An Efficient Algorithm For Simulating Fracture Using Large Fuse Networks

The high computational cost involved in modeling of the progressive fracture simulations using large discrete lattice networks stems from the requirement to solve {\it a new large set of linear equations} every time a new lattice bond is broken. To address this problem, we propose an algorithm that combines the multiple-rank sparse Cholesky downdating algorithm with the rank-p inverse updating algorithm based on the Sherman-Morrison-Woodbury formula for the simulation of progressive fracture in disordered quasi-brittle materials using discrete lattice networks. Using the present algorithm, the computational complexity of solving the new set of linear equations after breaking a bond reduces to the same order as that of a simple {\it backsolve} (forward elimination and backward substitution) {\it using the already LU factored matrix}. That is, the computational cost is $O(nnz({\bf L}))$, where $nnz({\bf L})$ denotes the number of non-zeros of the Cholesky factorization ${\bf L}$ of the stiffness matrix ${\bf A}$. This algorithm using the direct sparse solver is faster than the Fourier accelerated preconditioned conjugate gradient (PCG) iterative solvers, and eliminates the {\it critical slowing down} associated with the iterative solvers that is especially severe close to the critical points. Numerical results using random resistor networks substantiate the efficiency of the present algorithm.Comment: 15 pages including 1 figure. On page pp11407 of the original paper (J. Phys. A: Math. Gen. 36 (2003) 11403-11412), Eqs. 11 and 12 were misprinted that went unnoticed during the proof reading stag

Reproducible gene targeting in recalcitrant Escherichia coli isolates

<p>Abstract</p> <p>Background</p> <p>A number of allele replacement methods can be used to mutate bacterial genes. For instance, the Red recombinase system of phage Lambda has been used very efficiently to inactivate chromosomal genes in <it>E. coli </it>K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical <it>E. coli </it>isolates.</p> <p>Findings</p> <p>The procedure was modified by using longer homologous regions (85 bp and 500-600 bp), to inactivate genes in the uropathogenic <it>E. coli </it>strain UTI89. An <it>lrhA </it>regulator mutant, and deletions of the <it>lac </it>operon as well as the complete <it>type 1 </it>fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant <it>E. coli</it>, like the avian pathogenic <it>E. coli </it>strain APEC1. The <it>lrhA </it>regulator and <it>lac </it>operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic <it>E. coli </it>strains (APEC3E, APEC11A and APEC16A) it was very difficult or impossible to construct these mutants, with the original Red recombinase-based method, with a Red recombinase-based method using longer (85 bp) homologous regions or with our modified protocol, using 500 - 600 bp homologous regions.</p> <p>Conclusions</p> <p>The method using 500-600 bp homologous regions can be used reliably in some clinical isolates, to delete single genes or entire operons by homologous recombination. However, it does not invariably show a greater efficiency in obtaining mutants, when compared to the original Red-mediated gene targeting method or to the gene targeting method with 85 bp homologous regions. Therefore the length of the homology regions is not the only limiting factor for the construction of mutants in these recalcitrant strains.</p

Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

BACKGROUND: The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. RESULTS: Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. CONCLUSION: This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer

The role of SHDA in faecal shedding of Salmonella typhimurium in pigs

Prolonged faecal shedding of Salmonella in pigs contributes to contamination of carcasses. The shdA gene has been characterized as an important locus for persistency of Salmonella Typhimurium in mice. The aim of this study was to assess the contribution of ShdA in faecal shedding of S. Typhimurium in pigs. Pigs were orally inoculated with a S. Typhimurium strain or its isogenic shdA mutant strain. For the first few days after inoculation, the shdA mutant strain was more virulent than the wild type strain, as indicated by higher excretion levels, more pronounced diarrhea and higher numbers of infected organs. No effect on long-term shedding was found. Increased in vitro invasion levels of the shdA mutant strain were noticed in intestinal epithelial cells. In conclusion, a shdA mutant strain of S. Typhimurium is more virulent during the first days after inoculation and is not impaired in persistency or prolonged shedding in pigs

In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter

Campylobacteriosis is a widespread infectious disease, leading to a major health and economic burden. Chickens are considered as the most common infection source for humans. Campylobacter mainly multiplies in the mucus layer of their caeca. No effective control measures are currently available, but passive immunisation of chickens with pathogen-specific maternal IgY antibodies, present in egg yolk of immunised chickens, reduces Campylobacter colonisation. To explore this strategy further, anti-Campylobacter nanobodies, directed against the flagella and major outer membrane proteins, were fused to the constant domains of chicken IgA and IgY, combining the benefits of nanobodies and the effector functions of the Fc-domains. The designer chimeric antibodies were effectively produced in leaves of Nicotiana benthamiana and seeds of Arabidopsis thaliana. Stable expression of the chimeric antibodies in seeds resulted in production levels between 1% and 8% of the total soluble protein. These in planta produced antibodies do not only bind to their purified antigens but also to Campylobacter bacterial cells. In addition, the anti-flagellin chimeric antibodies are reducing the motility of Campylobacter bacteria. These antibody-containing Arabidopsis seeds can be tested for oral passive immunisation of chickens and, if effective, the chimeric antibodies can be produced in crop seeds

Plasmid parB contributes to uropathogenic Escherichia coli colonization in vivo by acting on biofilm formation and global gene regulation

