Pathogenic C9ORF72 Antisense Repeat RNA Forms a Double Helix with Tandem C:C Mismatches.

Expansion of a GGGGCC/CCCCGG repeat sequence in the first intron of the C9ORF72 gene is a leading cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this combined disorder, called c9FTD/ALS, the expansion is bidirectionally transcribed into sense and antisense repeat RNA associated with disease. To better understand the role of C9ORF72 repeat RNA in molecular disease pathology, we determined crystal structures of a [(CCCCGG)3(CCCC)] model antisense repeat RNA to 1.47 Å resolution. The RNA structure was an A-form-like double helix composed of repeating and regularly spaced tandem C:C mismatch pairs that perturbed helical geometry and surface charge. Solution studies revealed a preference for A-form-like helical conformations as the repeat number increased. Results provide a structural starting point for rationalizing the contribution of repeat RNA to c9FTD/ALS molecular disease mechanisms and for developing molecules to target C9ORF72 repeat RNA as potential therapeutics.

Intramolecular circularization increases efficiency of RNA sequencing and enables CLIP-Seq of nuclear RNA from human cells.

RNA sequencing (RNA-Seq) is a powerful tool for analyzing the identity of cellular RNAs but is often limited by the amount of material available for analysis. In spite of extensive efforts employing existing protocols, we observed that it was not possible to obtain useful sequencing libraries from nuclear RNA derived from cultured human cells after crosslinking and immunoprecipitation (CLIP). Here, we report a method for obtaining strand-specific small RNA libraries for RNA sequencing that requires picograms of RNA. We employ an intramolecular circularization step that increases the efficiency of library preparation and avoids the need for intermolecular ligations of adaptor sequences. Other key features include random priming for full-length cDNA synthesis and gel-free library purification. Using our method, we generated CLIP-Seq libraries from nuclear RNA that had been UV-crosslinked and immunoprecipitated with anti-Argonaute 2 (Ago2) antibody. Computational protocols were developed to enable analysis of raw sequencing data and we observe substantial differences between recognition by Ago2 of RNA species in the nucleus relative to the cytoplasm. This RNA self-circularization approach to RNA sequencing (RC-Seq) allows data to be obtained using small amounts of input RNA that cannot be sequenced by standard methods.

Wide-pulse-high-frequency neuromuscular stimulation of triceps surae induces greater muscle fatigue compared with conventional stimulation.

We compared the extent and origin of muscle fatigue induced by short-pulse-low-frequency [conventional (CONV)] and wide-pulse-high-frequency (WPHF) neuromuscular electrical stimulation. We expected CONV contractions to mainly originate from depolarization of axonal terminal branches (spatially determined muscle fiber recruitment) and WPHF contractions to be partly produced via a central pathway (motor unit recruitment according to size principle). Greater neuromuscular fatigue was, therefore, expected following CONV compared with WPHF. Fourteen healthy subjects underwent 20 WPHF (1 ms-100 Hz) and CONV (50 µs-25 Hz) evoked isometric triceps surae contractions (work/rest periods 20:40 s) at an initial target of 10% of maximal voluntary contraction (MVC) force. Force-time integral of the 20 evoked contractions (FTI) was used as main index of muscle fatigue; MVC force loss was also quantified. Central and peripheral fatigue were assessed by voluntary activation level and paired stimulation amplitudes, respectively. FTI in WPHF was significantly lower than in CONV (21,717 ± 11,541 vs. 37,958 ± 9,898 N·s P<0,001). The reductions in MVC force (WPHF: -7.0 ± 2.7%; CONV: -6.2 ± 2.5%; P < 0.01) and paired stimulation amplitude (WPHF: -8.0 ± 4.0%; CONV: -7.4 ± 6.1%; P < 0.001) were similar between conditions, whereas no change was observed for voluntary activation level (P > 0.05). Overall, our results showed a different motor unit recruitment pattern between the two neuromuscular electrical stimulation modalities with a lower FTI indicating greater muscle fatigue for WPHF, possibly limiting the presumed benefits for rehabilitation programs.

Digital quantitation of potential therapeutic target RNAs.

