Novel Mobile Phase to Control Charge States and Metal Adducts in the LC/MS for mRNA Characterization Assays.

Mass spectrometry is a widely used tool in the characterization of oligonucleotides. This analysis can be challenging due to the large number of possible charge states of oligonucleotides, which can limit the sensitivity of the assay, along with the propensity of oligonucleotides to readily form adducts with free alkali metals. To reduce the adduct formation, oligonucleotides are typically purified with desalting columns prior to analysis. We have developed a mobile phase that gives superior reduction in charge states and adduct formation compared to previously reported methods and, more importantly, obviates the requirement of desalting samples prior to mass spectrometric analysis, significantly decreasing the sample preparation time and amount of RNA required for analysis. We have applied this mobile phase to develop methods to quantify the 5'-capping efficiency and to characterize the polyadenosine (poly(A)) tail of mRNA synthesized in vitro: two critical quality attributes of mRNA therapeutics. Through this, we were able to demonstrate RNA that was co-transcriptionally capped to have capping efficiency equivalent (the percent total molecules that contain a cap) to other reports in the literature using materials that were generated using the same synthesis procedure. Furthermore, by using a mobile phase mixture comprised of hexafluoroisopropanol, triethylammonium acetate, triethylamine, and ethanol, we were able to determine the size distribution of the poly(A) tail in various mRNA samples from DNA templates that ranged from 50 to 150 nt poly(A) and verify that distribution with commercially available RNA standards, successfully demonstrating that this mobile phase composition could be used for characterization assays for both mRNA caps and tails.

Radiofrequency Identification Track for Tray Optimization: An Instrument Utilization Pilot Study in Surgical Oncology.

BACKGROUND: Surgical instrument tray reduction attempts to minimize intraoperative inefficiency and processing costs. Previous reduction methods relied on trained observers manually recording instrument use (i.e. human ethnography), and surgeon and/or staff recall, which are imprecise and inherently limited. We aimed to determine the feasibility of radiofrequency identification (RFID)-based intraoperative instrument tracking as an effective means of instrument reduction. METHODS: Instrument trays were tagged with unique RFID tags. A RFID reader tracked instruments passing near RFID antennas during 15 breast operations performed by a single surgeon; ethnography was performed concurrently. Instruments without recorded use were eliminated, and 10 additional cases were performed utilizing the reduced tray. Logistic regression was used to estimate odds of instrument use across cases. Cohen's Kappa estimated agreement between RFID and ethnography. RESULTS: Over 15 cases, 37 unique instruments were used (median 23 instruments/case). A mean 0.64 (median = 0, range = 0-3) new instruments were added per case; odds of instrument use did not change between cases (OR = 1.02, 95%CI 1.00-1.05). Over 15 cases, all instruments marked as used by ethnography were recorded by RFID tracking; 7 RFID-tracked instruments were never recorded by ethnography. Tray size was reduced 40%. None of the 25 eliminated instruments were required in 10 subsequent cases. Cohen's Kappa comparing RFID data and ethnography over all cases was 0.82 (95%CI 0.79-0.86), indicating near perfect agreement between methodologies. CONCLUSIONS: Intraoperative RFID instrument tracking is a feasible, data-driven method for surgical tray reduction. Overall, RFID tracking represents a scalable, systematic, and efficient method of optimizing instrument supply across procedures.

Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa.

Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT.IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

Nuclear Noncoding RNAs and Genome Stability.

In modern molecular biology, RNA has emerged as a versatile macromolecule capable of mediating an astonishing number of biological functions beyond its role as a transient messenger of genetic information. The recent discovery and functional analyses of new classes of noncoding RNAs (ncRNAs) have revealed their widespread use in many pathways, including several in the nucleus. This Review focuses on the mechanisms by which nuclear ncRNAs directly contribute to the maintenance of genome stability. We discuss how ncRNAs inhibit spurious recombination among repetitive DNA elements, repress mobilization of transposable elements (TEs), template or bridge DNA double-strand breaks (DSBs) during repair, and direct developmentally regulated genome rearrangements in some ciliates. These studies reveal an unexpected repertoire of mechanisms by which ncRNAs contribute to genome stability and even potentially fuel evolution by acting as templates for genome modification.

Electrostatic Localization of RNA to Protocell Membranes by Cationic Hydrophobic Peptides.

