Identification of human endogenous retrovirus-specific T cell responses in vertically HIV-1-infected subjects

Human endogenous retrovirus (HERV)-specific T cell responses in HIV-1-infected adults have been reported. Whether HERV-specific immunity exists in vertically HIV-1-infected children is unknown. We performed a cross-sectional analysis of HERV-specific T cell responses in 42 vertically HIV-1-infected children. HERV (-H, -K, and -L family)-specific T cell responses were identified in 26 of 42 subjects, with the greatest magnitude observed for the responses to HERV-L. These HERV-specific T cell responses were inversely correlated with the HIV-1 plasma viral load and positively correlated with CD4(+) T cell counts. These data indicate that HERV-specific T cells may participate in controlling HIV-1 replication and that certain highly conserved HERV-derived proteins may serve as promising therapeutic vaccine targets in HIV-1-infected children.     

A rapid column technique for trapping and collecting of volatile fungal hydrocarbons and hydrocarbon derivatives

A custom-made stainless steel column was designed to contain various materials that would trap the hydrocarbons and hydrocarbon derivatives during the processes of fungal fermentation ultimately yielding preparative amounts of volatile organic substances (VOCs). Trapping materials tested in the column were Carbotrap materials A and B (Supelco) as well as bentonite-shale from the oil bearing areas of Eastern Montana, the former allowed for the effective and efficient trapping of VOCs from purged cultures of Hypoxylon sp. Trapping efficiencies of various materials were measured by both gravimetric as well as proton transfer reaction mass spectroscopy with the Carbotraps A and B being 99% efficient when tested with known amounts of 1,8-cineole. Trapped fungal VOCs could effectively be removed and recovered via controlled heating of the stainless steel column followed by passage of the gases through a liquid nitrogen trap at a recovery rate of ca 65–70%. This method provides for the recovery of mg quantities of compounds normally present in the gas phase that may be needed for spectroscopy, bioassays and further separation and analysis and may have wide applicability for many other biological systems involving VOCs. Other available Carbotraps could be used for other applications.     

Nitric oxide-mediated vasodilation becomes independent of beta-adrenergic receptor activation with increased intensity of hypoxic exercise

Hypoxic vasodilation in skeletal muscle at rest is known to include β-adrenergic receptor-stimulated nitric oxide (NO) release. We previously reported that the augmented skeletal muscle vasodilation during mild hypoxic forearm exercise includes β-adrenergic mechanisms. However, it is unclear whether a β-adrenergic receptor-stimulated NO component exists during hypoxic exercise. We hypothesized that NO-mediated vasodilation becomes independent of β-adrenergic receptor activation with increased exercise intensity during hypoxic exercise. Ten subjects (7 men, 3 women; 23 ± 1 yr) breathed hypoxic gas to titrate arterial O(2) saturation to 80% while remaining normocapnic. Subjects performed two consecutive bouts of incremental rhythmic forearm exercise (10% and 20% of maximum) with local administration (via a brachial artery catheter) of propranolol (β-adrenergic receptor inhibition) alone and with the combination of propranolol and nitric oxide synthase inhibition [N(G)-monomethyl-l-arginine (l-NMMA)] under normoxic and hypoxic conditions. Forearm blood flow (FBF, ml/min; Doppler ultrasound) and blood pressure [mean arterial pressure (MAP), mmHg; brachial artery catheter] were assessed, and forearm vascular conductance (FVC, ml·min(-1)·100 mmHg(-1)) was calculated (FBF/MAP). During propranolol alone, the rise in FVC (Δ from normoxic baseline) due to hypoxic exercise was 217 ± 29 and 415 ± 41 ml·min(-1)·100 mmHg(-1) (10% and 20% of maximum, respectively). Combined propranolol-l-NMMA infusion during hypoxic exercise attenuated ΔFVC at 20% (352 ± 44 ml·min(-1)·100 mmHg(-1); P < 0.001) but not at 10% (202 ± 28 ml·min(-1)·100 mmHg(-1); P = 0.08) of maximum compared with propranolol alone. These data, when integrated with earlier findings, demonstrate that NO contributes to the compensatory vasodilation during mild and moderate hypoxic exercise; a β-adrenergic receptor-stimulated NO component exists during low-intensity hypoxic exercise. However, the source of the NO becomes less dependent on β-adrenergic mechanisms as exercise intensity increases.     

