Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding

The biogenesis of outer-membrane proteins (OMPs) in gram-negative bacteria involves delivery by periplasmic chaperones to the β-barrel assembly machinery (BAM), which catalyzes OMP insertion into the outer membrane. Here, we examine the effects of membrane thickness, the Escherichia coli periplasmic chaperones Skp and SurA, and BamA, the central subunit of the BAM complex, on the folding kinetics of a model OMP (tOmpA) using fluorescence spectroscopy, native mass spectrometry, and molecular dynamics simulations. We show that prefolded BamA promotes the release of tOmpA from Skp despite the nM affinity of the Skp:tOmpA complex. This activity is located in the BamA β-barrel domain, but is greater when full-length BamA is present, indicating that both the β-barrel and polypeptide transport-associated (POTRA) domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane. Consistent with this, molecular dynamics simulations reveal that increases in membrane thinning/disorder by the transmembrane domain of BamA is greatest in thicker bilayers. Finally, we demonstrate that cross-linking of the BamA barrel does not affect tOmpA folding kinetics in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, suggesting that lateral gating of the BamA barrel and/or hybrid barrel formation is not required, at least for the assembly of a small 8-stranded OMP in vitro

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Dynamic Capacity Allocation Algorithms for iNET Link Manager

ITC/USA 2014 Conference Proceedings / The Fiftieth Annual International Telemetering Conference and Technical Exhibition / October 20-23, 2014 / Town and Country Resort & Convention Center, San Diego, CAIn an iNET telemetry network, Link Manager (LM) dynamically allocates capacity to radio links to achieve desired QoS guarantees. Under the T&E S&T iMANPOL program, we developed an enhanced capacity allocation algorithm that can better cope with severe congestion and misbehaving users and traffic flows. We compare the E-LM with the LM baseline algorithm (B-LM), which employs priority-weighted allocation. The B-LM is expected to perform well for the majority of traffic patterns, but does not prevent an ill-behaved traffic class from causing excessive latency on other radio links. The E-LM ensures that each class has a "guaranteed" portion of the total available bandwidth that is proportional to the weight of the class. If the traffic loading of a class is lower than its quota, the difference can be flexibly shared by other classes across multiple links. If the traffic loading of a class is higher than its quota, its demand may still be satisfied, provided that the capacity is not taken away from well-behaved traffic classes that stay below their quotas. The qualitative analysis shows the E-LM provides lower latencies for the well-behaved links in overloading conditions and increases the overall system throughput when the traffic is unbalanced. We conducted extensive experiments to confirm that analysis, with the E-LM reducing latency of well-behaved flows up to 90%, and increasing overall throughput up to 65% over the B-LM.International Foundation for TelemeteringProceedings from the International Telemetering Conference are made available by the International Foundation for Telemetering and the University of Arizona Libraries. Visit http://www.telemetry.org/index.php/contact-us if you have questions about items in this collection

Immune plasma for the treatment of severe influenza: an open-label, multicentre, phase 2 randomised study

BackgroundInfluenza causes substantial morbidity and mortality despite available treatments. Anecdotal reports suggest that plasma with high antibody titres to influenza might be of benefit in the treatment of severe influenza.MethodsIn this randomised, open-label, multicentre, phase 2 trial, 29 academic medical centres in the USA assessed the safety and efficacy of anti-influenza plasma with haemagglutination inhibition antibody titres of 1:80 or more to the infecting strain. Hospitalised children and adults (including pregnant women) with severe influenza A or B (defined as the presence of hypoxia or tachypnoea) were randomly assigned to receive either two units (or paediatric equivalent) of anti-influenza plasma plus standard care, versus standard care alone, and were followed up for 28 days. The primary endpoint was time to normalisation of patients' respiratory status (respiratory rate of ≤20 breaths per min for adults or age-defined thresholds of 20-38 breaths per min for children) and a room air oxygen saturation of 93% or more. This study is registered with ClinicalTrials.gov, number NCT01052480.FindingsBetween Jan 13, 2011, and March 2, 2015, 113 participants were screened for eligibility and 98 were randomly assigned from 20 out of 29 participating sites. Of the participants with confirmed influenza (by PCR), 28 (67%) of 42 in the plasma plus standard care group normalised their respiratory status by day 28 compared with 24 (53%) of 45 participants on standard care alone (p=0·069). The hazard ratio (HR) comparing plasma plus standard care with standard care alone was 1·71 (95% CI 0·96-3·06). Six participants died, one (2%) from the plasma plus standard care group and five (10%) from the standard care group (HR 0·19 [95% CI 0·02-1·65], p=0·093). Participants in the plasma plus standard care group had non-significant reductions in days in hospital (median 6 days [IQR 4-16] vs 11 days [5-25], p=0·13) and days on mechanical ventilation (median 0 days [IQR 0-6] vs 3 days [0-14], p=0·14). Fewer plasma plus standard care participants had serious adverse events compared with standard care alone recipients (nine [20%] of 46 vs 20 [38%] of 52, p=0·041), the most frequent of which were acute respiratory distress syndrome (one [2%] vs two [4%] patients) and stroke (one [2%] vs two [4%] patients).InterpretationAlthough there was no significant effect of plasma treatment on the primary endpoint, the treatment seemed safe and well tolerated. A phase 3 randomised trial is now underway to further assess this intervention.FundingNational Institute of Allergy and Infectious Diseases, US National Institutes of Health