Network Coding an Secure and Efficient Process for Content Distribution

Abstract-Content distribution in network sometimes may be vulnerable henceforth unauthorized users can inject "bogus" data to corrupt the content during its distribution process in order to deplete the network resource. The direct application of network coding may be insecure. Content verification is the important and practical issue when maintaining integrity of the content. While random linear networking coding is used, it is impracticable for the source of the content to sign all the data, and also the traditional methods such as "hash and sign" are no longer applicable. This is achieved by on the fly verification, which employs a classical homomorphic hash function. In this method content is spitted and hashing is applied. Hashed content is sent to the destination through peer to peer network. Key is sent to the destination directly from the source. Without the key unauthorized users finds difficult to modify the content. Hence the content is secured, however this technique is very complex to be applied to network coding because of high computational and communication overhead. We analyzing this issue further by carefully for different types of overhead, and we propose methods to reducing both communication and computational cost, and also to achieving that providing provable security at the same time

Interaction of βA3-Crystallin with Deamidated Mutants of αA- and αB-Crystallins

<div><p>Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and β-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with βA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with βA3-crystallin. The deamidated mutants of αA–crystallin (αA N101D and αA N123D) displayed lower adherence propensity for βA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for βA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with βA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with βA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor βA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant’s large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.</p></div

Confocal images of expression of domains of αA- and αB-crystallins with βA3-crystallin in HeLa cells.

<p>Confocal microscopic images of HeLa cells co-transfected with: <b>A.</b> CFP βA3-crystallin -YFP αA NTD, YFP αA CD and YFP αA CTE crystallin. <b>B.</b> CFP βA3-crystallin-YFP αB NTD, YFP αB CD and YFP αB CTE crystalline. Note the co-expression of CFP- and YFP-tagged crystallins.Panel a: CFP channel image of cells co-transfected with pairs of CFP- and YFP-fusion crystallins. Panel b: YFP channel image of cells co-transfected with pairs of CFP- and YFP-fusion crystallins. Panel c: Merged images for CFP- and YFP-channels of cells co-transfected with pairs of CFP- and YFP-fusion crystallins.</p

SPR assay of the binding of WT αA-, αA N101D and αA N123D crystallins with βA3- crystallin.

<p>Binding responses (average RU obtained between 260–270 sec of association) of at 5, 10, 15, 20 and 25 μM of analytes (WTαA-crystallin, αA N101D and αA N123D) with βA3-crystallin.</p

A diagrammatic representation of the in vitro and in vivo interaction of WT α-crystallin and their deamidated mutants with βA3-crystallin.

<p>A diagrammatic representation of the in vitro and in vivo interaction of WT α-crystallin and their deamidated mutants with βA3-crystallin.</p

Confocal images of expression of αA- and WT αB- crystallin with βA3- crystallin in HeLa cells.

<p>Confocal microscopic images of transfected HeLa cells with: <b>A.</b> CFP βA3-crystallin alone and co-transfected with CFP βA3-crystallin-YFP WT αA-crystallin; and CFP βA3-crystallin-YFP WT αB-crystallin. Note the co-expression of CFP- and YFP-tagged crystallins. Panel a: CFP channel image of cells co-transfected with pairs of CFP- and YFP-fusion crystallins Panel b: YFP channel image of cells co-transfected with pairs of CFP- and YFP- fusion crystallins Panel c: Merged images for CFP- and YFP-channels of cells co-transfected with pairs of CFP- and YFP-fusion crystallins. Note that in case of CFP βA3-crystallin alone, no-bleed through expression of YFP was observed. <b>B.</b> Western blot analysis of the expression of <b>a.</b> CFP βA3-crystallin and YFP WT αA-crystallin Lanes 1 and 2: αA+ βA3, lanes 3 and 4: αA-, lane 5: βA3, Lane 6:Control. <b>b.</b> CFP βA3-crystallin and YFP WT αB-crystallin Lanes 1 and 2: αB+ βA3, lanes 3 and 4: αB-, lane 5: βA3, lane 6:Control. using βA3-1° antibody (green) and αA/ αB 1° antibody (red). β-actin was used as a loading control.</p

FLIM FRET images of HeLa cells showing life-time of the CFP βA3- crystallin in the presence of acceptor proteins (WT αA- and WT αB-crystallins and their deamidated and domain mutants).

<p>FRET measurement by fluorescence life time imaging microscopy of the HeLa cells transfected with <b>A.</b> Positive (YFP-CFP fusion), negative (CFP and YFP co-transfected) controls and CFP βA3-crystallin <b>B.</b> Co-transfected with CFP βA3-crystallin and YFP WT αA-crystallin and its mutants <b>C.</b> Co-transfected with CFP βA3-crystallin and YFP WT αB-crystallin and its mutants. The images were taken with Becker and Hickl FLIM system attached with the confocal miroscope. The life-time images are shown in pseudocolours (nanoseconds). The apparent mean life-time™ for each image is shown in the center at the bottom of each image.</p

Confocal images of expression of deamidated mutants of αA- and αB-crystallins with βA3- crystallin in HeLa cells.

<p>Confocal microscopic images of HeLa cells co-transfected with: <b>A.</b> CFP βA3-crystallin-YFP αA N101D crystallin and YFP αA N123D crystallin <b>B.</b> CFP βA3-crystallin-YFP αB N78D crystallin and YFP αB N146D crystallin. Note the co-expression of CFP- and YFP-tagged crystallins. Panel a: CFP channel image of cells co-transfected with pairs of CFP- and YFP-fusion crystallins. Panel b: YFP channel image of cells co-transfected with pairs of CFP- and YFP-fusion crystallins. Panel c: Merged images for CFP and YFP channels of cells co-transfected with pairs of CFP- and YFP- fusion crystallin.</p