Cooperative interactions between RNA and vesicle membranes on the prebiotic earth may have led to the emergence of primitive cells. The membrane surface offers a potential platform for the catalysis of reactions involving RNA, but this scenario relies upon the existence of a simple mechanism by which RNA could become associated with protocell membranes. Here, we show that electrostatic interactions provided by short, basic, amphipathic peptides can be harnessed to drive RNA binding to both zwitterionic phospholipid and anionic fatty acid membranes. We show that the association of cationic molecules with phospholipid vesicles can enhance the local positive charge on a membrane and attract RNA polynucleotides. This phenomenon can be reproduced with amphipathic peptides as short as three amino acids. Finally, we show that peptides can cross bilayer membranes to localize encapsulated RNA. This mechanism of polynucleotide confinement could have been important for primitive cellular evolution.

Regulation of histone methylation by noncoding RNAs.

Cells can adapt to their environment and develop distinct identities by rewiring their transcriptional networks to regulate the output of key biological pathways without concomitant mutations to the underlying genes. These alterations, called epigenetic changes, persist stably through mitotic or, in some instances, meiotic cell divisions. In eukaryotes, heritable changes to chromatin structure are a prominent, but not exclusive, mechanism by which epigenetic changes are mediated. These changes are initiated by sequence-specific events, which trigger a cascade of molecular interactions resulting in feedback mechanisms, alterations in chromatin structure, histone posttranslational modifications (PTMs), and ultimately establishment of distinct transcriptional states. In recent years, advances in next generation sequencing have led to the discovery of several novel classes of noncoding RNAs (ncRNAs). In addition to their well-established cytoplasmic roles in posttranscriptional regulation of gene expression, ncRNAs have emerged as key regulators of epigenetic changes via chromatin-dependent mechanisms in organisms ranging from yeast to man. They function by affecting chromatin structure, histone PTMs, and the recruitment of transcriptional activating or repressing complexes. Among histone PTMs, lysine methylation serves as the binding substrate for the recruitment of key protein complexes involved in the regulation of genome architecture, stability, and gene expression. In this review, we will outline the known mechanisms by which ncRNAs of different origins regulate histone methylation, and in doing so contribute to a variety of genome regulatory functions in eukaryotes.

Brain-penetrant LSD1 inhibitors can block memory consolidation.

Modulation of histone modifications in the brain may represent a new mechanism for brain disorder therapy. Post-translational modifications of histones regulate gene expression, affecting major cellular processes such as proliferation, differentiation, and function. An important enzyme involved in one of these histone modifications is lysine specific demethylase 1 (LSD1). This enzyme is flavin-dependent and exhibits homology to amine oxidases. Parnate (2-phenylcyclopropylamine (2-PCPA); tranylcypromine) is a potent inhibitor of monoamine oxidases and derivatives of 2-PCPA have been used for development of selective LSD1 inhibitors based on the ability to form covalent adducts with flavin adenine dinucleotide (FAD). Here we report the synthesis and in vitro characterization of LSD1 inhibitors that bond covalently to FAD. The two most potent and selective inhibitors were used to demonstrate brain penetration when administered systemically to rodents. First, radiosynthesis of a positron-emitting analog was used to obtain preliminary bio-distribution data and whole brain time-activity curves. Second, we demonstrate that this series of LSD1 inhibitors is capable of producing a cognitive effect in a mouse model. By using a memory formation paradigm, novel object recognition, we show that LSD1 inhibition can abolish long-term memory formation without affecting short-term memory, providing further evidence for the importance of reversible histone methylation in the function of the nervous system.

Enrolling eligible children and keeping them enrolled.

Coverage under Medicaid and the State Children's Health Insurance Program (SCHIP) provides low-income children with a vital link to needed health care, yet a significant proportion of children eligible for these programs remain uninsured. States have found that expanding eligibility and marketing new programs are not enough to increase enrollment of eligible uninsured children in public health programs. States also need to simplify enrollment and renewal procedures to make them more family-friendly. According to survey data, a key reason for underenrollment is that families find enrollment and renewal procedures too complex. This article details the efforts that states have made to increase enrollment in Medicaid and SCHIP, and it offers recommendations for strengthening these efforts. Although barriers to enrollment and renewal still exist, states are making progress in several ways, such as: Simplifying eligibility procedures. Using community-based application assistance. Eliminating procedural differences between Medicaid and separate SCHIP programs. The authors recommend that states continue to simplify program requirements and procedures, making it easier for children to enroll in Medicaid and SCHIP, retain coverage for as long as they qualify, and transfer between programs when necessary. In addition, outreach and community-based application assistance will continue to be essential activities, along with developing efforts to enroll children through other public programs, such as the food stamp program.