Accurate determination of the amount of a given RNA within a cell is necessary to gain a full understanding of the RNA's function and regulation. Typically, the abundance of RNA is measured by quantitative polymerase chain reaction (qPCR). With qPCR, however, absolute quantification is not possible unless an adequate reference standard curve is generated. The method is not well suited for detecting low copy number templates and values vary depending on the specific primers used. To overcome these drawbacks, digital PCR (dPCR) has been developed to obtain exact values for RNA copies in a sample. Here we report the characterization of droplet digital PCR (ddPCR). We used ddPCR to quantify long noncoding RNAs from various subcellular compartments within human cells and found that results obtained using ddPCR parallel those from qPCR. Mutant huntingtin (HTT) protein is the cause of Huntington's Disease, and we show that we can quantify human HTT messenger RNA and discriminate between the mutant and wild-type HTT alleles using ddPCR. These results reveal insights into the design of experiments using ddPCR and show that ddPCR can be a robust tool for identifying the number of RNA species inside of cells.

Transcriptional silencing by hairpin RNAs complementary to a gene promoter.

Double-stranded RNAs can target gene promoters and inhibit transcription. To date, most research has focused on synthetic RNA duplexes. Transcriptional silencing by hairpin RNAs would facilitate a better understanding of endogenous RNA-mediated regulation of transcription within cells. Here we examine transcriptional silencing of progesterone receptor (PR) expression by hairpin RNAs. We identify the guide strand as the strand complementary to an antisense transcript at the PR promoter and that hairpin RNAs are active transcriptional silencing agents. The sequence of the hairpin loop affects activity, with the highest activity achieved when the loop has the potential for full complementarity to the antisense transcript target. Introduction of centrally mismatched bases relative to the target transcript does not prevent transcriptional silencing unless the mismatches are present on both the guide and passenger strands. These data demonstrate that hairpin RNAs can cause transcriptional silencing and offer insights into the mechanism of gene modulation by RNAs that target gene promoters.

Safety and cost-effectiveness of a clinical protocol implemented to standardize the use of Crotalidae polyvalent immune Fab antivenom at an academic medical center.

STUDY OBJECTIVE: To evaluate the safety and cost-effectiveness of a clinical protocol adopted in June 2006 that included a comprehensive, objective assessment of snake bite envenomations and standardized the use of Crotalidae polyvalent immune Fab antivenom (FabAV). DESIGN: Retrospective medical record review. SETTING: Academic medical center that serves as the regional level I trauma center. PATIENTS: Seventy-five adults treated with FabAV for snake envenomations in the emergency department between June 1, 2003, and June 1, 2009; 30 patients received treatment according to the protocol (treatment group), and 45 patients received treatment that did not adhere to the protocol (control group). MEASUREMENTS AND MAIN RESULTS: Demographic and envenomation characteristics, as well as treatment details, were collected for all patients. In addition, information on quantity of FabAV vials required, length of hospital stay, and length of intensive care unit stay were compared between the treatment and control groups. In the treatment group, significantly fewer vials of FabAV were used (2.5 vs 4.727 vials, p=0.007). This decreased in usage correlated to a cost savings of approximately $2000/patient. Despite no significant difference in the severity of the envenomations between the two groups (p=0.379), the treatment group experienced a significantly shorter hospital length of stay (1.933 vs 2.791 days, p=0.030). No significant difference in the progression to fasciotomy or the development of allergic reactions was noted between the two groups. CONCLUSION: Use of a clinical protocol related to snake envenomations resulted in approximately two fewer vials of FabAV required for each patient. In addition, the treatment group experienced a shorter hospital length of stay without a corresponding increase in adverse events or envenomation progression. Data show that use of the protocol was cost-effective. The development of institution-specific multidisciplinary protocols regarding snake bite envenomations is recommended. Clinical pharmacists can play a vital role in the protocol development to ensure that optimal care is provided for this distinct patient population.

Electrogenerated chemiluminescence of triazole-modified deoxycytidine analogues in N,N-dimethylformamide.

Triazole-modified deoxycytidines have been prepared for incorporation into single-stranded deoxyribonucleic acid (ssDNA). Electrochemical responses and electrogenerated chemiluminescence (ECL) of these deoxycytidine (dC) analogues, 1-4, were investigated as the monomers. Cyclic voltammetry and differential pulse voltammetry techniques were used to determine the oxidation and reduction potentials of 1-4, along with the reversibility of their electrochemical reactions. The dC analogues, in N,N-dimethylformamide containing 0.1 M tetra-n-butylammonium perchlorate as electrolyte, exhibited weak relative ECL efficiencies following the annihilation mechanism, while these efficiencies were enhanced with the use of benzoyl peroxide following the coreactant mechanism. It was shown that these nucleosides could generate excited monomers, and excimers as seen by the red-shifted ECL maxima relative to their corresponding photoluminescence peak wavelengths.

