Continuous delivery of IFN-beta promotes sustained maturation of intratumoral vasculature

IFNs have pleiotropic antitumor mechanisms of action. The purpose of this study was to further investigate the effects of IFN-beta on the vasculature of human xenografts in immunodeficient mice. We found that continuous, systemic IFN-beta delivery, established with liver-targeted adeno-associated virus vectors, led to sustained morphologic and functional changes of the tumor vasculature that were consistent with vessel maturation. These changes included increased smooth muscle cell coverage of tumor vessels, improved intratumoral blood flow, and decreased vessel permeability, tumor interstitial pressure, and intratumoral hypoxia. Although these changes in the tumor vasculature resulted in more efficient tumor perfusion, further tumor growth was restricted, as the mature vasculature seemed to be unable to expand to support further tumor growth. In addition, maturation of the intratumoral vasculature resulted in increased intratumoral penetration of systemically administered chemotherapy. Finally, molecular analysis revealed increased expression by treated tumors of angiopoietin-1, a cytokine known to promote vessel stabilization. Induction of angiopoietin-1 expression in response to IFN-beta was broadly observed in different tumor lines but not in those with defects in IFN signaling. In addition, IFN-beta-mediated vascular changes were prevented when angiopoietin signaling was blocked with a decoy receptor. Thus, we have identified an alternative approach for achieving sustained vascular remodeling-continuous delivery of IFN-beta. In addition to restricting tumor growth by inhibiting further angiogenesis, maturation of the tumor vasculature also improved the efficiency of delivery of adjuvant therapy. These results have significant implications for the planning of combination anticancer therapy.     

Use of passive radio frequency identification technologies to monitor nest usage in the northern carmine bee‐eater (Merops n. nubicus)

The use of radio frequency identification (RFID) technology is common in animal‐monitoring applications in the wild and in zoological and agricultural settings. RFID is used to track animals and to collect information about movements and other behaviors, as well as to automate or improve husbandry. Disney's Animal Kingdom® uses passive RFID technology to monitor nest usage by a breeding colony of northern carmine bee‐eaters. We implemented RFID technologies in various equipment configurations, initially deploying low‐frequency (LF) 125 kHz RFID and later changing to high‐frequency (HF) 13.56 MHz RFID technology, to monitor breeding behavior in the flock. We installed antennas connected to RFID readers at the entrances of nest tunnels to detect RFID transponders attached to leg bands as birds entered and exited tunnels. Both LF‐RFID and HF‐RFID systems allowed the characterization of nest visitation, including the timing of nest activity, breeding pair formation, identification of egg‐laying females, participation by nonresidents, and detection of nest disruptions. However, we collected a substantially larger volume of data using the increased bandwidth and polling speed inherent with HF‐RFID, which permitted tag capture of multiple birds simultaneously and resulted in fewer missed nest visits in comparison to LF‐RFID. Herein, we describe the evolution of the RFID setups used to monitor nest usage for more than 7 years, the types of data that can be gained using RFID at nests, and how we used these data to gain insights into carmine bee‐eater breeding behavior and improve husbandry.     

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.     

The relative roles of passive surface forces and active ion transport in the modulation of airway surface liquid volume and composition

Two hypotheses have been proposed recently that offer different views on the role of airway surface liquid (ASL) in lung defense. The "compositional" hypothesis predicts that ASL [NaCl] is kept low (<50 mM) by passive forces to permit antimicrobial factors to act as a chemical defense. The "volume" hypothesis predicts that ASL volume (height) is regulated isotonically by active ion transport to maintain efficient mechanical mucus clearance as the primary form of lung defense. To compare these hypotheses, we searched for roles for: (1) passive forces (surface tension, ciliary tip capillarity, Donnan, and nonionic osmolytes) in the regulation of ASL composition; and (2) active ion transport in ASL volume regulation. In primary human tracheobronchial cultures, we found no evidence that a low [NaCl] ASL could be produced by passive forces, or that nonionic osmolytes contributed substantially to ASL osmolality. Instead, we found that active ion transport regulated ASL volume (height), and that feedback existed between the ASL and airway epithelia to govern the rate of ion transport and volume absorption. The mucus layer acted as a "reservoir" to buffer periciliary liquid layer height (7 microm) at a level optimal for mucus transport by donating or accepting liquid to or from the periciliary liquid layer, respectively. These data favor the active ion transport/volume model hypothesis to describe ASL physiology.     

Tau-mediated process outgrowth is differentially altered by the expression of MAP2b and MAP2c in Sf9 cells

It is well documented that the MAPs, MAP2 and tau, play pivotal roles in neurite outgrowth. Several isoforms of MAP2 and tau are coexpressed in neurons, suggesting that the pattern of neurite outgrowth results from a functional equilibrium among these isoforms. In the present study, by coexpressing two of these MAPs at the same time in Sf9 cells, we demonstrated that tau-mediated process outgrowth is affected differently by MAP2b and MAP2c. MAP2b impairs tau ability to induce process outgrowth. Tau affects MAP2c capacity to induce the formation of multiple processes. There is evidence that actin microfilaments (F-actin) are involved in the elaboration of tau-mediated process outgrowth in Sf9 cells. We compared the effects of MAP2b and MAP2c with the effects of tau on F-actin distribution and stability in Sf9 cells. In MAP2b- and MAP2c-expressing cells with processes, F-actin was redistributed. However, in MAP2b-expressing cells without processes, the distribution of F-actin appears to be similar to the one in wild-type infected cells. Collectively, these results indicate that MAP2b could impair the ability of MAP2c and tau to redistribute F-actin in Sf9 cells, thereby decreasing their capacity to induce process formation. Furthermore, MAP2b and MAP2c patterns of process outgrowth were differentially modified by depolymerization of F-actin by cytochalasin D (CD). As previously reported for tau-expressing cells, the MAP2b-expressing cells developed a higher number of processes per cell and a higher number of cells presented processes in the presence of CD. However, the number of cells with multiple processes was lower in MAP2b-expressing cells than in tau-expressing cells treated with CD at 24 h postinfection. This suggests that MAP2b exerts an effect on F-actin stability at an earlier stage of infection than tau. MAP2c had also some stabilizing effects on F-actin at an early stage of infection, since the percentage of cells presenting one process was similar to the nontreated cells. Therefore, MAP2b seems to have less capacity than MAP2c to redistribute F-actin but, nonetheless, both of these MAP2 isoforms exert a stabilizing effect on F-actin at an early stage of infection. Finally, by modifying phosphorylation we showed that MAP2c capacity to induce multiple processes is related to protein phosphorylation in Sf9 cells. Therefore, the differential effect of MAP2c and MAP2b on process outgrowth seems also to depend on protein phosphorylation.     

C-Mannosylation of MUC5AC and MUC5B Cys subdomains

We expressed recombinant Cys subdomains in COS-7 cells to examine the role of this highly conserved protein domain in mucin biosynthesis. The entire Cys1 and Cys5 and Cys1 and Cys3 subdomains in MUC5AC and MUC5B, respectively, each with six carboxyl terminal histidine residues, were pulse-labeled with [(35)S]cysteine/methionine, and the labeled proteins were examined in the culture medium. Under nonreducing conditions, secreted Cys subdomains were monomers, indicating the absence of interchain disulfide bonds. Cross-linking studies suggested the domains are able to interact through very weak noncovalent interactions. Though the domains had apparent M(r) consistent with the absence of N- and O-glycans, they could be purified with mannose-specific lectins. Lectin binding was prevented by mutation of the first tryptophan residue in the putative C-mannosylation acceptor motif WXXW, indicating that C-mannosylation is responsible for lectin binding. As judged by pulse-chase experiments, C-mannosylation occurred very early during the domain biosynthesis, likely in the endoplasmic reticulum (ER). Mutation of the WXXW motif or expression of the unmutated domain in CHO-Lec35.1 cells, a C-mannosylation-defective cell line, resulted in reduced secretion of the corresponding Cys subdomains. Live cell imaging of green fluorescent protein fused to the Cys subdomains clearly revealed increased presence of Cys subdomains in the ER of CHO-Lec35.1 cells when compared to the same domains expressed in CHO-K1 cells. Considered together, these studies suggest that the Cys subdomains of MUC5AC and MUC5B are C-mannosylated in their respective WXXW motifs. C-mannosylation is likely required for proper folding of the Cys subdomains and/or for some aspect of ER export during mucin biosynthesis.