How uromodulin helps flush out bacteria
Urinary tract infections (UTIs) are one of the most frequent bacterial infections in humans. The glycoprotein uromodulin is the most abundant urinary protein ...and can provide some protection from UTIs, but the precise mechanism has been unclear. Weiss
et al.
found that uromodulin forms stacked, fishbone-like filaments that act as a multivalent decoy for bacterial pathogens with adhesive pili that attach to the uromodulin glycans (see the Perspective by Kukulski). The resulting uromodulin-pathogen aggregates prevent bacterial adhesion to glycoproteins of the urinary epithelium and promote pathogen clearance as urine is excreted. This innate protection against UTIs is likely to be particularly important in infants and children.
Science
, this issue p.
1005
; see also p.
917
Uromodulin filaments in human urine associate with uropathogens and mediate bacterial aggregation and clearance.
Uromodulin is the most abundant protein in human urine, and it forms filaments that antagonize the adhesion of uropathogens; however, the filament structure and mechanism of protection remain poorly understood. We used cryo–electron tomography to show that the uromodulin filament consists of a zigzag-shaped backbone with laterally protruding arms. N-glycosylation mapping and biophysical assays revealed that uromodulin acts as a multivalent ligand for the bacterial type 1 pilus adhesin, presenting specific epitopes on the regularly spaced arms. Imaging of uromodulin-uropathogen interactions in vitro and in patient urine showed that uromodulin filaments associate with uropathogens and mediate bacterial aggregation, which likely prevents adhesion and allows clearance by micturition. These results provide a framework for understanding uromodulin in urinary tract infections and in its more enigmatic roles in physiology and disease.
Ligand-receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell-cell adhesion. They critically contribute to widespread urinary tract ...infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force.
Cancer cell lines selected for resistance to microtubule targeting agents (MTA) often have acquired mutations in the β-tubulin binding sites for these agents. Despite strong correlational evidence, ...the functional and quantitative significance of such mutations in the resistance to MTA remains unknown. We recently showed that peloruside A (PLA) and laulimalide (LAU)-resistant cancer cell lines, 1A9-R1 (R1) and 1A9-L4 (L4), generated through multi-step selection of human 1A9 ovarian cancer cells with high concentrations of either PLA (for R1) or LAU (for L4) have single distinct mutations in their βI-tubulin gene. The R1 cells have a mutation at amino acid position 296 (A296T), and the L4 cells have a mutation at position 306 (R306H/C), both of which lie at the putative binding sites of PLA and LAU. To gain insights on the functional role of these mutations in the resistance phenotype, R1 and L4 cells were transfected with wild type βI-tubulin. MTT cell proliferation assays revealed that restoration of wild type βI-tubulin expression partially sensitized the R1 and L4 cells to PLA and LAU. Cell cycle analysis and intracellular tubulin polymerization assays demonstrated that the increased sensitivity was correlated with an increased ability of PLA and LAU to induce G2-M arrest and tubulin polymerization in the cells. Unlike paclitaxel-selected clones of 1A9 cells, both R1 and L4 cells exhibited a functional p53 gene, and the abundance of the mismatch repair gene hMSH2 (human mutS homolog 2) was comparable to the parental 1A9 cells. This study provides the first direct evidence that A296 and R306 of βI-tubulin are important determinants of the PLA and LAU response in cancer cells.
The antibody Fv module which binds antigen consists of the variable domains V
and V
. These exhibit a conserved ß-sheet structure and comprise highly variable loops (CDRs). Little is known about the ...contributions of the framework residues and CDRs to their association. We exchanged conserved interface residues as well as CDR loops and tested the effects on two Fvs interacting with moderate affinities (K
s of ~2.5 µM and ~6 µM). While for the rather instable domains, almost all mutations had a negative effect, the more stable domains tolerated a number of mutations of conserved interface residues. Of particular importance for Fv association are V
P44 and V
L45. In general, the exchange of conserved residues in the V
/V
interface did not have uniform effects on domain stability. Furthermore, the effects on association and antigen binding do not strictly correlate. In addition to the interface, the CDRs modulate the variable domain framework to a significant extent as shown by swap experiments. Our study reveals a complex interplay of domain stability, association and antigen binding including an unexpected strong mutual influence of the domain framework and the CDRs on stability/association on the one side and antigen binding on the other side.
The complex between the bacterial type 1 pilus subunit FimG and the peptide corresponding to the N‐terminal extension (termed donor strand, Ds) of the partner subunit FimF (DsF) shows the strongest ...reported noncovalent molecular interaction, with a dissociation constant (KD) of 1.5×10−20 m. However, the complex only exhibits a slow association rate of 330 m−1 s−1 that limits technical applications, such as its use in affinity purification. Herein, a structure‐based approach was used to design pairs of FimGt (a FimG variant lacking its own N‐terminal extension) and DsF variants with enhanced electrostatic surface complementarity. Association of the best mutant FimGt/DsF pairs was accelerated by more than two orders of magnitude, while the dissociation rates and 3D structures of the improved complexes remained essentially unperturbed. A KD value of 8.8×10−22 m was obtained for the best mutant complex, which is the lowest value reported to date for a protein/ligand complex.
