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The barley powdery mildew fungus, Blumeria hordei (Bh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley nucleotide-binding leucine-rich-repeat (NLR) receptor MLA1 and therefore is designated AVR A1 . Here, we show that AVR A1 and the sequence-unrelated Bh effector BEC1016 (CSEP0491) suppress immunity in barley. We used yeast two-hybrid next-generation interaction screens (Y2H-NGIS), followed by binary Y2H and in planta protein-protein interactions studies, and identified a common barley target of AVR A1 and BEC1016, the endoplasmic reticulum (ER)-localized J-domain protein HvERdj3B. Silencing of this ER quality control (ERQC) protein increased Bh penetration. HvERdj3B is ER luminal, and we showed using split GFP that AVR A1 and BEC1016 translocate into the ER signal peptide-independently. Overexpression of the two effectors impeded trafficking of a vacuolar marker through the ER; silencing of HvERdj3B also exhibited this same cellular phenotype, coinciding with the effectors targeting this ERQC component. Together, these results suggest that the barley innate immunity, preventing Bh entry into epidermal cells, requires ERQC. Here, the J-domain protein HvERdj3B appears to be essential and can be regulated by AVR A1 and BEC1016. Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host NLR receptors. Previous work has shown that AVR A1 is directly recognized in the cytosol by the immune receptor MLA1. We speculate that the AVR A1 J-domain target being inside the ER, where it is inapproachable by NLRs, has forced the plant to evolve this challenging direct recognition., (© 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

A crucial step in functional genomics is identifying actively translated ORFs and linking them to biological functions. The challenge lies in identifying short ORFs, as their identification is greatly influenced by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis (Arabidopsis thaliana), revealing uncharacterized translation events for nuclear, chloroplastic, and mitochondrial genes. Assisted by a transcriptome assembly, we identified 7,751 unconventional translation events, comprising 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ORFs in presumed noncoding RNAs. Proteomic data confirmed the production of stable proteins from some of these unannotated translation events. We present evidence of active translation from primary transcripts of trans-acting small interfering RNAs (TAS1-4) and microRNAs (pri-MIR163 and pri-MIR169) and periodic ribosome stalling supporting cotranslational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORFs (AUG-stop), and 2,921 tiny uORFs (2 to 10 amino acids) and 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as do longer uORFs. We also systematically discovered 594 uORFs regulated by alternative splicing, suggesting widespread isoform-specific translational control. Finally, these prevalent uORFs are associated with numerous important pathways. In summary, our improved Arabidopsis translational landscape provides valuable resources to study gene expression regulation., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The plant pathogenic fungus Fusarium graminearum is the causal agent of Fusarium head blight (FHB) disease on small-grain cereals. F. graminearum produces trichothecene mycotoxins such as deoxynivalenol (DON) that are required for full virulence. DON must be exported outside the cell to cause FHB disease, a process that may require the involvement of membrane-bound transporters. In this study, we show that the deletion of membrane-bound transporters results in reduced DON accumulation as well as reduced FHB symptoms on wheat. Deletion of the ATP-binding cassette (ABC) transporter gene Abc1 results in the greatest reduction in DON accumulation and virulence. Deletion of another ABC transporter gene, Abc6 , also reduces FHB symptoms to a lesser degree. Combining deletions fails to reduce DON accumulation or virulence in an additive fashion, even when a ∆ abc1 deletion is included. Heterologous expression of F. graminearum transporters in a DON-sensitive strain of yeast confirms Abc1 as a major DON resistance mechanism; furthermore, it suggests that Abc1 is directly participating in DON transport rather than facilitating DON transport though other means. Yeast expression further indicates that multiple transporters, including Abc1, play an important role in resistance to the wheat phytoalexin 2-benzoxazolinone (BOA) and other xenobiotics. Thus, Abc1 may contribute to virulence on wheat both by facilitating export of DON and by providing resistance to the wheat phytoalexin BOA. This research provides useful information that may aid in designing novel management techniques of FHB or other destructive plant diseases., Competing Interests: The author(s) declare no conflict of interest.

