Techniques for the realization of ultrareliable spaceborne computers Interim scientific report

Reliability tests on spaceborne digital computer

Crystal Structure of the P Pilus Rod Subunit PapA

P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE) mechanism, whereby the chaperone's G1 β-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it. Author Summary. Bacterial adhesion to a host is a crucial step that determines the onset of bacterial infection. It is mediated through recognition of a receptor on the host cell surface by a protein called an adhesin displayed on the surface of the bacterium. Many adhesins are displayed at the tip of specialized organelles called pili, some of which are assembled by the ubiquitous chaperone-usher pathway. In this pathway, each pilus subunit is assisted in folding by a chaperone. The resulting chaperone-subunit complex is targeted to a pore located in the outer membrane, called the usher, that serves as assembly platform. There, pilus subunits dissociate from the chaperone and polymerize, resulting in a surface organelle, the pilus, that protrudes out of the usher. Here, we have determined the structure of the major subunit of the P pilus, PapA. The P pilus, produced in uropathogenic Escherichia coli, displays the adhesin PapG responsible for targeting the bacterium to the kidney epithelium. We have determined the structure of PapA either bound to its cognate chaperone, PapD, or bound to another PapA subunit. These structures provide a view of PapA before and after its assembly in the pilus and shed light on the mechanism of PapA assembly.National Institutes of Health (DE 09761, GM040388, DE 09161); Committee of Scientific Research (3 PO4A 003 24, 2 P05A 137 24); Foundation for Polish Science (SUBSYDIUM PROFESORSKIE award); Swedish Rheumatism Association; Nanna Svartz Foundation; King Gustaf V Foundatio

Structure of a bacterial type IV secretion core complex at subnanometre resolution

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived

Exploring Millions of 6-State FSSP Solutions: the Formal Notion of Local CA Simulation

In this paper, we come back on the notion of local simulation allowing to transform a cellular automaton into a closely related one with different local encoding of information. This notion is used to explore solutions of the Firing Squad Synchronization Problem that are minimal both in time (2n -- 2 for n cells) and, up to current knowledge, also in states (6 states). While only one such solution was proposed by Mazoyer since 1987, 718 new solutions have been generated by Clergue, Verel and Formenti in 2018 with a cluster of machines. We show here that, starting from existing solutions, it is possible to generate millions of such solutions using local simulations using a single common personal computer

Dosimetry characterization of 32^{32}P intravascular brachytherapy source wires using Monte Carlo codes PENELOPE and GEANT4

Monte Carlo calculations using the codes PENELOPE and GEANT4 have been performed to characterize the dosimetric parameters of the new 20 mm long catheter based $^{32}$P beta source manufactured by Guidant Corporation. The dose distribution along the transverse axis and the two dimensional dose rate table have been calculated. Also, the dose rate at the reference point, the radial dose function and the anisotropy function were evaluated according to the adapted TG-60 formalism for cylindrical sources. PENELOPE and GEANT4 codes were first verified against previous results corresponding to the old 27 mm Guidant $^{32}$P beta source. The dose rate at the reference point for the unsheathed 27 mm source in water was calculated to be $0.215 \pm 0.001$ cGy s$^{-1}$ mCi$^{-1}$, for PENELOPE, and $0.2312 \pm 0.0008$ cGy s$^{-1}$ mCi$^{-1}$, for GEANT4. For the unsheathed 20 mm source these values were $0.2908 \pm 0.0009$ cGy s$^{-1}$ mCi$^{-1}$ and $0.311 \pm 0.001$ cGy s$^{-1}$ mCi$^{-1}$, respectively. Also, a comparison with the limited data available on this new source is shown. We found non negligible differences between the results obtained with PENELOPE and GEANT4.Comment: 13 pages, 7 figures, 7 tables (accepted for publication in Medical Physics

Structural and Functional Diversity of Type IV Secretion Systems

Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host–pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections

Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones

Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for,10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e. g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 A crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5

Avenues of future research in homotransplantation of the liver with particular reference to hepatic supportive procedures, antilymphocyte serum, and tissue typing

Three general areas of research which bear on the developing field of liver transplantation are reviewed. These are: (1) the prospects of obtaining better immunosuppression with particular reference to heterologous antilymphocyte serum; (2) the possible use of antigen matching technics as an advanced indicator of donorrecipient histocompatibility; (3) a simlified system of extracorporeal transplntation designed to provide teporary hepatic support. © 1966

Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry

P pili are multisubunit fibers essential for the attachment of uropathogenic Escherichia coli to the kidney. These fibers are formed by the noncovalent assembly of six different homologous subunit types in an array that is strictly defined in terms of both the number and order of each subunit type. Assembly occurs through a mechanism termed “donor-strand exchange (DSE)” in which an N-terminal extension (Nte) of one subunit donates a β-strand to an adjacent subunit, completing its Ig fold. Despite structural determination of the different subunits, the mechanism determining specificity of subunit ordering in pilus assembly remained unclear. Here, we have used noncovalent mass spectrometry to monitor DSE between all 30 possible pairs of P pilus subunits and their Ntes. We demonstrate a striking correlation between the natural order of subunits in pili and their ability to undergo DSE in vitro. The results reveal insights into the molecular mechanism by which subunit ordering during the assembly of this complex is achieved