Megan Keniry

A new class of cancer-associated PTEN mutations defined by membrane translocation defects

Phosphatase and tensin homolog (PTEN), which negatively regulates tumorigenic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) signaling, is a commonly mutated tumor suppressor. The majority of cancer-associated PTEN mutations block its essential PIP3 phosphatase activity. However, there is a group of clinically identified PTEN mutations that maintain enzymatic activity, and it is unknown how these mutations contribute to tumor pathogenesis. Here, we show that these enzymatically competent PTEN mutants fail to translocate to the plasma membrane where PTEN converts PIP3 to PI(4,5)P2. Artificial membrane tethering of the PTEN mutants effectively restores tumor suppressor activity and represses excess PIP3 signaling in cells. Thus, our findings reveal a novel mechanism of tumorigenic PTEN deficiency.

Discovery and Analysis of Natural Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa

ABSTRACT Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa. This system has been used for high-throughput screening of a natural-compound library. Assays were developed for each component of the system to ascertain the specific target of inhibitory compounds. In high-throughput screens, 13 compounds were identified that inhibit protein synthesis with 50% inhibitory concentrations ranging from 0.3 to >80 μM. MICs were determined for the compounds against the growth of a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Moraxella catarrhalis, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. Three of the compounds were observed to have broad-spectrum activity and inhibited a hypersensitive strain of P. aeruginosa with MICs of 8 to 16 μg/ml. The molecular target of each of the three compounds was determined to be PheRS. One compound was found to be bacteriostatic, and one compound was bactericidal against both Gram-positive and Gram-negative pathogens. The third compound was observed to be bacteriostatic against Gram-positive and bactericidal against Gram-negative bacteria. All three compounds were competitive with the substrate ATP; however, one compound was competitive, one was uncompetitive, and one noncompetitive with the amino acid substrate. Macromolecular synthesis assays confirm the compounds inhibit protein synthesis. The compounds were shown to be more than 25,000-fold less active than the control staurosporine in cytotoxicity MTT testing in human cell lines.

Identification of Chemical Compounds That Inhibit the Function of Glutamyl-tRNA Synthetase from Pseudomonas aeruginosa

Pseudomonas aeruginosa glutamyl-tRNA synthetase (GluRS) was overexpressed in Escherichia coli. Sequence analysis indicated that P. aeruginosa GluRS is a discriminating GluRS and, similar to other GluRS proteins, requires the presence of tRNAGlu to produce a glutamyl-AMP intermediate. Kinetic parameters for interaction with tRNA were determined and the kcat and KM were 0.8 s–1 and 0.68 µM, respectively, resulting in a kcat/KM of 1.18 s–1 µM–1. A robust aminoacylation-based scintillation proximity assay (SPA) assay was developed and 800 natural products and 890 synthetic compounds were screened for inhibitory activity against P. aeruginosa GluRS. Fourteen compounds with inhibitory activity were identified. IC50s were in the low micromolar range. The minimum inhibitory concentration (MIC) was determined for each of the compounds against a panel of pathogenic bacteria. Two compounds, BT_03F04 and BT_04B09, inhibited GluRS with IC50s of 21.9 and 24.9 µM, respectively, and both exhibited promising MICs against Gram-positive bacteria. Time-kill studies indicated that one compound was bactericidal and one was bacteriostatic against Gram-positive bacteria. BT_03F04 was found to be noncompetitive with both ATP and glutamic acid, and BT_04B09 was competitive with glutamic acid but noncompetitive with ATP. The compounds were not observed to be toxic to mammalian cells in MTT assays.

New Frontiers for the NFIL3 bZIP Transcription Factor in Cancer, Metabolism and Beyond.

The bZIP transcription factor NFIL3 (Nuclear factor Interleukin 3 regulated, also known as E4 binding protein 4, E4BP4) regulates diverse biological processes from circadian rhythm to cellular viability. Recently, a host of novel roles have been identified for NFIL3 in immunological signal transduction, cancer, aging and metabolism. Elucidating the signaling pathways that are impacted by NFIL3 and the regulatory mechanisms that it targets, inhibits or activates will be critical for developing a clearer picture of its physiological roles in disease and normal processes. This review will discuss the recent advances and emerging issues regarding NFIL3-mediated transcriptional regulation of CEBPb and FOXO1 activated genes and signal transduction.