Peptide nucleic acid Pt(II) conjugates: a preliminary study of antisense effects in Xenopus laevis.

The avid hybridization of peptide nucleic acid (PNA) to DNA and RNA, coupled with the analogue's stability toward enzymatic degradation, has led to its investigation as an antigene/antisense agent. PNA targeted toward the 5'-UTR of an mRNA transcript can effect efficient silencing; however, if targeted to an area within the coding region, the PNA can be displaced by the moving ribosome and be an ineffective antisense agent. Platinum-appended and standard PNAs antisense to an area within the open-reading frame of the gene noggin, were injected into Xenopus laevis embryos. Phenotypic responses were observed and the preliminary results are reported herein.

Blue fluorescent deoxycytidine analogues: convergent synthesis, solid-state and electronic structure, and solvatochromism.

We report the synthesis and photospectroscopic characterisation of intrinsically fluorescent triazole-appended cytidines. Fluorescence was found to be highly dependent on solvent conditions. X-Ray crystallographic data show the proton of the exocyclic amine of the nucleobase and the triazole N(3) engaged in a H-bond.

Hydrogelation abilities of nucleobase-modified cytidines possessing substituted triazoles.

Nucleoside-derived hydrogelators have been sought for their potential biomedical applications, such as are found in tissue engineering and drug delivery. By judiciously adding a degree of hydrophobicity certain analogues are able to form micelles, bi-layers and gels in water. Research in this area has yet to lay down solid ground rules for the rational design of novel nucleoside gelators making further studies necessary. The synthesis and examination of a series of aryl-substituted 5-triazolylcytidines yielded an analogue that gelates water. 5-(1-(2,2'-bithiophen-3-yl)-1H-1,2,3-triazol-4-yl)-2'-deoxycytidine was found to form gels in water down to 0.3 wt%. The ribocytidine analogue failed to form gel in aqueous solution; but was able to form a hydrogel in the presence of guanosine. Images obtained by SEM show the different architectures of the gel; varying from cribriform to fibrous to lamellar. The present gelating compound studied may have potential as a component of a controlled-release drug delivery system.

Cellular localization and allele-selective inhibition of mutant huntingtin protein by peptide nucleic acid oligomers containing the fluorescent nucleobase [bis-o-(aminoethoxy)phenyl]pyrrolocytosine.

Peptide nucleic acid (PNA) is a successful DNA/RNA mimic. A major challenge for research is to invent chemically modified PNAs that retain the favorable properties of the parent compound while improving biological recognition. Here, we test modified PNAs containing [bis-o-(aminoethoxy)phenyl]pyrrolocytosine bases designed to engage guanine with an additional hydrogen bond. We observe elevated melting temperatures, localization to cellular compartments, and allele-selective inhibition of mutant huntingtin protein expression.

Gamma-radiation induced interstrand cross-links in PNA:DNA heteroduplexes.

