DSB sites, and clusters of vertical bars indicate the TR region. The DSB ends can be bound by two groups of proteins independently: the binding of DNA-dependent protein kinase (DNA-PK) complex (Ku 70 and Ku80) and the following ligase IV seals the gap by direct rejoin the broken ends, which is termed non-homologous end-joining (NHEJ) pathway (A); While the binding of MRN complex and Exo1 nuclease initiates the 5'-3' resection of the ends, which is followed by either a replication slippage pathway (B) or homologous recombination (HR) pathway (C, D). In the replication slippage pathway, the TR region forms a secondary structure and leads to mispairing between the template and the newly-synthesized DNA strand. In the HR pathway, the 3' overhang invades into the homologous template DNA (red lines) and primes DNA synthesis (dash lines) to form a structure called D-loop, which will result in a double Holliday junction (dHJ). dHJ can either be resolved by strand cleavage with or without crossover, which is referred as classical DSB repair (DSBR) pathway of HR (C), and dHJ can also be dissolved by helicases to generate a non-crossover (D). Alternatively, D-loop can be directly dissociated through a synthesis-dependent strand annealing (SDSA) pathway, which results in exclusively non-crossover products (D).</p

Type of clones in the examined DSTR TALEN individuals.

<p>Type of clones in the examined DSTR TALEN individuals.</p