Cellular Delivery of Locked Nucleic Acids (LNAs)

Locked nucleic acids (LNAs) are RNA derivatives that have an O‐methylene linkage between the 2 and 4 positions of the ribose. This leads to exceptionally high‐affinity binding to complementary sequences. They are synthesized using standard DNA/RNA synthesis methods, and have a negatively charged backbone that confers good solubility. This unit describes a method for the introduction of LNA oligomers into cells. A support protocol also describes the determination of melting temperatures for LNA oligomers.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143781/1/cpnc0413.pd

Synthesis and Purification of Peptide Nucleic Acids

Peptide nucleic acids (PNAs) are DNA analogs in which the normal phosphodiester backbone is replaced by 2‐aminoethyl glycine linkages. Hybridization of PNAs with RNA or DNA follows normal rules for Watson‐Crick base pairing and occurs with high affinity. Thus, PNAs are a promising choice for applications that benefit from high‐affinity hybridization. They are assembled using techniques adapted from peptide chemistry. Protocols are given for both automated and manual synthesis of PNAs as well as their purification. The advantages of each method are discussed, as are the different monomers and reagents that are required. Additionally, protocols are given for adding peptides to PNAs (which can enhance hybridization or cell uptake of the PNA) and for adding a biotin label.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143745/1/cpnc0411.pd

Loss of cell viability is tracked by decreased cytoplasmic conductivity

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Current Topics Novel Antisense and Peptide Nucleic Acid Strategies for Controlling Gene Expression †

ABSTRACT: Antisense oligonucleotides have the potential to make revolutionary contributions to basic science and medicine. Oligonucleotides can bind mRNA and inhibit translation. Because they can be rapidly synthesized to be complementary to any sequence, they offer ideal tools for exploiting the massive amount of genome information now available. However, until recently, this potential was largely theoretical, and antisense experiments often produced inconclusive or misleading outcomes. This review will discuss the chemical and biological properties of some of the different types of oligomers now available and describe the challenges confronting in vitro and in vivo use of oligonucleotides. Oligomers with improved chemical properties, combined with advances in cell biology and success in clinical trials, are affording powerful new options for basic research, biotechnology, and medicine

Acute-Phase CD8 T cell responses that select for escape variants are needed to control live attenuated simian immunodeficiency virus

The overall CD8 T cell response to human/simian immunodeficiency virus (HIV/SIV) targets a collection of discrete epitope specificities. Some of these epitope-specific CD8 T cells emerge in the weeks and months following infection and rapidly select forsequence variants, whereas other CD8 T cell responses develop during the chronic infection phase and rarely select for sequence variants. In this study, we tested the hypothesis that acute-phase CD8 T cell responses that do not rapidly select for escapevariants are unable to control viral replication in vivo as well as those that do rapidly select for escape variants. We created a derivative of live attenuated SIV (SIVmac239Δnef) in which we ablated five epitopes that elicit early CD8 T cell responses and rapidly accumulate sequence variants in SIVmac239-infected Mauritian cynomolgus macaques (MCMs) that are homozygous for the M3 major histocompatibility complex (MHC) haplotype. This live attenuated SIV variant was called m3KOΔnef. Viremia was significantly higher in M3 homozygous MCMs infected withm3KOΔnef than in either MHC-mismatched MCMs infected with m3KOΔ nef or MCMs infected with SIVmac239Δnef. Three CD8 T cell responses, including two that do not rapidly select for escape variants, predominated during earlym3KOΔnef infection in the M3 homozygous MCMs, but these animals were unable to control viral replication. These re-sults provide evidence that acute-phase CD8 T cell responses that have the potential to rapidly select for escape variants in the early phase ofinfection are needed to establish viral control in vivo

The Development of an Open Source Intelligence Gathering Exercise for Teaching Information Security & Privacy

This research-in-progress paper describes the development of a pedagogical exercise on open source intelligence gathering (OSINT). Exercise materials will include instructions, teaching notes, assessment criteria, and a preconfigured virtual machine (VM), which acts as a local web server. The VM will host multiple websites containing vulnerable information pertinent to a fictitious target organization, in effect creating a capture the flag (CTF) scenario. The exercise will not only teach students how to find public information, but also help students realize the importance of protecting such information. While this exercise is primarily geared towards those pursuing a career in information security, the exercise is appropriate for all students as it shows how personal information could be used against them, as well as their organizations

Destruction of Opportunistic Pathogens Via Polymer Nanoparticle-Mediated Release of Plant-Based Antimicrobial Payloads

The synthesis of antimicrobial thymol/carvacrol‐loaded polythioether nanoparticles (NPs) via a one‐pot, solvent‐free miniemulsion thiol‐ene photopolymerization process is reported. The active antimicrobial agents, thymol and carvacrol, are employed as “solvents” for the thiol‐ene monomer phase in the miniemulsion to enable facile high capacity loading (≈50% w/w), excellent encapsulation efficiencies (\u3e95%), and elimination of all postpolymerization purification processes. The NPs serve as high capacity reservoirs for slow‐release and delivery of thymol/carvacrol‐combination payloads that exhibit inhibitory and bactericidal activity (\u3e99.9% kill efficiency at 24 h) against gram‐positive and gram‐negative bacteria, including both saprophytic (Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 25922) and pathogenic species (E. coli ATCC 43895, Staphylococcus aureus RN6390, and Burkholderia cenocepacia K56‐2). This report is among the first to demonstrate antimicrobial efficacy of essential oil‐loaded nanoparticles against B. cenocepacia – an innately resistant opportunistic pathogen commonly associated with debilitating respiratory infections in cystic fibrosis. Although a model platform, these results point to promising pathways to particle‐based delivery of plant‐derived extracts for a range of antimicrobial applications, including active packaging materials, topical antiseptics, and innovative therapeutics