The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS

The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species

Divergent Regulation of Actin Dynamics and Megakaryoblastic Leukemia-1 and -2 (Mkl1/2) by cAMP in Endothelial and Smooth Muscle Cells.

Proliferation and migration of vascular smooth muscle cells (VSMCs) or endothelial cell (ECs) promote or inhibit, respectively, restenosis after angioplasty, vein graft intimal thickening and atherogenesis. Here we investigated the effects of cAMP-induced cytoskeletal remodelling on the serum response factor (SRF) co-factors Megakaryoblastic Leukemia-1 and -2 (MKL1 and MKL2) and their role in controlling VSMC and EC proliferation and migration. Elevation of cAMP using forskolin, dibutyryl-cAMP (db-cAMP), BAY60-6583 or Cicaprost induced rapid cytoskeleton remodelling and inhibited proliferation and migration in VSMCs but not EC. Furthermore, elevated cAMP inhibited mitogen-induced nuclear-translocation of MKL1 and MKL2 in VSMCs but not ECs. Forskolin also significantly inhibited serum response factor (SRF)-dependent reporter gene (SRE-LUC) activity and mRNA expression of pro-proliferative and pro-migratory MKL1/2 target genes in VSMCs but not in ECs. In ECs, MKL1 was constitutively nuclear and MKL2 cytoplasmic, irrespective of mitogens or cAMP. Pharmacological or siRNA inhibition of MKL1 significantly inhibited the proliferation and migration of VSMC and EC. Our new data identifies and important contribution of MKL1/2 to explaining the strikingly different response of VSMCs and ECs to cAMP elevation. Elucidation of these pathways promises to identify targets for specific inhibition of VSMC migration and proliferation

Finite Difference Approximations for the Fractional Fokker-Planck Equation

The fractional Fokker–Planck equation has been used in many physical transport problems which take place under the influence of an external force field. In this paper we examine some practical numerical methods to solve a class of initial-boundary value problems for the fractional Fokker–Planck equation on a finite domain. The solvability, stability, consistency, and convergence of these methods are discussed. Their stability is proved by the energy method. Two numerical examples are also presented to evaluate these finite difference methods against the exact analytical solutions

Dissection of pilus tip assembly by the FimD usher monomer

Type 1 pili are representative of a class of bacterial surface structures assembled by the conserved chaperone/usher pathway and used by uropathogenic Escherichia coli to attach to bladder cells during infection. The outer membrane assembly platform – the usher – is critical for the formation of pili, catalysing the polymerization of pilus subunits and enabling the secretion of the nascent pilus. Despite extensive structural characterisation of the usher, a number of questions about its mechanism remain, notably its oligomerization state, and how it orchestrates the ordered assembly of pilus subunits. We demonstrate here that the FimD usher is able to catalyse in vitro pilus assembly effectively in its monomeric form. Further, by establishing the kinetics of usher-catalysed reactions between various pilus subunits, we establish a complete kinetic model of tip fibrillum assembly, able to account for the order of subunits in native type 1 pili

Analyzing enhanced real-time uplink scheduling algorithm in 3GPP LTE-advanced networks using multimedia systems

Third Generation Partnership Project (3GPP) standardizes the Long-Term Evolution (LTE) to improve the quality of service in modern communication systems using 3GPP LTE-advanced (LTE-A) networks. As this technology is converging with modern devices, efficient resource allocation schemes are essential for minimization of the communication delay for the sensitive real-time devices. To achieve the demands of latest technologies, this paper proposes two novel mechanisms, enhanced real-time polling system with continuous time-batch Markovian arrival process and proactive resource allocation framework. The objective of the former mechanism is to improvise the service connectivity between the multimedia devices in LTE-A networks and the latter is to exploit the transmission bits in each user's buffer in addition to the state information of wireless channel. The proposed mechanisms consider the constraints enforced by 3GPP standard in terms of radio resource blocks allocation for LTE-A networks. In addition, the proposed mechanisms use LTE-A networks to investigate the Voice over LTE traffic in order to analyze various quality-of-service measures, namely, sum throughput, session setup delay, packet delivery ratio, resource utilization ratio, and throughput improvement via real-time multimedia client-server systems. The probing results show that the proposed algorithm is superior in comparison to other typical services of IEEE 802.16d/e-based systems as well as LTE-A system with 7 orthogonal frequency-division multiplexing symbols over a duration slot better throughput performance in comparison with existing mechanisms