Single Stranded Fully Modified-Phosphorothioate Oligonucleotides can Induce Structured Nuclear Inclusions, Alter Nuclear Protein Localization and Disturb the Transcriptome In Vitro.

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2' O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects.

The Biology of Casmara subagronoma (Lepidoptera: Oecophoridae), a Stem-Boring Moth of Rhodomyrtus tomentosa (Myrtaceae): Descriptions of the Previously Unknown Adult Female and Immature Stages, and Its Potential as a Biological Control Candidate.

Rhodomyrtus tomentosa is a perennial shrub native to Southeast Asia and is invasive in South Florida and Hawai'i, USA. During surveys of R. tomentosa in Hong Kong from 2013-2018 for potential biological control agents, we collected larvae of the stem borer, Casmara subagronoma. Larvae were shipped in stems to a USDA-ARS quarantine facility where they were reared and subjected to biology studies and preliminary host range examinations. Casmara subagronoma is the most recent Casmara species to be described from males collected in Vietnam and Indonesia. Because the original species description was based on only two male specimens, we also provide a detailed description of the female, egg, larva, and pupa. Finally, we conducted preliminary host range trials utilizing Myrtus communis, Myrcianthes fragrans, and Camellia sinensis. Casmara subagronoma emerged from M. fragrans, a Florida-native shrub, and larvae were able to survive in non-target stems for over a year (>400 days). Based on these findings and difficulty in rearing, we do not believe C. subagronoma is a suitable insect for biological control of R. tomentosa at this time, but may warrant further study. This investigation also illustrates the importance of host surveys for conservation and taxonomic purposes.

Multifaceted Substrate-Ligand Interactions Promote the Copper-Catalyzed Hydroboration of Benzylidenecyclobutanes and Related Compounds.

A unified synthetic strategy to access tertiary four-membered carbo/heterocyclic boronic esters is reported. Use of a Cu(I) catalyst in combination with a modified dppbz ligand enables regioselective hydroboration of various trisubstituted benzylidenecyclobutanes and carbo/heterocyclic analogs. The reaction conditions are mild, and the method tolerates a wide range of medicinally relevant heteroarenes. The protocol can be conveniently conducted on gram-scale, and the tertiary boronic ester products undergo facile diversification into valuable targets. Reaction kinetics and computational studies indicate that the migratory insertion step is turnover-limiting and accelerated by electron-withdrawing groups on the dppbz ligand. Energy decomposition analysis (EDA) calculations reveal that electron-deficient P-aryl groups on the dppbz ligand enhance the T-shaped π/π interactions with the substrate and stabilize the migratory insertion transition state.

Nickel-Catalyzed 1,2-Diarylation of Alkenyl Carboxylates: A Gateway to 1,2,3-Trifunctionalized Building Blocks.

A nickel-catalyzed conjunctive cross-coupling of alkenyl carboxylic acids, aryl iodides, and aryl/alkenyl boronic esters is reported. The reaction delivers the desired 1,2-diarylated and 1,2-arylalkenylated products with excellent regiocontrol. To demonstrate the synthetic utility of the method, a representative product is prepared on gram scale and then diversified to eight 1,2,3-trifunctionalized building blocks using two-electron and one-electron logic. Using this method, three routes toward bioactive molecules are improved in terms of yield and/or step count. This method represents the first example of catalytic 1,2-diarylation of an alkene directed by a native carboxylate group.

Interplay of orbital effects and nanoscale strain in topological crystalline insulators.

Orbital degrees of freedom can have pronounced effects on the fundamental properties of electrons in solids. In addition to influencing bandwidths, gaps, correlation strength and dispersion, orbital effects have been implicated in generating novel electronic and structural phases. Here we show how the orbital nature of bands can result in non-trivial effects of strain on band structure. We use scanning-tunneling microscopy to study the effects of strain on the electronic structure of a heteroepitaxial thin film of a topological crystalline insulator, SnTe. By studying the effects of uniaxial strain on the band structure we find a surprising effect where strain applied in one direction has the most pronounced influence on the band structure along the perpendicular direction. Our theoretical calculations indicate that this effect arises from the orbital nature of the conduction and valence bands. Our results imply that a microscopic model capturing strain effects must include a consideration of the orbital nature of bands.

