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Klotho is an antiaging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels and signaling modulate inflammation in diabetic kidneys. Renal Klotho expression was determined by quantitative real-time PCR and immunoblot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-κB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-α in the presence or absence of Klotho, followed by immunoblot analysis to evaluate inhibitor of κB (IκB)α degradation, IκB kinase (IKK) and p38 activation, RelA nuclear translocation, and phosphorylation. A chromatin immunoprecipitation assay was performed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine (Ser)(536). Renal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of soluble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-κB activation and subsequent production of inflammatory cytokines in response to TNF-α stimulation. Klotho specifically inhibited RelA Ser(536) phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IκBα degradation, total RelA nuclear translocation, and total RelA DNA binding. These findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the production of NF-κB-linked inflammatory proteins via a mechanism that involves phosphorylation of Ser(536) in the transactivation domain of RelA.

BackgroundConstitutional mismatch repair deficiency (CMMRD) syndrome is a childhood cancer predisposition syndrome involving biallelic germline mutations of MMR genes, poorly recognised by clinicians so far.MethodsRetrospective review of all 31 patients with CMMRD diagnosed in French genetics laboratories in order to describe the characteristics, treatment and outcome of the malignancies and biological diagnostic data.Results67 tumours were diagnosed in 31 patients, 25 (37%) Lynch syndrome-associated malignancies, 22 (33%) brain tumours, 17 (25%) haematological malignancies and 3 (5%) sarcomas. The median age of onset of the first tumour was 6.9 years (1.2–33.5). Overall, 22 patients died, 9 (41%) due to the primary tumour. Median survival after the diagnosis of the primary tumour was 27 months (0.26–213.2). Failure rate seemed to be higher than expected especially for T-cell non-Hodgkin's lymphoma (progression/relapse in 6/12 patients). A familial history of Lynch syndrome was identified in 6/23 families, and consanguinity in 9/23 families. PMS2 mutations (n=18) were more frequent than other mutations (MSH6 (n=6), MLH1 (n=4) and MSH2 (n=3)).ConclusionsIn conclusion, this unselected series of patients confirms the extreme severity of this syndrome with a high mortality rate mostly related to multiple childhood cancers, and highlights the need for its early detection in order to adapt treatment and surveillance.

PurposeTo assess clinical impact and perform cost-consequence analysis of the broadest multiplex PCR panels available for the rapid diagnosis of bloodstream infections (BSI).MethodsSingle-center, randomized controlled trial conducted from June 2019 to February 2021 at a French University hospital with an institutional antimicrobial stewardship program. Primary endpoint was the percentage of patients with optimized antimicrobial treatment 12 h after transmission of positivity and Gram stain results from the first positive BC.ResultsThis percentage was significantly higher in the multiplex PCR (mPCR) group (90/105 = 85.7% %, CI95% [77.5 ; 91.8] vs. 68/107 = 63.6%, CI95% [53.7 ; 72.6]; p < 10− 3) at interim analysis, resulting in the early termination of the study after the inclusion of 309 patients. For patients not optimized at baseline, the median time to obtain an optimized therapy was much shorter in the mPCR group than in the control group (6.9 h, IQR [2.9; 17.8] vs. 26.4 h, IQR [3.4; 47.5]; p = 0.001). Early optimization of antibiotic therapy resulted in a non-statistically significant decrease in mortality from 12.4 to 8.8% (p = 0.306), with a trend towards a shorter median length of stay (18 vs. 20 days; p = 0.064) and a non-significant reduction in the average cost per patient of €3,065 (p = 0.15). mPCR identified all the bacteria present in 88% of the samples.ConclusionDespite its higher laboratory cost, the use of multiplex PCR for BSI diagnosis leads to early-optimised therapy, seems cost-effective and could reduce mortality and length of stay. Their impact could probably be improved if implemented 24/7.

Drops loaded in calcium ions detach from stalactites and impact the underlying stalagmites, thereby allowing these latter to grow through calcite precipitation. Nevertheless, little is known about the influence of the drop free fall and splash dynamics on stalagmite shape and width. Through high-speed imaging of impacting drops on stalagmites from several caves, we observed that the impact point position of the drops is scattered, sometimes over several centimetres. We show that this dispersal has no external cause and must, therefore, be self-induced. Using a Langevin-like equation, we then propose a prediction of the impact point dispersal as a function of the falling height travelled by the drops. We finally show that measured stalagmite widths are correlated to the dispersal in the impact point position of the drop.

We report the association of CDH1/E-cadherin mutations with cleft lip, with or without cleft palate (CLP), in two families with hereditary diffuse gastric cancer (HDGC). In each family, the CDH1 mutation was a splicing mutation generating aberrant transcripts with an in-frame deletion, removing the extracellular cadherin repeat domains involved in cell-cell adhesion. Such transcripts might encode mutant proteins with trans-dominant negative effects. We found that CDH1 is highly expressed at 4 and 5 weeks in the frontonasal prominence, and at 6 weeks in the lateral and medial nasal prominences of human embryos, and is therefore expressed during the critical stages of lip and palate development. These findings suggest that alteration of the E-cadherin pathway can contribute to human clefting.

Background: Microsatellite instability (MSI) is a molecular phenotype due to defective DNA mismatch repair (MMR) system. It is used to predict outcome of colorectal tumours and to screen tumours for Lynch syndrome (LS). A pentaplex panel composed of five mononucleotide markers has been largely recommended for determination of the MSI status. However, its sensitivity may be taken in default in occasional situations. The aim of the study was to optimise this panel for the detection of MSI. Methods: We developed an assay allowing co-amplification of six mononucleotide repeat markers (BAT25, BAT26, BAT40, NR21, NR22, NR27) and one polymorphic dinucleotide marker (D3S1260) in a single reaction. Performances of the new panel were evaluated on a cohort of patients suspected of LS. Results: We demonstrate that our assay is technically as easy to use as the pentaplex assay. The hexaplex panel shows similar performances for the identification of colorectal and non-MSH6-deficient tumours. On the other hand, the hexaplex panel has higher sensitivity for the identification of MSH6-deficient tumours (94.7% vs 84.2%) and MMR-deficient tumours other than colorectal cancer (92.9% vs 85.7%). Conclusion: The hexaplex panel could thus be an attractive alternative to the pentaplex panel for the identification of patients with LS.

Local variability in total electron content can seriously affect the accuracy of GNSS real-time applications. We have developed software to compute the positioning error due to the ionosphere for all baselines of the Belgian GPS network, called the Active Geodetic Network (AGN). In a first step, a reference day has been chosen to validate the methodology by comparing results with the nominal accuracy of relative positioning at centimeter level. Then, the effects of two types of ionospheric disturbances on the positioning error have been analyzed: (1) Traveling ionospheric disturbances (TIDs) and (2) noise-like variability due to geomagnetic storms. The influence of baseline length on positioning error has been analyzed for these three cases. The analysis shows that geomagnetic storms induce the largest positioning error (more than 2 m for a 20 km baseline) and that the positioning error depends on the baseline orientation. Baselines oriented parallel to the propagation direction of the ionospheric disturbances are more affected than others. The positioning error due to ionospheric small-scale structures can be so identified by our method, which is not always the case with the modern ionosphere mitigation techniques. In the future, this ionospheric impact formulation could be considered in the development of an integrity monitoring service for GNSS relative positioning users.

OBJECTIVE--Klotho is an anti-aging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels and signaling modulate inflammation in diabetic kidneys. RESEARCH DESIGN AND METHODS--Renal Klotho expression was determined by quantitative real-time PCR and immunoblot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-κB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-α in the presence or absence of Klotho, followed by immunoblot analysis to evaluate inhibitor of κB (IκB)α degradation, IκB kinase (IKK) and p38 activation, RelA nuclear translocation, and phosphorylation. A chromatin immunoprecipitation assay was performed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine [(Set).sup.536]. RESULTS--Renal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of soluble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-κB activation and subsequent production of inflammatory cytokines in response to TNF-α stimulation. Klotho specifically inhibited RelA [Ser.sup.536] phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IκBα degradation, total RelA nuclear translocation, and total RelA DNA binding. CONCLUSIONS--These findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the production of NF-κB-linked inflammatory proteins via a mechanism that involves phosphorylation of [Ser..sup.536] in the transactivation domain of RelA. Diabetes 60:1907-1916, 2011

Diabetes-Induced Activation of Canonical and Noncanonical Nuclear Factor-κB Pathways in Renal Cortex Jonathan M. Starkey 1 , Sigmund J. Haidacher 1 , Wanda S. LeJeune 1 , Xiaoquan Zhang 1 , Brian C. Tieu 1 2 , Sanjeev Choudhary 1 , Allan R. Brasier 1 2 3 , Larry A. Denner 1 4 and Ronald G. Tilton 1 4 1 Division of Endocrinology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 2 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 3 Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, Texas 4 Stark Diabetes Center, University of Texas Medical Branch, Galveston, Texas Address correspondence and reprint requests to Ronald G. Tilton, PhD, Division of Endocrinology, Department of Internal Medicine, Stark Diabetes Center, 8.138 Medical Research Building, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1060. E-mail: rgtilton{at}utmb.edu Abstract Evidence of diabetes-induced nuclear factor-κB (NF-κB) activation has been provided with DNA binding assays or nuclear localization with immunohistochemistry, but few studies have explored mechanisms involved. We examined effects of diabetes on proteins comprising NF-κB canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-κB–regulated cytokines were increased after 1 month of hyperglycemia, but most returned to control levels or lower by 3 months, when the same cytokines were increased significantly in renal cortex. Cytosolic content of NF-κB canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-κB noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of κB kinase-α and several fold increases in NF-κB–inducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-κB pathway proteins was decreased by diabetes, with the largest change in RelB and p50 (approximately twofold decrease). Despite this decrease, measurable increases in protein binding to DNA in diabetic versus control nuclear extracts were observed with electrophoretic mobility shift assay. These results provide evidence for chronic NF-κB activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-κB pathway proteins. DTT, dithiothreitol EMSA, electrophoretic mobility shift assay IκB, inhibitor of κB IKK, inhibitor of κB kinase IL, interleukin NF-kB, nuclear factor-κB NIK, NF-κB–inducing kinase TNF, tumor necrosis factor Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted February 16, 2006. Received November 30, 2005. DIABETES