Yeast two-hybrid next-generation interaction screening (Y2H-NGIS) uses the output of next-generation sequencing to mine for novel protein-protein interactions. Here, we outline the analytics underlying Y2H-NGIS datasets. Different systems, libraries, and experimental designs comprise Y2H-NGIS methodologies. We summarize the analysis in several layers that comprise the characterization of baits and preys, quantification, and identification of true interactions for subsequent secondary validation. We present two software designed for this purpose, NGPINT and Y2H-SCORES, which are used as front-end and back-end tools in the analysis. Y2H-SCORES software can be used and adapted to analyze different datasets not only from Y2H-NGIS but from other techniques ruled by similar biological principles., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Yeast two-hybrid is a powerful approach to discover new protein-protein interactions. Traditional methods involve screening a target protein against a cDNA expression library and assaying individual positive colonies to identify interacting partners. Here we describe a simple approach to perform yeast two-hybrid screens of a cDNA expression library in batch liquid culture. Positive yeast cell populations are enriched under selection and then harvested en masse. Prey cDNAs are amplified and used as input for next-generation sequencing libraries for identification, quantification, and ranking., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Brassinosteroids (BRs) and Target of Rapamycin Complex (TORC) are two major actors coordinating plant growth and stress responses. Brassinosteroids function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies have revealed connections between these two pathways, but a system-wide view of their interplay is still missing. We quantified the level of 23 975 transcripts, 11 183 proteins, and 27 887 phosphorylation sites in wild-type Arabidopsis thaliana and in mutants with altered levels of either BRASSINOSTEROID INSENSITIVE 2 (BIN2) or REGULATORY ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Furthermore, we used the multi-omic data to reconstruct an integrated signaling network. We screened 41 candidate genes identified from the reconstructed network and found that loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results establish a predictive network that defines different layers of molecular interactions between BR- or TORC-regulated growth and autophagy., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

The barley MLA nucleotide-binding leucine-rich-repeat (NLR) receptor and its orthologs confer recognition specificity to many fungal diseases, including powdery mildew, stem-, and stripe rust. We used interolog inference to construct a barley protein interactome (Hordeum vulgare predicted interactome, HvInt) comprising 66,133 edges and 7,181 nodes, as a foundation to explore signaling networks associated with MLA. HvInt was compared with the experimentally validated Arabidopsis interactome of 11,253 proteins and 73,960 interactions, verifying that the 2 networks share scale-free properties, including a power-law distribution and small-world network. Then, by successive layering of defense-specific "omics" datasets, HvInt was customized to model cellular response to powdery mildew infection. Integration of HvInt with expression quantitative trait loci (eQTL) enabled us to infer disease modules and responses associated with fungal penetration and haustorial development. Next, using HvInt and infection-time-course RNA sequencing of immune signaling mutants, we assembled resistant and susceptible subnetworks. The resulting differentially coexpressed (resistant - susceptible) interactome is essential to barley immunity, facilitates the flow of signaling pathways and is linked to mildew resistance locus a (Mla) through trans eQTL associations. Lastly, we anchored HvInt with new and previously identified interactors of the MLA coiled coli + nucleotide-binding domains and extended these to additional MLA alleles, orthologs, and NLR outgroups to predict receptor localization and conservation of signaling response. These results link genomic, transcriptomic, and physical interactions during MLA-specified immunity., (Published by Oxford University Press on behalf of Genetics Society of America 2022. This work is written by a US Government employee and is in the public domain in the US.)

The Mla ( Mildew resistance locus a ) of barley ( Hordeum vulgare L.) is an effective model for cereal immunity against fungal pathogens. Like many resistance proteins, variants of the MLA coiled-coil nucleotide-binding leucine-rich repeat (CC-NLR) receptor often require the HRS complex (HSP90, RAR1, and SGT1) to function. However, functional analysis of Sgt1 has been particularly difficult, as deletions are often lethal. Recently, we identified rar3 ( required for Mla6 resistance 3 ), an in-frame Sgt1 ΔKL308-309 mutation in the SGT1-specific domain, that alters resistance conferred by MLA but without lethality. Here, we use autoactive MLA6 and recombinant yeast-two-hybrid strains with stably integrated HvRar1 and HvHsp90 to determine that this mutation weakens but does not entirely disrupt the interaction between SGT1 and MLA. This causes a concomitant reduction in MLA6 protein accumulation below the apparent threshold required for effective resistance. The ΔKL308-309 deletion had a lesser effect on intramolecular interactions than alanine or arginine substitutions, and MLA variants that display diminished interactions with SGT1 appear to be disproportionately affected by the SGT1 ΔKL308-309 mutation. We hypothesize that those dimeric plant CC-NLRs that appear unaffected by Sgt1 silencing are those with the strongest intermolecular interactions with it. Combining our data with recent work in CC-NLRs, we propose a cyclical model of the MLA-HRS resistosome interactions.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.