Pharmacokinetics of nebulized and subcutaneously implanted terbinafine in cottonmouths (Agkistrodon piscivorus).

Ophidiomyces ophiodiicola, the causative agent of snake fungal disease, is proposed as a serious threat to the conservation of several snake populations. The objective of this study was to determine the pharmacokinetic parameters of terbinafine administered through nebulization and a sustained subcutaneous implant as potential treatments of Ophidiomyces in reptiles. Seven adult cottonmouths (Agkistrodon piscivorus) were used in single-dose trials. Each snake was nebulized with terbinafine (2 mg/ml) for 30 min and had blood collected before nebulization and up to 12 hr after nebulization. Following a 5-month washout, the same snakes were administered a subcutaneous implant containing 24.5 mg terbinafine; blood was collected at baseline, 1 day postimplant placement, and then once weekly for 9 weeks. Plasma for both studies was analyzed by high-performance liquid chromatography. The mean plasma concentrations of nebulized terbinafine peaked between 0.5 and 4 hr. The subcutaneously implanted terbinafine reached therapeutic concentrations on day 1 and maintained therapeutic for over 6 weeks. These methods and doses are recommended as potential treatment options for snake fungal disease in reptiles.

Estimating Energy Expenditure using Individualized, Power-Specific Gross Efficiencies.

Our purpose was to determine if using an individual's power-specific gross efficiency improves the accuracy of estimating energy expenditure from cycling power. 30 subjects performed a graded cycling test to develop 4 gross efficiencies: individual power-specific gross efficiencies, a group mean power-specific gross efficiency, individual fixed gross efficiencies, and a group mean fixed gross efficiency. Energy expenditure was estimated from power using these different gross efficiencies and compared to measured energy expenditure during moderate- and hard-intensity constant-power and 2 variable-power cycling bouts. Estimated energy expenditures using individual or group mean power-specific gross efficiencies were not different from measured energy expenditure across all cycling bouts (p>0.05). To examine the intra-individual variability of the estimates, absolute difference scores (absolute value of estimated minus measured energy expenditure) were compared, where values closer to zero represent more accurate individual estimates. The absolute difference score using individual power-specific gross efficiencies was significantly lower compared to the other gross efficiencies across all cycling bouts (p<0.01). Significant and strong correlations (r≥0.97, p<0.001) were found across all cycling bouts between estimated and measured energy expenditures using individual power-specific gross efficiencies. In conclusion, using an individual's power-specific gross efficiency significantly improves their energy expenditure estimate across different power outputs.

Ten Days of Intermittent, Low-dose Carbon Monoxide Inhalation does not Significantly Alter Hemoglobin Mass, Aerobic Performance Predictors, or Peak-power Exercise Tolerance.

Carbon monoxide (CO) rebreathing procedures are used to assess hemoglobin mass (Hbmass) but recent evidence suggests that CO is a signaling molecule that may alter physiological functions. We examined the effects of 10 days of intermittent, low-dose CO inhalation on Hbmass, aerobic performance predictors, and peak-power exercise tolerance. 18 recreationally-active men were randomized to either CO or placebo inhalation groups in a single-blind, pre-post parallel-groups trial. Primary outcomes were assessed before and after an intervention period during which subjects inhaled a bolus of 1.2 ml kg(-1) CO or placebo (room air) for 30 s, once per day on 10 days over a 12-day period. Cycling tests were performed >16 h following CO inhalation to exclude acute effects of CO exposure. CO inhalation elevated carboxyhemoglobin by 4.4±0.4% (mean±SD) following each exposure. Compared to placebo, chronic CO inhalation did not significantly alter Hbmass (p=0.99), peak oxygen uptake (p=0.59), peak power output (p=0.10), submaximal oxygen uptake (p=0.91), submaximal RER (p=0.22), lactate threshold (p=0.65), or peak-power exercise tolerance (p=0.60). In conclusion, our data support the ability to perform repeated measurements of Hbmass using CO rebreathing over a 12-day period without altering physiological responses.

Acute, Low-dose CO Inhalation does not Alter Energy Expenditure during Submaximal Exercise.

