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.

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.

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.

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.

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.

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.

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.

Disruption of Akt kinase activation is important for immunosuppression induced by measles virus.

Surface-contact-mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2-dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

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.

Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER.

The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.

Fas-induced caspase denitrosylation.

Only a few intracellular S-nitrosylated proteins have been identified, and it is unknown if protein S-nitrosylation/denitrosylation is a component of signal transduction cascades. Caspase-3 zymogens were found to be S-nitrosylated on their catalytic-site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol. Protein S-nitrosylation/denitrosylation can thus serve as a regulatory process in signal transduction pathways.

Fatigue in non-alcoholic fatty liver disease (NAFLD) is significant and associates with inactivity and excessive daytime sleepiness but not with liver disease severity or insulin resistance.

OBJECTIVE: To quantify fatigue in non-alcoholic fatty liver disease (NAFLD), to determine whether perceived fatigue reflects impairment of physical function and to explore potential causes. PATIENTS AND METHODS: A cohort study was carried out on 156 consecutive patients with histologically proven NAFLD studied in two cohorts. Phase 1 determined the perceived fatigue experienced by NAFLD patients (assessed using the Fatigue Impact Scale (FIS)) in comparison with normal and liver disease controls, and the relationship to physical function (actigraphy). In phase 2, biological associations of fatigue in NAFLD were explored. RESULTS: Fatigue was markedly higher in NAFLD patients than in controls (mean (SD) FIS 51 (38) vs 8 (12), p<0.001). NAFLD patients showed significantly lower physical activity over 6 days (7089 (2909) mean steps/day vs 8676 (2894), p = 0.02). A significant inverse correlation was seen between FIS and physical activity (r = 0.1, p = 0.02). Fatigue experienced by NAFLD patients was similar to that in primary biliary cirrhosis (n = 36) (FIS 64 (9) vs 61 (2), p = NS). No association was seen between FIS and biochemical and histological markers of liver disease severity or insulin resistance (homeostasis model assessment (HOMA)) (r < 0.005). Significant association was seen between fatigue severity and daytime somnolence (Epworth Sleepiness Scale) (r = 0.2, p < 0.001). CONCLUSION: Fatigue is a significant problem in NAFLD, is similar in degree to that in primary biliary cirrhosis patients and is associated with impairment in physical function. Fatigue in NAFLD appears to be unrelated to either severity of underlying liver disease or insulin resistance, but is associated with significant daytime somnolence.

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.

Induction of NF-kappaB by the Akt/PKB kinase.

The serine/threonine kinase Akt (also known as protein kinase B, PKB) is activated by numerous growth-factor and immune receptors through lipid products of phosphatidylinositol (PI) 3-kinase. Akt can couple to pathways that regulate glucose metabolism or cell survival [1]. Akt can also regulate several transcription factors, including E2F, CREB, and the Forkhead family member Daf-16 [2] [3] [4]. Here, we show that Akt can regulate signaling pathways that lead to induction of the NF-kappaB family of transcription factors in the Jurkat T-cell line. This induction occurs, at least in part, at the level of degradation of the NF-kappaB inhibitor IkappaB, and is specific for NF-kappaB, as other inducible transcription factors are not affected by Akt overexpression. Furthermore, the effect requires the kinase activity and pleckstrin homology (PH) domain of Akt. Also, Akt does not act alone to induce cytokine promoters and NF-kappaB reporters, because signals from other pathways are required to observe the effect. These studies uncover a previously unappreciated connection between Akt and NF-kappaB induction that could have implications for the control of T-cell growth and survival.

Signal transduction by the TCR for antigen.

The past several years have seen the beginning of a shift in the way that TCR signal transduction is studied. Although many investigators continue to identify new molecules, particularly adaptor proteins, others have attempted to look at signaling events in a larger cellular context. Thus the identification of distinct formations of signaling molecules at junctions between T cells and antigen-presenting cells, the role of the cytoskeleton and the partitioning of molecules into specialized lipid subdomains have been the subjects of many publications. Such concepts are helping to assemble a blueprint of how the myriad adaptors and kinases fit together to effect T cell activation.

Feasibility study of a head and neck (upper aero-digestive tract) cancer examination.

Nearly all of the requirements for a cancer screening program can be met for head and neck--upper aero-digestive tract (UADT)--cancers. These UADT squamous cancers have a clearly definable high-risk group (smokers, greater than or equal to 40 yr), known etiology (tobacco and alcohol), identifiable premalignant lesions, a high prevalence rate worldwide, and high curability with "early" diagnosis. However, a need exists for an efficient detection examination suitable for physicians and dentists alike. Such an examination was designed and field tested as a feasibility study. The examination consisted of a six-step, dentist-oriented, site-targeted, 10-minute procedure, including the use of a fiberoptic pharyngoscope. Two practicing dentists were instructed in the procedure and used it on a randomly selected, high-risk Health America, Inc.--Park DuValle Community Health Center population, who were voluntary responders to a questionnaire and a phone call. Analysis of results showed easy and reproducible teachability, a high degree of acceptance by dentists and examinees, accuracy, and low cost. Associated findings were the following: Of 6,206 respondents, ages 40 and over, 27% were current smokers; 29% were ex-smokers; and 33% never smoked. Of invited current and ex-smokers, ages 40 and over, 51% appeared for a single examination. Compliance emerged as the major problem. The feasibility demonstrated in this study appears to justify a larger, controlled investigation.