BTB-Kelch protein Krp1 regulates proliferation and differentiation of myoblasts

The BTB-Kelch protein Krp1 is highly and specifically expressed in skeletal muscle, where it is proposed to have a role in myofibril formation. We observed significant upregulation of Krp1 in C2 cells early in myoblast differentiation, well before myofibrillogenesis. Krp1 has a role in cytoskeletal organization and cell motility; since myoblast migration and elongation/alignment are important events in early myogenesis, we hypothesized that Krp1 is involved with earlier regulation of differentiation. Krp1 protein levels were detectable by 24 h after induction of differentiation in C2 cells and were significantly upregulated by 48 h, i.e., following the onset myogenin expression and preceding myosin heavy chain (MHC) upregulation. Upregulation of Krp1 required a myogenic stimulus as signaling derived from increased myoblast cell density was insufficient to activate Krp1 expression. Examination of putative Krp1 proximal promoter regions revealed consensus E box elements associated with myogenic basic helix-loop-helix binding. The activity of a luciferase promoter-reporter construct encompassing this 2,000-bp region increased in differentiating C2 myoblasts and in C2 cells transfected with myogenin and/or MyoD. Knockdown of Krp1 via short hairpin RNA resulted in increased C2 cell number and proliferation rate as assessed by bromodeoxyuridine incorporation, whereas overexpression of Krp1-myc had the opposite effect; apoptosis was unchanged. No effects of changed Krp1 protein levels on cell migration were observed, either by scratch wound assay or live cell imaging. Paradoxically, both knockdown and overexpression of Krp1 inhibited myoblast differentiation assessed by expression of myogenin, MEF2C, MHC, and cell fusion.     

An outcome-based model for predicting recovery pathways in restored ecosystems: The Recovery Cascade Model

Restoration is increasingly the focus of ecosystem management. Few conceptual models exist for predicting the consequences of restoration, especially those that predict the stages of recovery following restoration. Existing models focus either on defining endpoints for recovery or on defining ecosystem processes, but often do not identify barriers to recovery or potential negative effects of restoration. We describe a conceptual model that identifies the outcomes of the recovery pathways following flow restoration in rivers: the Recovery Cascade Model. The model identifies six general aspects of recovery following restoration: physical ecosystem change; creation of, or improvement in habitat condition; reconnection of the restored area to adjacent ecosystems; recolonization of the restored area; resumption of ecological processes; re-establishment of biotic interactions and reproduction by colonists in the restored area. These aspects may occur in sequence, such that recovery is blocked by a single barrier. The model accommodates feedback loops and includes strong connections between physical processes and ecosystem processes, but also identifies factors that are important in achieving endpoints such as potential barriers to further recovery. Identification of barriers to recovery enables improved planning to maximise the positive effects of restoration. By focussing on outcomes, the model provides a planning tool for managers that can be adapted for different ecosystems and restoration methods and which can be used to identify the amenities that an ecosystem will deliver at different stages of recovery. Ecosystem recovery is as much about overcoming barriers as it is about restorative actions.     

Experimental and theoretical investigation of the scope of enantioselective ketone allylations employing Nakamura's allylzinc-bisoxazoline reagent

The scope of enantioselective allylations employing Nakamura’s allylzinc-bisoxazoline reagent was examined by performing allylations of a selection of readily available ketones. Low-to-moderate ee’s were observed, and a computational study was conducted to rationalize the results. Examination of transition structures of previously performed allylations that proceeded with high ee revealed the importance of both local and global control elements in these successful reactions. The ability of density functional theory methods to estimate the enantioselectivity of these asymmetric ketone allylations was established. All allylations that were studied computationally exhibited low (<5 kcal/mol) activation barriers, a result that is consistent with the highly reactive nature of Nakamura’s reagent.     

Optimisation of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors

Rho kinase is an important target implicated in a variety of cardiovascular diseases. Herein, we report the optimisation of the fragment derived ATP-competitive ROCK inhibitors 1 and 2 into lead compound 14A. The initial goal of improving ROCK-I potency relative to 1, whilst maintaining a good PK profile, was achieved through removal of the aminoisoquinoline basic centre. Lead 14A was equipotent against both ROCK-I and ROCK-II, showed good in vivo efficacy in the spontaneous hypertensive rat model, and was further optimised to demonstrate the scope for improving selectivity over PKA versus hydroxy Fasudil 3.     

Triamino pyrimidines and pyridines as histamine H(4) receptor modulators

Two series of triamino pyrimidines and a series of triamino pyridines have been synthesized and their structure-activity relationships evaluated for activity at the H₄ receptor in competitive binding and functional assays. Small structural changes in these three hetereoaromatic cores influenced the functional activity of these compounds.     

Synthesis of magnetic resonance-, X-ray- and ultrasound-visible alginate microcapsules for immunoisolation and noninvasive imaging of cellular therapeutics

Cell therapy has the potential to treat or cure a wide variety of diseases. Non-invasive cell tracking techniques are, however, necessary to translate this approach to the clinical setting. This protocol details methods to create microcapsules that are visible by X-ray, ultrasound (US) or magnetic resonance (MR) for the encapsulation and immunoisolation of cellular therapeutics. Three steps are generally used to encapsulate cellular therapeutics in an alginate matrix: (i) droplets of cell-containing liquid alginate are extruded, using an electrostatic generator, through a needle tip into a solution containing a dissolved divalent cation salt to form a solid gel; (ii) the resulting gelled spheres are coated with polycations as a cross-linker; and (iii) these complexes are then incubated in a second solution of alginate to form a semipermeable membrane composed of an inner and an outer layer of alginate. The microcapsules can be rendered visible during the first step by adding contrast agents to the primary alginate layer. Such contrast agents include superparamagnetic iron oxide for detection by (1)H MR imaging (MRI); the radiopaque agents barium or bismuth sulfate for detection by X-ray modalities; or perfluorocarbon emulsions for multimodal detection by (19)F MRI, X-ray and US imaging. The entire synthesis can be completed within 2 h.