Carbon monoxide, a gas known most widely for its toxic effects at high doses, is receiving increased attention for its role as a physiological signaling molecule and potential therapeutic agent when administered in low doses. We sought to quantify any changes to oxygen consumption and energy expenditure during submaximal exercise after low-dose CO inhalation. 9 active individuals completed 4 graded submaximal exercise tests, with each test occurring during a separate visit. For their first exercise test, subjects inhaled CO or room air (1.2 mL·kg(-1) body mass) in a randomized, subject-blind fashion. A second test was repeated 24 h later when the inhaled gas should have cleared the system. Subjects repeated study procedures with the alternate dose after a washout period of at least 2 days. Low-dose CO administration did not affect oxygen consumption or energy expenditure during submaximal exercise immediately or 24 h following its administration. Increases in heart rate, blood [lactate], and perceived exertion were observed following acute CO inhalation but these effects were absent after 24 h. The results of this study suggest that low-dose CO administration does not influence the energetics of submaximal exercise, but it acutely increases the relative intensity associated with absolute workloads below the lactate threshold.

Strain engineering Dirac surface states in heteroepitaxial topological crystalline insulator thin films.

The unique crystalline protection of the surface states in topological crystalline insulators has led to a series of predictions of strain-generated phenomena, from the appearance of pseudo-magnetic fields and helical flat bands to the tunability of Dirac surface states by strain that may be used to construct 'straintronic' nanoswitches. However, the practical realization of this exotic phenomenology via strain engineering is experimentally challenging and is yet to be achieved. Here, we have designed an experiment to not only generate and measure strain locally, but also to directly measure the resulting effects on Dirac surface states. We grew heteroepitaxial thin films of topological crystalline insulator SnTe in situ and measured them using high-resolution scanning tunnelling microscopy to determine picoscale changes in the atomic positions, which reveal regions of both tensile and compressive strain. Simultaneous Fourier-transform scanning tunnelling spectroscopy was then used to determine the effects of strain on the Dirac electrons. We find that strain continuously tunes the momentum space position of the Dirac points, consistent with theoretical predictions. Our work demonstrates the fundamental mechanism necessary for using topological crystalline insulators in strain-based applications.

Age and sex differences in c-Fos expression and serum corticosterone concentration following LPS treatment.

Exposure to an immune challenge during peripuberty/adolescence, but not in adulthood, can cause enduring alterations in reproductive and non-reproductive behaviors. This suggests that the peripubertal/adolescent brain might respond differently to a stressor, like an immune challenge, than the adult brain. The goal of this study was to examine whether there are age and sex differences in the acute response to an immune challenge. To examine this research question, we investigated c-Fos expression in various brain regions. Corticosterone (CORT) concentration in the serum was quantified to examine hypothalamic-pituitary-adrenal axis (HPA-axis) responsiveness. Results showed that lipopolysaccharide (LPS; a bacterial endotoxin) treatment, induced a significant increase in the number of c-Fos immunoreactive cells in adult male and female mice compared to their saline controls. However, in peripubertal/adolescent mice, LPS treatment failed to increase the number of c-Fos immunoreactive cells in both male and female mice compared to their saline controls. LPS treatment also significantly increased serum CORT concentration in all mice regardless of sex and age. However, adult female mice treated with LPS showed significantly greater serum CORT concentration compared to adult and peripubertal/adolescent males and peripubertal/adolescent females treated with LPS. These findings support our hypothesis and suggest that there are important age and sex differences in acute immune response, which may allude to mechanisms for the enduring behavioral alterations, observed previously in mice exposed to an immune challenge during puberty but not in adulthood.

Nanoscale determination of the mass enhancement factor in the lightly doped bulk insulator lead selenide.

Bismuth chalcogenides and lead telluride/selenide alloys exhibit exceptional thermoelectric properties that could be harnessed for power generation and device applications. Since phonons play a significant role in achieving these desired properties, quantifying the interaction between phonons and electrons, which is encoded in the Eliashberg function of a material, is of immense importance. However, its precise extraction has in part been limited due to the lack of local experimental probes. Here we construct a method to directly extract the Eliashberg function using Landau level spectroscopy, and demonstrate its applicability to lightly doped thermoelectric bulk insulator PbSe. In addition to its high energy resolution only limited by thermal broadening, this novel experimental method could be used to detect variations in mass enhancement factor at the nanoscale level. This opens up a new pathway for investigating the local effects of doping and strain on the mass enhancement factor.