Peptide nucleic acids (PNAs) efficiently hybridize with DNA and are promoted as versatile gene-targeting analytical tools and pharmaceuticals. However, PNAs have never been exploited as radiopharmaceuticals, and radiation-induced physicochemical modifications of PNA:DNA heteroduplexes have not been studied. Drug- and radiation-induced creation of covalent cross-links in DNA obstruct crucial cell survival processes such as transcription and replication and are thus considered genotoxic events with a high impact in anticancer therapies. Here we report that gamma-irradiation of complementary PNA:DNA heteroduplexes, wherein the PNA contains l-lysine, free amino, or N-methylmorpholinium N- and C-capping groups, results in the formation of irreversible interstrand cross-links (ICL). The number of detected ICL corresponds to the number of available amino functional groups on the PNA. The effect of DNA sequence on the formation of ICL was studied by modifying the terminal nucleotides of the DNA oligonucleotide to create deletions and overhangs. The involvement of abasic sites (ABS) on the DNA strand in the cross-linking reaction was confirmed by independent experiments with synthetic ABS-containing oligonucleotides. Molecular modeling and molecular dynamics (MD) simulations were applied to elucidate the conformation of the N- and C-capping groups of the PNA oligomer and their interactions with the proximal terminus of the DNA. Good agreement between experimental and modeling results was achieved. Modeling indicated that the presence of positively charged capping groups on the PNA increases the conformational flexibility of the PNA:DNA terminal base pairs and often leads to their melting. This disordered orientation of the duplex ends provides conditions for multiple encounters of the short (amino) and bulky (Lys) side chains with nucleobases and the DNA backbone up to the third base pair along the duplex stem. Dangling duplex ends offer favorable conditions for increased accessibility of the radiation-induced free radicals to terminal nucleotides and their damage. It is suggested that the ICL are produced by initial formation of Schiff base adducts between the PNA amino functions and the opposed DNA oxidation-damaged bases or abasic 2'-deoxyribose-derived aldehydic groups. The subsequent reduction by solvated electrons (e(-)(aq)) or other radiation-produced reducing species results in irreversible covalent interstrand cross-links. The simultaneous involvement of oxidizing, (*)OH, and reducing, e(-)(aq), radicals presents a case in which multiple ionization events along a gamma-particle path lead to DNA injuries that also encompass ICL as part of the multiply damaged sites (MDS). The obtained results may find applications in the development of a new generation of gene-targeted radiosensitizers based on PNA vectors.

A continuing medical education approach to improve sexual boundaries of physicians.

INTRODUCTION: Physician sexual boundary violations are a public health problem. Few resources exist to address physicians who behave inappropriately with patients. In response, the Center for Professional Health at Vanderbilt University developed a three-day continuing medical education (CME) course about proper professional sexual boundaries in 2000. The mission of this CME course is to offer an educational intervention for those physicians whose professional sexual misconduct has required such education as part of a larger accountability sanction. Previous studies suggest that when such education is offered through non-traditional medical education, it is effective in promoting behavioral change. This paper describes the three-day intensive educational experience offered by a CME course with a particular focus on lessons learned from more than 7 years of experience working with these physicians. METHODS: Over 381 physicians from 40 states and Canada have attended. Data about course participants was collected by self-report and aggregated into three categories: demographics, results of assessment tools administered, and quality of the experience. Assessment tools used include the Family Adaptability and Cohesion Evaluation Scale II (FACES II), the Trauma Symptom Inventory (TSI) and the Sexual Addiction Screening Test (SAST). RESULTS: Most physicians were referred to the course from physician health programs and boards of medical examiners. The majority of physician participants were male and in group or solo practice. A full range of medical specialties was represented with most physicians being internists, psychiatrists, obstetricians and surgeons. Results of assessment tools administered indicate that physicians referred for sexual boundary violations often come from dysfunctional families and demonstrate symptoms indicative of trauma related problems and possible sexual addiction. Physician attendees report being highly satisfied with the new knowledge attained in this course. DISCUSSION: Curriculum aimed at addressing sexual boundary violations should address family of origin issues, trauma coping skills and sexual acting out. Satisfaction data continues to support a small group, experiential, and confidential format as an effective means for intervention. CONCLUSION: A CME course offers a model for future training experiences for faculty, residents, medical students and community physicians to teach skills that may help prevent and remediate professional boundary crossings.

Analysis and purification of peptide nucleic acids by denaturing PAGE.

A flexible and convenient protocol for the analysis and purification of peptide nucleic acid (PNA) oligomers and PNA-peptide chimeras by denaturing PAGE is described. Vertical slab gel electrophoresis, 26% in polyacrylamide and 8 M urea at pH 3, was suitable for analysis of oligomers ranging in size from tetramers (4-mers) to tetradodecamers (24-mers). Single-base resolution of oligomers was achieved and separations are generally superior to those given by standard RP-HPLC techniques. The separation of a related series of PNA oligomers showed the distance migrated was linearly dependent on the logarithm of the molecular weight. The migration of oligomers through the gel is dependent on the number of basic functional groups present, such as amino groups, and the A and C content of the oligomer. PNAs are amenable to detection by UV-shadowing technique illuminated at 260 nm or Coomassie blue staining, both with similar, sub-microgram per band detection limits.