The endogenous plasmid pUTI89 harbored by the uropathogenic Escherichia coli (UPEC) strain UTI89 plays an important role in the acute stage of infection. The partitioning gene parB is important for stable inheritance of pUTI89. However, the function of partitioning genes located on the plasmid in pathogenesis of UPEC still needs to be further investigated. In the present study, we observed that disruption of the parB gene leads to a deficiency in biofilm formation in vitro. Moreover, in a mixed infection with the wild type strain and the parB mutant, in an ascending UTI mouse model, the mutant displayed a lower bacterial burden in the bladder and kidneys, not only at the acute infection stage but also extending to 72 hours post infection. However, in the single infection test, the reduced colonization ability of the parB mutant was only observed at six hpi in the bladder, but not in the kidneys. The colonization capacity in vivo of the parB-complemented strain was recovered. qRT-PCR assay suggested that ParB could be a global regulator, influencing the expression of genes located on both the endogenous plasmid and chromosome, while the gene parA or the operon parAB could not. Our study demonstrates that parB contributes to the virulence of UPEC by influencing biofilm formation and proposes that the parB gene of the endogenous plasmid could regulate gene expression globally

Удосконалення комерційної діяльності як фактор підвищення конкурентоспроможності підприємства

Additional file 5. ELISA to assess the interaction between Campylobacter -specific nanobodies and purified MOMP. The saturation binding curve of the interaction between coated MOMP (1 µg/mL) and a His-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) was obtained via ELISA. The dose-dependent inhibitory effect of a strep-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) on the interaction between His-tagged Nb84 (5.10−2 µg/mL) and MOMP (1 µg/mL), is demonstrated in the competition binding curve. Inhibition by strep-tagged (A) Nb5, (B) Nb22, (C) Nb23, (D) Nb24, (E) Nb49, (F) 84, (G) Nb15, (H) Nb32, (I) Nb34, (J) Nb45, (K) Nb48 and (L) Nb63, was assessed. The ELISA was developed with mouse anti-Histidine tag monoclonal antibody and goat anti-mouse IgG conjugated to alkaline phosphatase. The error bars represent the standard deviations

The dynamic transcriptome during maturation of biofilms formed by methicillin-resistant Staphylococcus aureus

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA), a leading cause of chronic infections, forms prolific biofilms which afford an escape route from antibiotic treatment and host immunity. However, MRSA clones are genetically diverse, and mechanisms underlying biofilm formation remain under-studied. Such studies form the basis for developing targeted therapeutics. Here, we studied the temporal changes in the biofilm transcriptome of three pandemic MRSA clones: USA300, HEMRSA-15, and ST239.MethodsBiofilm formation was assessed using a static model with one representative strain per clone. Total RNA was extracted from biofilm and planktonic cultures after 24, 48, and 72 h of growth, followed by rRNA depletion and sequencing (Illumina Inc., San Diego, CA, United States, NextSeq500, v2, 1 × 75 bp). Differentially expressed gene (DEG) analysis between phenotypes and among early (24 h), intermediate (48 h), and late (72 h) stages of biofilms was performed together with in silico co-expression network construction and compared between clones. To understand the influence of SCCmec and ACME on biofilm formation, isogenic mutants containing deletions of the entire elements or of single genes therein were constructed in USA300.ResultsGenes involved in primarily core genome-encoded KEGG pathways (transporters and others) were upregulated in 24-h biofilm culture compared to 24-h planktonic culture. However, the number of affected pathways in the ST239 24 h biofilm (n = 11) was remarkably lower than that in USA300/EMRSA-15 biofilms (USA300: n = 27, HEMRSA-15: n = 58). The clfA gene, which encodes clumping factor A, was the single common DEG identified across the three clones in 24-h biofilm culture (2.2- to 2.66-fold). In intermediate (48 h) and late (72 h) stages of biofilms, decreased expression of central metabolic and fermentative pathways (glycolysis/gluconeogenesis, fatty acid biosynthesis), indicating a shift to anaerobic conditions, was already evident in USA300 and HEMRSA-15 in 48-h biofilm cultures; ST239 showed a similar profile at 72 h. Last, SCCmec+ACME deletion and opp3D disruption negatively affected USA300 biofilm formation.ConclusionOur data show striking differences in gene expression during biofilm formation by three of the most important pandemic MRSA clones, USA300, HEMRSA-15, and ST239. The clfA gene was the only significantly upregulated gene across all three strains in 24-h biofilm cultures and exemplifies an important target to disrupt early biofilms. Furthermore, our data indicate a critical role for arginine catabolism pathways in early biofilm formation

Intervening with Urinary Tract Infections Using Anti-Adhesives Based on the Crystal Structure of the FimH–Oligomannose-3 Complex

Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and alpha3beta1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections.We demonstrate that alpha-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl alpha-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl alpha-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl alpha-D-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Man alpha1,3Man beta1,4GlcNAc beta1,4GlcNAc in an extended binding site. The interactions along the alpha1,3 glycosidic bond and the first beta1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl alpha-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group.The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection

The SlyB outer membrane lipoprotein of Burkholderia multivorans contributes to membrane integrity.

SlyB is a small lipoprotein of 158 amino acids which is conserved in different Gram-negative bacteria. In contrast to other bacteria, where slyB is monocistronic, in Burkholderia multivorans and in B. cenocepacia, slyB is the last gene of an operon comprising three open reading frames encoding a putative thiol peroxidase, a putative sugar kinase and SlyB. B. multivorans slyB mutants produced elongated cells and filaments which were never observed in cultures of wild-type or slyB-complemented cells. The slyB mutant also showed increased sensitivity to EDTA and SDS, and decreased siderophore production. Proteome analysis of a fraction enriched for membrane proteins suggested that SlyB, like the peptidoglycan-associated protein OpcL, is a major protein of the outer membrane. Taken together, these phenotypes suggest that SlyB contributes to the integrity of the cell envelope. By PCR amplification we were also able to demonstrate the conservation of slyB in all B. cepacia complex species tested.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe