Silencing disease genes in the laboratory and the clinic

Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past 20 years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology

Functional characterization of synthetic leukotriene B and its stereochemical isomers.

Leukotriene B (LTB), a potent lipid chemotactic factor for neutrophils, is 5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid (Fig 1), based upon direct comparison of natural LTB with synthetic 5S,12R-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-di-HETE) stereoisomers in three biological assays. Of the six synthetic stereoisomers evaluated, only the 5S,12R,6,14-cis,8,10-trans compound had chemotactic potency for human neutrophils in vitro that was comparable to that of natural LTB, with a concentration of 3 X 10(9-9) M eliciting a one-half maximum response. In contrast, the racemic mixture of 5R,12R- and 5S,12S-6,10-trans,8,14-cis, the racemic mixture of 5S,12R- and 5R,12S-6,10-trans,8,14-cis, the 5S,12R-6,8-trans,10,14-cis, the 5S,12R-6,8,10-trans,14-cis, and the 5S,12S-6,8,10-trans,14-cis stereoisomers required concentrations of 3 X 10(-7) to 1 X 10(-6) M to elicit comparable responses. Only natural LTB and its synthetic counterpart elicited a local neutrophil infiltration when injected into the skin of the rhesus monkey at 10 ng and 100 ng per site. Natural and synthetic LTB at a concentration of 3 X 10(-8) M each provoked an EC25 contractile response of guinea pig pulmonary parenchymal strips in vitro, whereas the other four tested stereoisomers of 5,12-di-HETE were inactive at this concentration. Structure-function analyses suggest that the neutrophil chemotactic activity depends critically upon the C-1 to C-12 domain, including the stereochemistry of the 6-,8-,and 10-olefinic bonds and the presence of both hydroxyl groups

Damage areas on selected LDEF aluminum surfaces

With the U.S. about to embark on a new space age, the effects of the space environment on a spacecraft during its mission lifetime become more relevant. Included among these potential effects are degradation and erosion due to micrometeoroid and debris impacts, atomic oxygen and ultraviolet light exposure as well as material alteration from thermal cycling, and electron and proton exposure. This paper focuses on the effects caused by micrometeoroid and debris impacts on several LDEF aluminum plates from four different bay locations: C-12, C-10, C-01, and E-09. Each plate was coated with either a white, black, or gray thermal paint. Since the plates were located at different orientations on the satellite, their responses to the hypervelocity impacts varied. Crater morphologies range from a series of craters, spall zones, domes, spaces, and rings to simple craters with little or no spall zones. In addition, each of these crater morphologies is associated with varying damage areas, which appear to be related to their respective bay locations and thus exposure angles. More than 5% of the exposed surface area examined was damaged by impact cratering and its coincident effects (i.e., spallation, delamination and blow-off). Thus, results from this analysis may be significant for mission and spacecraft planners and designers

Antibacterial Activity of and Resistance to Small Molecule Inhibitors of the ClpP Peptidase

There is rapidly mounting evidence that intracellular proteases in bacteria are compelling targets for antibacterial drugs. Multiple reports suggest that the human pathogen Mycobacterium tuberculosis and other actinobacteria may be particularly sensitive to small molecules that perturb the activities of self-compartmentalized peptidases, which catalyze intracellular protein turnover as components of ATP-dependent proteolytic machines. Here, we report chemical syntheses and evaluations of structurally diverse β-lactones, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase. β-Lactones with certain substituents on the α- and β-carbons were found to be toxic to M. tuberculosis. Using an affinity-labeled analogue of a bioactive β-lactone in a series of chemical proteomic experiments, we selectively captured the ClpP1P2 peptidase from live cultures of two different actinobacteria that are related to M. tuberculosis. Importantly, we found that the growth inhibitory β-lactones also inactivate the M. tuberculosis ClpP1P2 peptidase in vitro via formation of a covalent adduct at the ClpP2 catalytic serine. Given the potent antibacterial activity of these compounds and their medicinal potential, we sought to identify innate mechanisms of resistance. Using a genome mining strategy, we identified a genetic determinant of β-lactone resistance in Streptomyces coelicolor, a non-pathogenic relative of M. tuberculosis. Collectively, these findings validate the potential of ClpP inhibition as a strategy in antibacterial drug development and define a mechanism by which bacteria could resist the toxic effects of ClpP inhibitors.National Institutes of Health (U.S.) (Grant GM-101988

Balance-Related Outcome Measures of Acquired Brain Injury Patients in a Student-Led Onsite Physical Therapy Clinic: A Retrospective Records Review

