Complications and Short-Term Explantation Rate Following Artificial Urinary Sphincter Implantation: Results from a Large Middle European Multi-Institutional Case Series

Background/Aims/Objectives: To analyze perioperative complication and short-term explantation rates after perineal or penoscrotal single-cuff and double-cuff artificial urinary sphincter (AUS) implantation in a large middle European multi-institutional patient cohort. Methods: 467 male patients with stress urinary incontinence underwent implantation of a perineal single-cuff (n = 152), penoscrotal single-cuff (n = 99), or perinea! double-cuff (n = 216) AUS between 2010 and 2012. Postoperative complications and 6-month explantation rates were assessed. For statistical analysis, Fisher's exact test and Kruskal Wallis rank sum test, and a multiple logistic regression model were used (p < 0.05). Results: Compared to perineal single-cuff AUS, penoscrotal single-cuff implantation led to significantly increased short-term explantation rates (8.6% (perinea)) vs. 19.2% (penoscrotal), p = 0.019). The postoperative infection rate was significantly higher after double-cuff compared to single cuff implantation (6.0% (single-cuff) vs. 13.9% (double-cuff), p = 0.019). The short-term explantation rate after primary double-cuff placement was 6.5% (p = 0.543 vs. perineal single -cuff). In multivariate analysis, the penoscrotal approach (p = 0.004), intraoperative complications (p = 0.005), postoperative bleeding (p = 0.011), and perioperative infection (p < 0.001) were independent risk factors for short-term explantation. Conclusions: Providing data from a large contemporary multi-institutional patient cohortfrom high-volume and low-volume institutions, our results reflect the current standard of care in middle Europe. We indicate that the penoscrotal approach is an independent risk factor for increased short-term explantation rates. (C) 2016 S. Karger AG, Base

Epigenetic Signatures at AQP3 and SOCS3 Engage in Low-Grade Inflammation across Different Tissues

Background Elevated levels of C-reactive protein (CRP, determined by a high-sensitivity assay) indicate low-grade inflammation which is implicated in many age-related disorders. Epigenetic studies on CRP might discover molecular mechanisms underlying CRP regulation. We aimed to identify DNA methylation sites related to CRP concentrations in cells and tissues regulating low-grade inflammation. Results Genome-wide DNA methylation was measured in peripheral blood in 1,741 participants of the KORA F4 study using Illumina HumanMethylation450 BeadChip arrays. Four CpG sites (located at BCL3, AQP3, SOCS3, and cg19821297 intergenic at chromosome 19p13.2, P <= 1.01E-07) were significantly hypomethylated at high CRP concentrations independent of various confounders including age, sex, BMI, smoking, and white blood cell composition. Findings were not sex-specific. CRP-related top genes were enriched in JAK/STAT pathways (Benjamini-Hochberg corrected P < 0.05). Results were followed-up in three studies using DNA from peripheral blood (EPICOR, n = 503) and adipose tissue (TwinsUK, n = 368) measured as described above and from liver tissue (LMU liver cohort, n = 286) measured by MALDI-TOF mass spectrometry using EpiTYPER. CpG sites at the AQP3 locus (significant p-values in peripheral blood = 1.72E-03 and liver tissue = 1.51E-03) and the SOCS3 locus (p-values in liver < 2.82E-05) were associated with CRP in the validation panels. Conclusions Epigenetic modifications seem to engage in low-grade inflammation, possibly via JAK/STAT mediated pathways. Results suggest a shared relevance across different tissues at the AQP3 locus and highlight a role of DNA methylation for CRP regulation at the SOCS3 locus

Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana

Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for the functionality of the tomato LRR-receptor-like protein Cf-4, which detects the secreted effector Avr4 of the pathogenic fungus Fulvia fulva. Here, we show that the kinase domains of SOBIR1 and BAK1 directly phosphorylate each other and that residues Thr522 and Tyr469 of the kinase domain of Nicotiana benthamiana SOBIR1 are required for its kinase activity and for interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic kinase (RLCK)-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, -7, and -8 differentially regulate the Avr4/Cf-4-triggered biphasic burst of reactive oxygen species. In addition, members of RLCK-VII-7 play an essential role in resistance against the oomycete pathogen Phytophthora palmivora. Our study provides molecular evidence for the specific roles of RLCKs downstream of SOBIR1/BAK1-containing immune complexes

EDS1 complexes are not required for PRR responses and execute TNL‐ETI from the nucleus in Nicotiana benthamiana

Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in Arabidopsis thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in Nicotiana benthamiana. We did not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad could abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation was sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown

Targeting Moderate and Severe Male Stress Urinary Incontinence With Adjustable Male Slings and the Perineal Artificial Urinary Sphincter: Focus on Perioperative Complications and Device Explantations

Purpose: To analyze perioperative complications and postoperative explantation rates for selected readjustable male sling systems and the perineal single-cuff artificial urinary sphincter (AUS) in a large, contemporary, multi-institutional patient cohort. Methods: Two hundred eighty-two male patients who underwent implantation between 2010 and 2012 in 13 participating institutions were included in the study (n = 127 adjustable male sling [n = 95 Argus classic, n = 32 Argus T], n = 155 AUS). Perioperative characteristics and postoperative complications were analyzed. The explantation rates of the respective devices were assessed using the Fisher exact test and the Mann-Whitney U-test. A Kaplan-Meier curve was generated. Potential features associated with device explantation were analyzed using a multiple logistic regression model (P < 0.05). Results: We found significantly increased intraoperative complication rates after adjustable male sling implantation (15.9% [adjustable male sling] vs. 4.2% [AUS], P = 0.003). The most frequent intraoperative complication was bladder perforation (n = 17). Postoperative infection rates did not vary significantly between the respective devices (P = 0.378). Device explantation rates were significantly higher after AUS implantation (9.7% [adjustable male sling] vs. 21.5% [AUS], P = 0.030). In multivariate analysis, postoperative infection was a strong independent predictor of decreased device survival (odds ratio, 6.556;P = 0.001). Conclusions: Complication profiles vary between adjustable male slings and AUS. Explantation rates are lower after adjustable male sling implantation. Any kind of postoperative infections are independent predictors of decreased device survival. There is no significant effect of the experience of the implanting institution on device survival

An EDS1-SAG101 Complex is Essential for TNL-mediated Immunity in Nicotiana benthamiana

International audienceHeterodimeric complexes containing the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) are regarded as central regulators of plant innate immunity. In this context, a complex of EDS1 with PHYTOALEXIN DEFICIENT4 (PAD4) is required for basal resistance and signaling downstream of immune receptors containing an N-terminal Toll-interleukin-1 receptor-like domain (TNLs) in Arabidopsis thaliana. Here we analyze EDS1 functions in the model Solanaceous plant Nicotiana benthamiana (Nb). Stable Nb mutants deficient in EDS1 complexes are not impaired in basal resistance, a finding which contradicts a general role for EDS1 in immunity. In Nb, PAD4 demonstrated no detectable immune functions, but TNL-mediated resistance responses required EDS1 complexes incorporating a SENESCENCE ASSOCIATED GENE101 (SAG101) isoform. Intriguingly, SAG101 is restricted to those genomes also encoding TNL receptors, and we propose it may be required for TNL-mediated immune signaling in most plants, except the Brassicaceae. Transient complementation in Nb was used for accelerated mutational analyses while avoiding complex biotic interactions. We identify a large surface essential for EDS1-SAG101 immune functions, which extends from the N-terminal lipase domains to the C-terminal EP domains and might mediate interaction partner recruitment. Further, this work demonstrates the value of genetic resources in Nb, which will facilitate elucidation of EDS1 functions

Targeted gene deletion with Sp Cas9 and multiple guide RNAs in Arabidopsis thaliana : four are better than two

Abstract Background In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the fragment to be deleted. Experimental approaches for deletion induction have not been systematically evaluated. Results We designed three pairs of guide RNAs for deletion of the Arabidopsis WRKY30 locus (~2.2 kb). We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal deletions. Conclusions Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal deletions, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects

Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two

Abstract Background In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated. Results We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions. Conclusions Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects

Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70&nbsp;kb deletion, which occurred at a frequency of ~\u20091.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation

Differential requirement for the EDS1 catalytic triad in A. thaliana and N. benthamiana

Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in A. thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Biochemical activities underlying these mechanistic frameworks remain unknown. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in N. benthamiana. We do not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, mutations within the catalytic triad of Solanaceae EDS1 can abolish or enhance TNL immunity in N. benthamiana. Furthermore, nuclear EDS1 accumulation is sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Dependency of Solanaceae but not A. thaliana EDS1 on catalytic triad residues raises the possibility that a TNL-derived small molecule binds to the Solanaceae EDS1 lipase-like domain, and that EDS1 lipase-like domain pocket contributions to TNL immune responses vary between lineages. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown