PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

The Malaria Secretome: From Algorithms to Essential Function in Blood Stage Infection

The malaria agent Plasmodium falciparum is predicted to export a “secretome” of several hundred proteins to remodel the host erythrocyte. Prediction of protein export is based on the presence of an ER-type signal sequence and a downstream Host-Targeting (HT) motif (which is similar to, but distinct from, the closely related Plasmodium Export Element [PEXEL]). Previous attempts to determine the entire secretome, using either the HT-motif or the PEXEL, have yielded large sets of proteins, which have not been comprehensively tested. We present here an expanded secretome that is optimized for both P. falciparum signal sequences and the HT-motif. From the most conservative of these three secretome predictions, we identify 11 proteins that are preserved across human- and rodent-infecting Plasmodium species. The conservation of these proteins likely indicates that they perform important functions in the interaction with and remodeling of the host erythrocyte important for all Plasmodium parasites. Using the piggyBac transposition system, we validate their export and find a positive prediction rate of ∼70%. Even for proteins identified by all secretomes, the positive prediction rate is not likely to exceed ∼75%. Attempted deletions of the genes encoding the conserved exported proteins were not successful, but additional functional analyses revealed the first conserved secretome function. This gave new insight into mechanisms for the assembly of the parasite-induced tubovesicular network needed for import of nutrients into the infected erythrocyte. Thus, genomic screens combined with functional assays provide unexpected and fundamental insights into host remodeling by this major human pathogen

The WEBT Campaign on the Intermediate BL Lac Object 3C66A in 2007-2008

Prompted by a high optical state in September 2007, the Whole Earth Blazar Telescope (WEBT) consortium organized an intensive optical, near-IR (JHK) and radio observing campaign on the intermediate BL Lac object 3C 66A throughout the fall and winter of 2007 -- 2008. The source remained in a high optical state throughout the observing period and exhibited several bright flares on time scales of ~ 10 days. This included an exceptional outburst around September 15 - 20, 2007, reaching a peak brightness at R ~ 13.4. Our campaign revealed microvariability with flux changes up to |dR/dt| ~ 0.02 mag/hr. Our observations do not reveal evidence for systematic spectral variability or spectral lags. We infer a value of the magnetic field in the emission region of B ~ 19 e_B^{2/7} \tau_h^{-6/7} D_1^{13/7} G. From the lack of systematic spectral variability, we can derive an upper limit on the Doppler factor, D <= 28 \tau_h^{-1/8} e_B^{3/16}. This is in agreement with superluminal motion measurements of \beta_{app} \le 27 and argues against models with very high Lorentz factors of \Gamma > 50, required for a one-zone SSC interpretation of some high-frequency-peaked BL Lac objects detected at TeV gamma-ray energies.Comment: Accepted for publication in The Astrophysical Journa

Electrochemically synthesized polymers in molecular imprinting for chemical sensing

This critical review describes a class of polymers prepared by electrochemical polymerization that employs the concept of molecular imprinting for chemical sensing. The principal focus is on both conducting and nonconducting polymers prepared by electropolymerization of electroactive functional monomers, such as pristine and derivatized pyrrole, aminophenylboronic acid, thiophene, porphyrin, aniline, phenylenediamine, phenol, and thiophenol. A critical evaluation of the literature on electrosynthesized molecularly imprinted polymers (MIPs) applied as recognition elements of chemical sensors is presented. The aim of this review is to highlight recent achievements in analytical applications of these MIPs, including present strategies of determination of different analytes as well as identification and solutions for problems encountered

The whole earth blazar telescope campaign on the intermediate BL Lac object 3C 66A in 2007-2008

Prompted by a high optical state in 2007 September, the Whole Earth Blazar Telescope consortium organized an intensive optical, near-IR (JHK) and radio observing campaign on the intermediate BL Lac object 3C 66A throughout the fall and winter of 2007-2008. In this paper, we present data from 28 observatories in 12 countries, covering the observing season from late 2007 July through 2008 February. The source remained in a high optical state throughout the observing period and exhibited several bright flares on timescales of 10 days. This included an exceptional outburst around 2007 September 15-20, reaching a peak brightness at R 13.4. Our campaign revealed microvariability with flux changes up to |dR/dt| 0.02 mag hr-1. Our observations do not reveal evidence for systematic spectral variability in the overall high state covered by our campaign, in agreement with previous results. In particular, we do not find evidence for spectral hysteresis in 3C 66A for which hints were found in an earlier campaign in a somewhat lower flux state. We also did not find any evidence for spectral lags in the discrete correlation functions between different optical bands. We infer a value of the magnetic field in the emission region of B 19 e 2/7B τ-6/7h D 13/71 G, where eB is the magnetic field equipartition fraction, τh is the shortest observed variability timescale in units of hours, and D 1 is the Doppler factor in units of 10. From the lack of systematic spectral variability, we can derive an upper limit on the Doppler factor, D ≤ 28 τ-1/8h e 3/16B. This is in perfect agreement with superluminal motion measurements with the VLBI/VLBA of βapp ≤ 27 and argues against models with very high Lorentz factors of Γ ≳ 50, required for a one-zone synchrotron-self-Compton interpretation of some high-frequency-peaked BL Lac objects detected at TeV γ-ray energies. © 2009 The American Astronomical Society