Accelerated binding of the protein FimGt to its natural peptide ligand DsF was achieved by rational design of FimGt and DsF variants with enhanced electrostatic interactions. Binding was accelerated by two orders of magnitude without structural perturbation of the complex. The most stable and fastest associating complex (see picture, shown in green) showed a dissociation constant of 8.8×10−22 m (wild‐type complex shown in gray).
The complex between the bacterial type 1 pilus subunit FimG and the peptide corresponding to the N‐terminal extension (termed donor strand, Ds) of the partner subunit FimF (DsF) shows the strongest ...reported noncovalent molecular interaction, with a dissociation constant (KD) of 1.5×10−20 m. However, the complex only exhibits a slow association rate of 330 m−1 s−1 that limits technical applications, such as its use in affinity purification. Herein, a structure‐based approach was used to design pairs of FimGt (a FimG variant lacking its own N‐terminal extension) and DsF variants with enhanced electrostatic surface complementarity. Association of the best mutant FimGt/DsF pairs was accelerated by more than two orders of magnitude, while the dissociation rates and 3D structures of the improved complexes remained essentially unperturbed. A KD value of 8.8×10−22 m was obtained for the best mutant complex, which is the lowest value reported to date for a protein/ligand complex.
Eine Beschleunigung der Bindung des Proteins FimGt an seinen natürlichen Peptidliganden DsF gelang durch Herstellung von FimGt‐ und DsF‐Varianten mit stärkeren elektrostatischen Wechselwirkungen. Der Bindungsvorgang war mit ihnen um zwei Größenordnungen schneller, ohne dass der Komplex strukturell beeinträchtigt wurde. Der stabilste und am schnellsten assoziierende Komplex (grün im Bild) zeigt eine Dissoziationskonstante von 8.8×10−22 m (Wildtypkomplex in Grau).
Taking a broad perspective of livelihoods, this book draws on more than ninety case studies from thirty-six countries across Asia, Africa and Latin America to examine how people are engaging and ...living with modernity. This extends from changes in the ways that households operate, to how and why people take on new work and acquire new skills, how migration and mobility have become increasingly common features of existence, and how aspirations and expectations are being reworked under the influence of modernization.
To date, this is the only book which takes such an approach to building an understanding of the global South. By using the experience of the non-Western world to illuminate and inform mainstream debates in geography, and in beginning from the lived experiences of ‘ordinary’ people, this book provides an alternative insight into a range of geographical debates. The clarity of argument and its use of detailed case studies makes this book an invaluable resource for students.
Jonathan Rigg is Professor of Geography at Durham University. His research interests include development in the South-East Asian region, rural and agrarian change, and political ecology.
1. What's With the Everyday?: The Everyday, Globalization and the Global South 2. Structures and Agencies: Lives, Living and Livelihoods 3. Life Styles and Life Courses: The Structures and Rhythms of Everyday Life 4. Making a Living in the Global South: Livelihood Transitions 5. Living with Modernity 6. Living on the Move 7. Governing the Everyday 8. Alternatives: The Everyday and Resistance 9. The Structures of the Everyday
" Jonathan Rigg draws from over 90 case studies in 36 countries to challenge standard topdown approaches to understanding the dynamics of poverty, development, and globalization in the Global South. He approaches his subject with a refreshing humility toward the characters—rickshaw wallas, migrant shrimp-farmers, single-mother traders, subsistence farmers—who people this investigation." -- JOURNAL OF REGIONAL SCIENCE , VOL. 50, NO. 2, 2010
Abstract
Background
It is unknown whether renal pathology lesions in immunoglobulin A nephropathy (IgAN) correlate with renal outcomes over decades of follow-up.
Methods
In 1130 patients of the ...original Validation Study of the Oxford Classification for IgA Nephropathy (VALIGA) cohort, we studied the relationship between the MEST score (mesangial hypercellularity, M; endocapillary hypercellularity, E; segmental glomerulosclerosis, S; tubular atrophy/interstitial fibrosis, T), crescents (C) and other histological lesions with both a combined renal endpoint 50% estimated glomerular filtration rate (eGFR) loss or kidney failure and the rate of eGFR decline over a follow-up period extending to 35 years median 7 years (interquartile range 4.1–10.8).
Results
In this extended analysis, M1, S1 and T1–T2 lesions as well as the whole MEST score were independently related with the combined endpoint (P < 0.01), and there was no effect modification by age for these associations, suggesting that they may be valid in children and in adults as well. Only T lesions were associated with the rate of eGFR loss in the whole cohort, whereas C showed this association only in patients not treated with immunosuppression. In separate prognostic analyses, the whole set of pathology lesions provided a gain in discrimination power over the clinical variables alone, which was similar at 5 years (+2.0%) and for the whole follow-up (+1.8%). A similar benefit was observed for risk reclassification analyses (+2.7% and +2.4%).
