Validation of computational fluid dynamics methodology used for human upper airway flow simulations

An anatomically accurate human upper airway model was constructed from multiple magnetic resonance imaging axial scans. This model was used to conduct detailed Computational Fluid Dynamics (CFD) simulations during expiration, to investigate the fluid flow in the airway regions where obstruction could occur. An identical physical model of the same airway was built using stereo lithography. Pressure and velocity measurements were conducted in the physical model. Both simulations and experiments were performed at a peak expiratory flow rate of 200 L/min. Several different numerical approaches within the FLUENT commercial software framework were used in the simulations; unsteady Large Eddy Simulation (LES), steady Reynolds-Averaged Navier-Stokes (RANS) with two-equation turbulence models (i.e. k?ε, standard k?ω, and k?ω Shear Stress Transport (SST)) and with one-equation Spalart–Allmaras model. The CFD predictions of the average wall static pressures at different locations along the airway wall were favorably compared with the experimental data. Among all the approaches, standard k?ω turbulence model resulted in the best agreement with the static pressure measurements, with an average error of ～20% over all ports. The highest positive pressures were observed in the retroglossal regions below the epiglottis, while the lowest negative pressures were recorded in the retropalatal region. The latter is a result of the airflow acceleration in the narrow retropalatal region. The largest pressure drop was observed at the tip of the soft palate. This location has the smallest cross section of the airway. The good agreement between the computations and the experimental results suggest that CFD simulations can be used to accurately compute aerodynamic flow characteristics of the upper airway.     

Determination of growth inhibitory action point of interferon gamma on WISH cells in cell cycle progression and the window of responsiveness of the cells to the interferon

We had earlier shown that human foetal epithelial cells (WISH), growth-inhibited by interferon gamma (IFNgamma), were reversibly detained at a point prior to DNA synthesis. In the present study, we determined the window of action of IFNgamma in the G1 phase duration and the exact point of detention of WISH cells in cell cycle progression with respect to the known points of detention by the inhibitors of DNA replication initiation (aphidicolin and carbonyl diphosphonate) and of activation of replication protein A (6-dimethylaminopurine), of which RPA activation being the earlier event compared to DNA replication initiation in cell cycle progression. WISH cells, which were released from IFNgamma-induced arrest, permeabilised and exposed independently to these inhibitors show that IFNgamma detains WISH cells prior to initiation of DNA synthesis. Further, exposure of IFNalpha-synchronized (at G0/G1) or mimosine-synchronized (at G1/S) WISH cells to IFNgamma, which was added at different time points post-release from the synchronizing agent, showed that the cells were promptly responsive to the growth inhibitory action of IFNgamma only during the first 11h in G1 phase. Taken together, these results suggest that IFNgamma inhibits growth of WISH cells by detaining them at a point prior to initiation of DNA synthesis and that the IFN acts within the first 11h in G1 phase of the cell cycle.     

Acylideneoxoindoles: a new class of reversible inhibitors of human transglutaminase 2

Inhibitors of human transglutaminase 2 (TG2) are anticipated to be useful in the therapy of a variety of diseases including celiac sprue as well as certain CNS disorders and cancers. A class of 3-acylidene-2-oxoindoles was identified as potent reversible inhibitors of human TG2. Structure-activity relationship analysis of a lead compound led to the generation of several potent, competitive inhibitors. Analogs with significant non-competitive character were also identified, suggesting that the compounds bind at one or more allosteric regulatory sites on this multidomain enzyme. The most active compounds had K_i values below 1.0 μM in two different kinetic assays for human TG2, and may therefore be suitable for investigations into the role of TG2 in physiology and disease in animals.     

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃‐S‐ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.     

The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.     

Human immunodeficiency virus envelope protein Gp120 induces proliferation but not apoptosis in osteoblasts at physiologic concentrations

Patients with HIV infection have decreased numbers of osteoblasts, decreased bone mineral density and increased risk of fracture compared to uninfected patients; however, the molecular mechanisms behind these associations remain unclear. We questioned whether Gp120, a component of the envelope protein of HIV capable of inducing apoptosis in many cell types, is able to induce cell death in bone-forming osteoblasts. We show that treatment of immortalized osteoblast-like cells and primary human osteoblasts with exogenous Gp120 in vitro at physiologic concentrations does not result in apoptosis. Instead, in the osteoblast-like U2OS cell line, cells expressing CXCR4, a receptor for Gp120, had increased proliferation when treated with Gp120 compared to control (P<0.05), which was inhibited by pretreatment with a CXCR4 inhibitor and a G-protein inhibitor. This suggests that Gp120 is not an inducer of apoptosis in human osteoblasts and likely does not directly contribute to osteoporosis in infected patients by this mechanism.     

Mechanisms of sex steroid effects on bone

Sex steroids play a major role in the regulation of bone turnover. Thus, gonadectomy in either sex is associated with an increase in bone remodeling, increased bone resorption, and a relative deficit in bone formation, resulting in accelerated bone loss. Recent physiological studies have established an important role for estrogen in regulating bone turnover not only in females, but also in males. Studies in mice with knock out of the estrogen receptor, aromatase, or androgen receptor have provided important insights into the in vivo mechanisms of sex steroid action on bone. The cellular and molecular mediators of sex steroid effects on the bone-forming osteoblasts and bone-resorbing osteoclasts are also being increasingly better defined. Estrogen inhibits bone remodeling by concurrently suppressing osteoblastogenesis and osteoclastogenesis from marrow precursors. Both estrogen and androgens inhibit bone resorption via effects on the receptor activator of NF-kappaB ligand (RANKL)/RANK/osteoprotegerin system, as well as by reducing the production of a number of pro-resorptive cytokines, along with direct effects on osteoclast activity and lifespan. Sex steroid effects on bone formation are also likely mediated by multiple mechanisms, including a prolongation of osteoblast lifespan via non-genotropic mechanisms, as well as effects on osteoblast differentiation and function. These pleiotropic actions of sex steroids on virtually all aspects of bone metabolism belie the importance of the skeleton not only in providing structural support for the body and in locomotion, but also as a dynamic tissue responsive, among other things, to the reproductive needs of the organism for calcium.