Oxidative insults disrupt OPA1-mediated mitochondrial dynamics in cultured mammalian cells

ABSTRACT Objective: To explore the impact of oxidative insults on mitochondrial dynamics. In mammalian cells, oxidative insults activate stress response pathways including inflammation, cytokine secretion, and apoptosis. Intriguingly, mitochondria are emerging as a sensitive network that may function as an early indicator of subsequent cellular stress responses. Mitochondria form a dynamic network, balancing fusion, mediated by optic atrophy-1 (OPA1), and fission events, mediated by dynamin-related protein-1 (DRP1), to maintain homeostasis. Methods: Here, we examine the impact of oxidative insults on mitochondrial dynamics in 143B osteosarcoma and H9c2 cardiomyoblast cell lines via confocal microscopy, flow cytometry, and protein-based analyses. Results: When challenged with hydrogen peroxide (H2O2), a ROS donor, both cell lines display fragmentation of the mitochondrial network and loss of fusion-active OPA1 isoforms, indicating that OPA1-mediated mitochondrial fusion is disrupted by oxidative damage in mammalian cells. Consistent with this, cells lacking OMA1, a key protease responsible for cleavage of OPA1, are protected against OPA1 cleavage and mitochondrial fragmentation in response to H2O2 challenge. Discussion: Taken together, these findings indicate that oxidative insults damage OPA1-mediated mitochondrial dynamics in mammalian cells via activation of OMA1, consistent with an emerging role for mitochondrial dynamics as an early indicator of cellular stress signaling. Abbreviations: Δψm: transmembrane potential; ROS: reactive oxygen species; H2O2: hydrogen peroxide; OPA1: optic atrophy-1; MFN1: mitofusin1; DRP1: dynamin-related protein 1; DMEM: Dulbecco’s Modified Eagle’s Medium; PBS: phosphate buffer saline; TOM20: translocase of the outer mitochondrial membrane-20; DAPI: diaminophenylindole; TMRE: tetramethylrhodamine ethyl ester; TBST: Tris-Buffered Saline Tween-20; MEF: mouse embryonic fibroblast.

Identification of Chemical Compounds That Inhibit Protein Synthesis in Pseudomonas aeruginosa

Four inhibitory compounds were identified using a poly-uridylic acid (polyU) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa in an in vitro screen of a synthetic compound library. The compounds were specific for inhibition of bacterial protein synthesis. In enzymatic assays, the compounds inhibited protein synthesis with IC50 values ranging from 20 to 60 μM. Minimum inhibitory concentrations (MICs) were determined in cultures for a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. All the compounds were observed to have broad-spectrum activity and inhibited an efflux pump mutant strain of P. aeruginosa with MICs of 0.5–16 μg/mL. The molecular target of two compounds was determined to be PheRS. These two compounds were bacteriostatic against both Gram-positive and Gram-negative pathogens. In competition assays, they were not observed to compete with the natural substrates ATP or phenylalanine for active site binding. The other two compounds directly inhibited the ribosome and were bactericidal against both Gram-positive and Gram-negative pathogens. In cytotoxicity MTT testing in human cell lines, the compounds were shown to be from 2500- to 30,000-fold less active than the control staurosporine.

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

BackgroundClustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.ResultsWe describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.ConclusionsHere we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).