Title: Balance-Related Outcome Measures of Acquired Brain Injury Patients in a Student-Led Onsite Physical Therapy Clinic: A Retrospective Records Review Authors: Parke Humphrey, SPT; Corey Kaleshnik, SPT; Lauren Wilson, SPT; Ann Wilson, PT, MEd, GCS Affiliation(s): 1. Physical Therapy Program, University of Puget Sound Purpose: Individuals with Acquired Brain Injury (ABI) often have balance impairments. Interventions targeting these impairments may improve balance and thus increase overall societal participation. The purpose of this review was to identify the types of balance-related outcome measures used to assess balance impairments in patients with ABI in a student-led onsite physical therapy clinic. Subjects: 13 individuals with an ABI receiving care at a student-run clinic. Materials & Methods: 23 records were selected for this review; however, only 13 of these met inclusion criteria. 12 subjects had a primary diagnosis of CVA and 1 subject had a primary diagnosis of TBI. The average number of balance measures per subject was 2.25 with all subjects having at least one measure taken. The mean age of the subjects was 59.8 years with a range of 35 to 85 years. The mean amount of time since ABI onset to the episode of care was 5.2 years with a range of 8 months to 21 years and a standard deviation of 4.8 years. The mean number of visits for our subjects in each episode of care was 12.4 with a range of 4 to 19 visits and a standard deviation of 4.9. The named balance measures we evaluated were the Berg Balance Scale, Dynamic Gait Index, Timed Up and Go, and the Mini-BESTest. Additionally there were subjects that were evaluated based on general static and dynamic balance. Results: The most commonly used recognized outcome measures were the Berg Balance Scale (N=8), Dynamic Gait Index (N=3), Timed up and Go (N=2) and the Mini-BESTest (N=2). In addition 8 records identified that either static or dynamic balance or both were assessed using other methods (N=8). Of the 13 subjects, 9 improved by a score greater than or equal to the MDC or MCID. Conclusions: Although the majority of student therapists are using validated measures to assess balance, a number are assessing balance in other ways that make it difficult to determine which components of balance are being assessed or how successful the targeted interventions were. Despite this, meaningful change was seen in 69.2% (N=9) of subjects that were evaluated with recognized outcome measures. Clinical Relevance: Meaningful change can be seen in patients with ABI who may be several years out from their original injury in relatively short episodes of care or with infrequent visits. While general balance measures might be appropriate to help inform a therapist’s decision-making process to work on specific deficits, this review highlights the importance of also including a named outcome measure to detect significant changes across many case studies. References 1. What is the Difference Between an Acquired Brain Injury and a Traumatic Brain Injury. Brain Injury Association of America website. http://www.biausa.org/FAQRetrieve.aspx?ID=43913. Updated 2014. Accessed 8 November 2014. 2. Caeyenberghs K, Leemans A, Geurts M, Taymans T, Vander Linden C, Smits-Engelsman BC, Sunaert S, Swinnen SP. Brain-behavior relationships in young traumatic brain injury patients: fractional anisotropy measures are highly correlated with dynamic visuomotor tracking performance. Neuropsychologia 2010 Apr;48(5):1472-82. Doi: 10.1016/j.neuropsychologia.2010.01.017. 3. Hoffer ME, Balough BJ, Gottshall KR. Posttraumatic balance disorders. Int Tinnitus J. 2007;13(1):69-72. 4. Perry SB, Woollard J, Little S, Shroyer K. Relationships among measures of balance, gait, and community integration in people with brain injury. J Head Trauma Rehabil 2014;29(2):117-124. doi:10.1097/HTR.0b013e3182864f2f. 5. Sartor-Glittenberg C, Brickner L. A multidimensional physical therapy program for individuals with cerebellar ataxia secondary to traumatic brain injury: a case series. Physiother Theory Pract 2014;30(2):138-148. doi:10.3109/09593985.2013.819952

Enabling Complex Fibre Geometries Using 3D Printed Axon-Mimetic Phantoms

Purpose: To introduce a method to create 3D-printed axon-mimetic phantoms with complex fibre orientations to characterise the performance of diffusion magnetic resonance imaging (MRI) models and representations in the presence of orientation dispersion. Methods: An extension to an open-source 3D printing package was created to produce a set of five 3D-printed axon-mimetic (3AM) phantoms with various combinations of bending and crossing fibre orientations. A two-shell diffusion MRI scan of the five phantoms in water was performed at 9.4T. Diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), the ball and stick model, neurite orientation density and dispersion imaging (NODDI), and Bingham-NODDI were all fit to the resulting diffusion MRI data. A ground truth map of that phantom’s crossing angles and/or arc radius was registered to the diffusion-weighted images. Metrics from each model and representation were compared to the ground-truth maps, and a quadratic regression model was fit to each combination of output metric and ground-truth metric. Results: The mean diffusivity (MD) metric defined by DTI was insensitive to crossing angle but increased with fibre curvature. Axial diffusivity (AD) decreased with increasing crossing angle. DKI’s diffusivity metrics replicated the trends seen in DTI, and its mean kurtosis (MK) metric decreased with fibre curvature, except in regions with high crossing angles. The estimated stick volume fraction in the ball and stick model decreased with increasing fibre curvature and crossing angle. NODDI’s intra-neurite volume fraction was insensitive to crossing angle, and its orientation dispersion index (ODI) was correlated to crossing angle. Bingham-NODDI’s intra-neurite volume fraction was also insensitive to crossing angle, while its primary ODI (ODIP) was also correlated to crossing angle and its secondary ODI (ODIS) was insensitive to crossing angle. For both NODDI models, the volume fractions of the extra-neurite and CSF compartments had low reliability with no clear relationship to crossing angle. Conclusion: Inexpensive 3D-printed axon-mimetic phantoms can be used to investigate the effect of fibre curvature and crossings on diffusion MRI representations and models of diffusion signal. The dependence of several representations and models on fibre dispersion/crossing was investigated. As expected, Bingham-NODDI was best able to characterise planar fibre dispersion in the phantoms