Targeted exon skipping to correct exon duplications in the dystrophin gene.

Duchenne muscular dystrophy is a severe muscle-wasting disease caused by mutations in the dystrophin gene that ablate functional protein expression. Although exonic deletions are the most common Duchenne muscular dystrophy lesion, duplications account for 10-15% of reported disease-causing mutations, and exon 2 is the most commonly duplicated exon. Here, we describe the in vitro evaluation of phosphorodiamidate morpholino oligomers coupled to a cell-penetrating peptide and 2'-O-methyl phosphorothioate oligonucleotides, using three distinct strategies to reframe the dystrophin transcript in patient cells carrying an exon 2 duplication. Differences in exon-skipping efficiencies in vitro were observed between oligomer analogues of the same sequence, with the phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide proving the most effective. Differences in exon 2 excision efficiency between normal and exon 2 duplication cells, were apparent, indicating that exon context influences oligomer-induced splice switching. Skipping of a single copy of exon 2 was induced in the cells carrying an exon 2 duplication, the simplest strategy to restore the reading frame and generate a normal dystrophin transcript. In contrast, multiexon skipping of exons 2-7 to generate a Becker muscular dystrophy-like dystrophin transcript was more challenging and could only be induced efficiently with the phosphorodiamidate morpholino oligomer chemistry.

A comparison of measured and calculated free 25(OH) vitamin D levels in clinical populations.

OBJECTIVE: Our goal was to compare direct quantitation of circulating free 25-hydroxyvitamin D (25(OH)D)levels to calculated free 25(OH)D levels and their relationships to intact PTH (iPTH), a biomarker of 25(OH)D effect, in humans with a range of clinical conditions. PATIENTS AND METHODS: Serum samples and clinical data were collected from 155 people: 111 without cirrhosis or pregnancy (comparison group), 24 cirrhotic patients with albumin <2.9 g/dL, and 20 pregnant women (second and third trimester). Total 25(OH)D (LC/MS/MS), free 25(OH)D (immunoassay), vitamin D binding protein (DBP) (immunoassay), albumin, and iPTH (immunoassay) were measured. RESULTS: Total 25(OH)D, DBP, and albumin were lowest in patients with cirrhosis, but measured free 25(OH)D was highest in this group (P < .001). DBP was highest in pregnant women (P < .001), but measured free 25(OH)D did not differ from the comparison group. Calculated free 25(OH)D was positively correlated with measured free 25(OH)D (P < .0001) but explained only 13% of the variability with calculated values higher than measured. African Americans had lower DBP than other ethnic populations within all clinical groups (P < .03), and differences between measured and calculated free 25(OH)D were greatest in African Americans (P < .001). Measured free 25(OH)D was correlated with total 25(OH)D (P < .0001; r(2) = 0.51), but calculated free 25(OH)D was not. Similarly, both measured free 25(OH)D (P < .02) and total 25(OH)D (P < .05) were correlated with iPTH, but calculated free 25(OH)D was not. CONCLUSIONS: Calculated free 25(OH)D levels varied considerably from direct measurements of free 25(OH)D with discrepancies greatest in the data for African Americans. Differences in DBP binding affinity likely contributed to estimation errors between the races. Directly measured free 25(OH)D concentrations were related to iPTH, but calculated estimates were not. Current algorithms to calculate free 25(OH)D may not be accurate. Further evaluation of directly measured free 25(OH)D levels to determine its role in research and clinical management of patients is needed.

Variability in free 25(OH) vitamin D levels in clinical populations.

