Summary
The OmpF porin from the Escherichia coli outer membrane folds into a trimer of β-barrel, each forming a wide aqueous pore allowing the passage of ions and small solutes. A long loop (L3) carrying multiple acidic residues folds into the β-barrel pore to form a narrow “constriction zone”. A strong and highly conserved charge asymmetry is observed at the constriction zone, with multiple basic residues attached to the wall of the β-barrel (Lys16, Arg42, Arg82 and Arg132) on one side, and multiple acidic residues of L3 (Asp107, Asp113, Glu117, Asp121, Asp126, Asp127) on the other side. Several computational studies have suggested that a strong transverse electric field could exist at the constriction zone as a result of such charge asymmetry, giving rise to separate permeation pathways for cations and anions. To examine this question, OmpF was expressed, purified and crystallized in the P63 space group and two different data sets were obtained at 2.6 Å and 3.0 Å resolution with K+ and Rb+, respectively. The Rb+ soaked crystals were collected at the rubidium anomalous wavelength of 0.8149 Å and cation positions were determined. A PEG molecule was observed in the pore region for both the K+ and Rb+ soaked crystals, where it is interacting with the loop L3. The results reveal the separate pathways of anions and cations across the constriction zone of the OmpF pore.
Summary
During infection, Legionella pneumophila creates a replication vacuole within eukaryotic cells and this process requires a Type IVb secretion system (T4bSS). IcmQ is a critical component of this translocase and associates with IcmR. In this report, we show that the N-terminal domain of IcmQ (Qn) mediates IcmQ dimerization, while the C-terminal domain with a linker region promotes a stable membrane association of IcmQ. We then determined crystal structures of Qn with the interacting domain of IcmR. In this complex, each protein forms an α-helical hairpin within a parallel 4-helix bundle. We find that IcmR binding to IcmQ prevents dimerization of IcmQ and blocks membrane permeabilization. However, IcmR does not completely block membrane association of IcmQ. The amphipathic nature of Qn suggests two models for how IcmQ may permeabilize membranes. The Rm-Qn structure also suggests how hyper-variable IcmR-like proteins in other Legionellae may interact with their IcmQ partners to regulate IcmQ function.
Objectives
The US Department of Veterans Affairs (VA) is the largest integrated health care provider for HIV‐infected patients in the USA. VA data for HIV‐specific clinical and quality improvement research are an important resource. We sought to determine the accuracy of using the VA Corporate Data Warehouse (CDW), a fully automated medical records database for all VA users nationally, to identify HIV‐infected patients compared with a gold‐standard VA HIV Clinical Case Registry (CCR).
Methods
We assessed the test performance characteristics of each of our CDW criteria‐based algorithms (presence of one, two or all of the following: diagnostic codes for HIV, positive HIV laboratory tests, and prescription for HIV medication) by calculating their sensitivity (proportion of HIV‐positive patients in the CCR accurately detected as HIV‐positive by the CDW algorithm) and positive predictive value (PPV; the proportion of patients identified by the CDW algorithm who were classified as HIV‐positive from the CCR).
Results
We found that using a CDW algorithm requiring two of three HIV diagnostic criteria yielded the highest sensitivity (95.2%) with very little trade‐off in PPV (93.5%).
Conclusions
A two diagnostic criteria‐based algorithm can be utilized to accurately identify HIV‐infected cohorts seen in the nationwide VA health care system.
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