An evaluation of Gout visits in the United States for the years 2007 to 2011

Abstract Background This study analyzed visits for and factors associated with gout and gout medication treatment trends for the years 2007–2011 in the United States given the introduction of febuxostat, the first new treatment option for gout in over 40 years, which was introduced to the market in 2009. Methods This study was a retrospective, cross-sectional, observational study of patients age 20 and older seen by providers who participated in the National Ambulatory Medical Care Survey (NAMCS), the National Hospital Ambulatory Medical Care Survey Outpatient Department (NHAMCS-OPD) or Emergency Department (NHAMCS-ED) in the United States. The outcome of interest was visits for gout diagnosis and visits where a gout medication was prescribed. Results Approximately 1.2% of visits had a diagnosis of gout. There was a significant increase in the percentage of visits with a diagnosis of gout in years 2009–2011 compared to 2007–2008, which remained after adjusting for covariates of interest. Groups more likely to have a visit with gout included those ≥65 and 45–64 (both as compared to those 20–44), the African-American and ‘Other’ race groups (as compared to Caucasians) and those on a diuretic. Groups less likely to have a visit with gout included females, Hispanic/Latinos, those with insurance type of ‘Other’ and Medicaid (both as compared to private insurance) and visits to a hospital emergency setting (as compared to physician’s office visits). Conclusion Although there was a significant increase in visits where gout is diagnosed across study years, the overall percentage of visits with a gout diagnosis is low in the US population. Treatment trends over the study years has remained consistent, with the introduction of febuxostat appearing to have little impact for the study years through 2011

Summer habitat use and movements of invasive wild pigs (Sus scrofa) in Canadian agro-ecosystems

Resource selection informs understanding of a species’ ecology and is especially pertinent for invasive species. Since introduced to Canada, wild pigs (Sus scrofa Linnaeus, 1978) remain understudied despite recognized negative impacts on native and agricultural systems globally. Elsewhere in North America, pigs typically use forests and forage in agricultural crops. We hypothesized Canadian wild pigs would behave similarly, and using GPS locations from 15 individuals, we examined diel and seasonal resource selection and movement in the Canadian prairie region. Forests were predominately selected during the day, while corn (Zea mays L.), oilseeds, and wheat (Triticum aestivum L.) were predominately selected at night. Forests and corn were consistently selected throughout the growing season.Wetlands and forests showed greater use rates than other habitats, with evident trade-offs as crop use increased with the timing of maturation. Activity was consistent with foraging in growing crops. Results indicate diel patterns were likely a function of short-term needs to avoid daytime anthropogenic risk, while seasonal patterns demonstrate how habitats that fill multiple functional roles——food, cover, and thermoregulation——can be optimized. Understanding selection by invasive species is an important step in understanding their potential environmental impacts in novel environments and informs their management

Triazole Inhibitors of Cryptosporidium parvum Inosine 5?-Monophosphate Dehydrogenase

Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5?-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Because C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series of 1,2,3-triazole containing ether CpIMPDH inhibitors are described. A structure?activity relationship study revealed that a small alkyl group on the ?-position of the ether was required, with the (R)-enantiomer significantly more active than the (S)-enantiomer. Electron-withdrawing groups in the 3- and/or 4-positions of the pendent phenyl ring were best, and conversion of the quinoline containing inhibitors to quinoline-N-oxides retained inhibitory activity both in the presence and absence of bovine serum albumin. The 1,2,3-triazole CpIMPDH inhibitors provide new tools for elucidating the role of IMPDH in C. parvum and may serve as potential therapeutics for treating cryptosporidiosis

Effective Temperatures of a Driven System Near Jamming

Fluctuations in a model of a sheared, zero-temperature foam are studied numerically. Five different quantities that reduce to the true temperature in an equilibrium thermal system are calculated. All five have the same shear-rate dependence, and three have the same value. Near the onset of jamming, the relaxation time is the same function of these three temperatures in the sheared system as of the true temperature in an unsheared system. These results imply that statistical mechanics is useful for the system and provide strong support for the concept of jamming.Comment: 4 pages, 4 postscript figure