Radio Frequency Electromagnetic waves induce cancer cell death

The primary objective of this research is to study and interpret the natural physics phenomenon of electromagnetic resonance in one end closed cavity for the eventual purpose of cancer treatment. Radio Frequency waves are released into a coaxial cavity filled with a small amount (1.6 mL) of breast cancer cells (BT549) and the reflection as well as the power input is measured to determine the absorption power into cancer cells. When the reflection of the RF waves from the uploaded sample of cancer cells is at its lowest power, the RF Frequency is noted and seen to be approximately close to the resonant frequency of that cavity. This cavity can potentially be used as a control method of testing RF frequencies on various types of cancer cells, such as the available BT549 cancer cell line obtained from the UTRGV Biology Department. 70% confluent basal breast cancer BT549 cells were grown in RPMI mammalian cell culture media with 10% fetal bovine serum (FBS) and 5% penicillin/streptomycin (P/S: from 10,000 U/mL stock solution) in 5% CO2. Samples were treated with 2 mL of 0.25% trypsin solution to detach cells from petri plates; cells were centrifuged at 100 x g for 5 minutes at room temperature to pellet. After this, cells were then re-suspended in fresh RPMI media (with 10% FBS and P/S). The cell density was 250,000 cells per mL at the time of RF treatment. The determined frequency for 1.6 mL of sample article was found to be within the range of radio frequency, but there is much room for improvement in our method, depending on the coaxial cavity design such as length and the radii of the coaxial tubes which is currently under investigation. Some initial results were obtained which showed that the electromagnetic waves induced cancer cell death as assessed by MTT cytotoxicity assays. These assays measure the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by mitochondrial reductases leading to the formation of purple formazan. MTT assays are commonly employed to detect decreased mitochondrial metabolic activity and cell death. The ability of RF waves to induce death in BT549 cells highlights a potential new intervention for poor prognosis basal breast cancer cells in the future. Introduction: Radio frequency is a measurement which represents the oscillation rate of electromagnetic radiation spectrum, or electromagnetic radio waves, from frequencies ranging from 300 GHz to as low as 9 kHz and radiofrequency ablation for cancer is a minimally invasive procedure that uses electrical energy and heat to destroy the cancer cells. Radiofrequency ablation is sometimes used to treat cancers in the: adrenal gland, breast, bone, kidney, liver, lung, pancreas, and thyroid. Electromagnetic resonance is produced by applying steady magnetic field simultaneously and electromagnetic radiation, usually radio waves to a sample of electrons and adjusting both the strength of the magnetic field and the frequency of the radiation to produce absorption of the radiation. While chemotherapy and other treatments that are taken by mouth or injection usually expose the whole body to cancerfighting drugs, radiation therapy is usually a local treatment, which means it is usually aimed at and affects only the part of the body that requires treatment. Radiation treatments are planned in such a way that they damage cancer cells with as little harm as possible to nearby healthy cells. Some cancers are very sensitive to radiation. Radiation may be used by itself in these cases to make the cancer shrink or completely disappear. In some cases, chemotherapy or other anti-cancer drugs may be given first and for other cancers, radiation may be used before surgery to shrink the tumor (this is called pre-operative therapy or neoadjuvant therapy), or after surgery to help keep the cancer from coming back (known as adjuvant therapy). Radiation not only kills or slows the growth of cancer cells; it can also affect nearby healthy cells and the damage to healthy cells can cause side effects. Cancer cells harbor diverse genetic mutational spectra and gene expression profiles that enable uncontrolled growth and survival. Breast cancer can be classified into at least five subgroups luminal A, luminal B, HER2 positive, basal and normal-like based on gene expression profiling. These subtypes of breast cancer are associated with distinctive characteristics such as cell fate and prognosis. Basal breast cancer typically lacks expression of the progesterone, estrogen and HER2 receptors and is associated with poor prognosis. Basal breast cancer is more commonly found in younger and African American patients. Oftentimes, breast cancer is treated with hormonal-based therapies or targeted therapy to HER2. However, basal breast cancer is resistant to hormonal and HER2-targeted therapies as these cancers lack the receptors necessary to elicit a response. Therefore, innovative therapies are needed to effectively treat basal breast cancer. On a cell biological level, the ultra-high frequency RF waves are thought to broadly disrupt cellular processes including metabolism, migration, mitotic spindle function and differentiation. Some studies implicate RF

A CRISPR View of Biological Mechanisms

A decade ago, only six manuscripts would be found on a PubMed search for “CRISPR,” compared to 2,011 manuscripts in 2016. The purpose of this review is to discuss this emergent technology that has revolutionized molecular biological research in just a few years. Endogenous CRISPR mechanisms are harbored by bacteria and archaea as an adaptive defense system that targets foreign DNA from viruses and plasmids. CRISPR has been adapted as a genome editing tool in a plethora of organisms ranging from yeast to humans. This tool has been employed to create loss of function mutations, gain of function mutations, and tagged alleles in a wide range of settings. CRISPR is now extensively employed for genetic screens. CRISPR has also been adapted to study transcriptional regulation. This versatile and relatively facile technique has, and will be, tremendously impactful in research areas such as biomedical sciences, agriculture, and the basic sciences.