UNLABELLED: Our goal was to determine total and directly measured free 25-hydroxy vitamin D (25(OH)D) serum levels in humans with a range of 25(OH)D levels and clinical conditions associated with low and high vitamin D binding protein levels. Serum samples and clinical data were collected from 106 subjects: 62 without cirrhosis or pregnancy, 24 cirrhotic patients with albumin <2.9g/dL, and 20 pregnant women. Total 25(OH)D (LC/MS/MS) and "free" 25(OH)D (immunoassay) were measured. Total 25(OH)D was significantly lower in liver disease patients but free 25(OH)D concentrations were significantly higher in this group (p<.001). Neither total nor free 25(OH)D concentrations were significantly different in pregnant women vs. the comparator group. There were significant direct positive relationships between free 25(OH)D and total 25(OH)D concentrations for the entire dataset and for each group (p<.0001), however slopes of relationships differed in the cirrhotic group compared to pregnant women or the comparator group. In cirrhotics: y (free 25(OH)D)=2.52+0.29×X(total 25 (OH)D), r(2)=.51, p<.001; y=1.45+0.09×X; r(2)=.77, p<.0001 for pregnant women; and y=1.11+0.12×X; r(2)=.72, p<.0001 for the comparator group). CONCLUSIONS: directly measured free 25(OH)D serum concentrations and relationships between total and free 25(OH)D vary with clinical conditions, and may differ from those predicted by indirect estimation methods. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Lenalidomide enhancement of human T cell functions in human immunodeficiency virus (HIV)-infected and HIV-negative CD4 T lymphocytopenic patients.

Suppressed T cell functions in human immunodeficiency virus (HIV) infection were identified and corrected by lenalidomide in middle-aged HIV-infected patients. Chemotaxis of T cells from HIV-infected men (n = 6, mean 43 years) to sphingosine 1-phosphate (S1P) and CCL21 was significantly lower than that of HIV-negative men (n = 6, mean 41 years), and was enhanced significantly up to control levels by 100 and 1000 nM lenalidomide. Generation of interleukin (IL)-2, but not interferon (IFN)-γ, by T cells of middle-aged HIV-infected men was significantly lower than that for controls and was increased significantly by 10-1000 nM lenalidomide up to a maximum of more than 300%. CD4 and CD8 T cells isolated from healthy middle-aged men and reconstituted in vitro at a low CD4 : CD8 ratio typical of HIV infection had depressed chemotaxis to S1P, but not CCL21, and generation of IL-2, but not IFN-γ. Significant enhancement of chemotaxis to S1P and CCL21 was induced by 100-1000 nM lenalidomide only for normal T cells at a low CD4 : CD8 ratio. T cells from HIV-negative middle-aged CD4 T lymphocytopenic patients (n = 3), with a CD4 : CD8 ratio as low as that of HIV-infected patients, had similarly diminished chemotaxis to S1P and CCL21, and depressed generation of IL-2, but not IFN-γ. Lenalidomide at 30-1000 nM significantly enhanced chemotaxis to S1P and IL-2 generation for T cells from HIV-negative CD4 T lymphocytopenic patients as from HIV-infected patients, with less effect on CCL21-elicited chemotaxis and none for IFN-γ generation. Defects in functions of T cells from middle-aged HIV-infected men are partially attributable to CD4 T lymphocytopenia and are corrected by lenalidomide.

TIM polymorphisms--genetics and function.

The transmembrane immunoglobulin and mucin domain (TIM) family was identified more than a decade ago. Although the founding member of the family was first described in a rat model of ischemia-reperfusion injury, much of the recent interest in the TIM family members has focused on their potential roles in immunity. There are now a large number of genetic studies that have investigated the possible association of various TIM1 and TIM3 polymorphisms with different diseases. Here, we review this body of literature, and highlight some of the most interesting studies.

Contextual effects on perceived three-dimensional shape.

Binocular disparity is a powerful cue for the perception of depth. The accuracy with which observers can judge depth from disparity can, however, be very poor. This has been attributed to difficulties associated with the scaling of disparity to take account of distance (Johnston, 1991). We test potential strategies that could be used to improve this scaling. Using the depth-to-width ratio task introduced by Bradshaw, Parton, and Eagle (1998), observers adjusted a depth interval to match the vertical distance between two points. The first experiment examined the effect of placing additional visual stimuli between the observer and the target. Despite the potential of these stimuli to provide reliable distance information, the accuracy of depth settings did not change. The second experiment demonstrated that the degree of binocular correlation present in natural images provides useful distance information, and investigated whether this is used by observers in scaling disparity. To do this, we measured whether varying the magnitude of relative disparity presented in the surround of the target affected depth settings. No such effect was observed. We conclude that the effect of information presented in the surrounding context on settings of depth is limited to those situations in which it provides direct information about the distance to the target.