Conclusion
Long-term follow-up analyses of the VALIGA cohort showed that the independent relationship between kidney biopsy findings and the risk of progression towards kidney failure in IgAN remains unchanged across all age groups and decades after the renal biopsy.
Characterising the correlates of HIV persistence improves understanding of disease pathogenesis and guides the design of curative strategies. This study investigated factors associated with ...integrated HIV-1 DNA load during consistently suppressive first-line antiretroviral therapy (ART).
Total, integrated, and 2-long terminal repeats (LTR) circular HIV-1 DNA, residual plasma HIV-1 RNA, T-cell activation markers, and soluble CD14 (sCD14) were measured in peripheral blood of 50 patients that had received 1–14years of efavirenz-based or nevirapine-based therapy.
Integrated HIV-1 DNA load (per 106 peripheral blood mononuclear cells) was median 1.9 log10 copies (interquartile range 1.7–2.2) and showed a mean difference of 0.2 log10 copies per 10years of suppressive ART (95% confidence interval −0.2, 0.6; p=0.28). It was positively correlated with total HIV-1 DNA load and frequency of CD8+HLA-DR/DP/DQ+ cells, and was also higher in subjects with higher sCD14 levels, but showed no correlation with levels of 2-LTR circular HIV-1 DNA and residual plasma HIV-1 RNA, or the frequency of CD4+CD38+ and CD8+CD38+ cells. Adjusting for pre-ART viral load, duration of suppressive ART, CD4 cell counts, residual plasma HIV-1 RNA levels, and sCD14 levels, integrated HIV-1 DNA load was mean 0.5 log10 copies higher for each 50% higher frequency of CD8+HLA-DR/DP/DQ+ cells (95% confidence interval 0.2, 0.9; p=0.01).
The observed positive association between integrated HIV-1 DNA load and frequency of CD8+DR/DP/DQ+ cells indicates that a close correlation between HIV persistence and immune activation continues during consistently suppressive therapy. The inducers of the distinct activation profile warrant further investigation.
•Data from a homogenously treated population with consistent virological suppression•Integrated HIV-1 DNA load did not vary significantly by duration of therapy•Integrated HIV-1 DNA load was not associated with markers of recent virus replication•Integrated HIV-1 DNA load and CD8+HLA-DR/DP/DQ+ frequency were positively associated•Subjects with top quartile integrated HIV-1 DNA load showed high sCD14 levels
Integrated HIV-1 DNA load remains constant in the peripheral blood of individuals receiving long-term suppressive antiretroviral therapy (ART). However, the mechanisms preventing decay of the reservoir remain unclear. We studied a cross-sectional population, defined by the duration of suppressive ART. Integrated HIV-1 DNA load did not differ significantly according to the duration of suppressive ART, and showed no association with direct or indirect markers of ongoing virus replication. Rather, there was an independent, positive association between integrated HIV-1 DNA load and the frequency of CD8 cells expressing the activation marker HLA-DR/DP/DQ. These cells appear to have important regulatory and effector function. Our findings add to growing evidence that immune activation sustains the HIV-1 reservoir during long-term suppressive ART.
Persistence of plasma HIV-1 RNA during seemingly effective antiretroviral thereapy (ART) is incompletely understood. Using an ultrasensitive assay, this cross-sectional study investigated residual ...plasma HIV-1 RNA in subjects maintained on firstline ART with continuous viral load suppression <50 copies/mL for ≤15 years without recognized viral load blips or treatment interruptions and explored its relationship with the duration of suppressive ART, efavirenz concentrations in plasma, 2-LTR circular HIV-1 DNA (2-LTRc DNA) in peripheral blood mononuclear cells, and cellular (CD4 plus CD26/CD38/CD69; CD8 plus CD38/HLA-DR/DP/DQ) and soluble (sCD14, sCD27, sCD30, IL-6) markers of immune activation in peripheral blood.
Residual plasma HIV-1 RNA, total HIV-1 DNA and 2-LTRc DNA were quantified by real-time and digital droplet PCR. Cellular (CD4 plus CD26/CD38/CD69; CD8 plus CD38/HLA-DR/DP/DQ) and soluble (sCD14, sCD27, sCD30, IL-6) markers of immune activation were measured by flow cytometry and ELISA.
Residual plasma HIV-1 RNA and 2-LTRc DNA were detected in 52/104 (50%) and 24/104 (23%) subjects, respectively. Among subjects with detectable HIV-1 RNA, 50/52 showed levels ≤11 copies/mL. In adjusted analyses, HIV-1 RNA levels were 0.37 log
copies/mL higher with each log
U/mL increase in sCD27 (95% confidence interval, 0.01-0.73;
= .02). No significant association was found between residual plasma HIV-1 RNA and other explored parameters.
These findings point to an ongoing relationship between plasma HIV-1 RNA and selected markers of immune activation during continuously suppressive ART. The novel direct association with levels of sCD27 warrants further investigation.
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