Pathogen infection triggers complex signaling networks in plant cells that ultimately result in either susceptibility or resistance. We have made substantial progress in dissecting many of these signaling events, and it is becoming clear that changes in proteome composition and protein activity are major drivers of plant-microbe interactions. Here, we highlight different approaches to analyze the functional proteomes of hosts and pathogens and discuss how they have been used to further our understanding of plant disease. Global proteome profiling can quantify the dynamics of proteins, posttranslational modifications, and biological pathways that contribute to immune-related outcomes. In addition, emerging techniques such as enzyme activity-based profiling, proximity labeling, and kinase-substrate profiling are being used to dissect biochemical events that operate during infection. Finally, we discuss how these functional approaches can be integrated with other profiling data to gain a mechanistic, systems-level view of plant and pathogen signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

ER bodies are endoplasmic reticulum-derived organelles present in plants belonging to the Brassicales order. In Arabidopsis thaliana , ER bodies are ubiquitous in cotyledons and roots and are present only in certain cell types in rosette leaves. However, both wounding and jasmonic acid treatment induce the formation of ER bodies in leaves. Formation of this structure is dependent on the transcription factor NAI1 . The main components of the ER bodies are β-glucosidases (BGLUs), enzymes that hydrolyze specialized compounds. In Arabidopsis , PYK10 (BGLU23) and BGLU18 are the most abundant ER body proteins. In this work, we found that ER bodies are downregulated as a consequence of the immune responses induced by bacterial flagellin perception. Arabidopsis mutants defective in ER body formation show enhanced responses upon flagellin perception and enhanced resistance to bacterial infections. Furthermore, the bacterial toxin coronatine induces the formation of de novo ER bodies in leaves and its virulence function is partially dependent on this structure. Finally, we show that performance of the polyphagous beet armyworm herbivore Spodoptera exigua increases in plants lacking ER bodies. Altogether, we provide new evidence for the role of the ER bodies in plant immune responses.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mapping protein-protein interactions at a proteome scale is critical to understanding how cellular signaling networks respond to stimuli. Since eukaryotic genomes encode thousands of proteins, testing their interactions one-by-one is a challenging prospect. High-throughput yeast-two hybrid (Y2H) assays that employ next-generation sequencing to interrogate complementary DNA (cDNA) libraries represent an alternative approach that optimizes scale, cost and effort. We present NGPINT, a robust and scalable software to identify all putative interactors of a protein using Y2H in batch culture. NGPINT combines diverse tools to align sequence reads to target genomes, reconstruct prey fragments and compute gene enrichment under reporter selection. Central to this pipeline is the identification of fusion reads containing sequences derived from both the Y2H expression plasmid and the cDNA of interest. To reduce false positives, these fusion reads are evaluated as to whether the cDNA fragment forms an in-frame translational fusion with the Y2H transcription factor. NGPINT successfully recognized 95% of interactions in simulated test runs. As proof of concept, NGPINT was tested using published data sets and it recognized all validated interactions. NGPINT can process interaction data from any biosystem with an available genome or transcriptome reference, thus facilitating the discovery of protein-protein interactions in model and non-model organisms., (Published by Oxford University Press 2020.)

Spectinomycin, an aminocyclitol antibiotic, is subject to inactivation by aminoglycoside modifying enzymes (AMEs) through adenylylation or phosphorylation of the 6-hydroxy group position. In this study, the effects of deoxygenation of the 2- and 6-hydroxy group positions on the spectinomycin actinamine ring are probed to evaluate their relationship to ribosomal binding and the antimicrobial activities of spectinomycin, semisynthetic aminomethyl spectinomycins (amSPCs), and spectinamides. To generate these analogs, an improved synthesis of 6-deoxyspectinomycin was developed using the Barton deoxygenation reaction. 6-Dehydrospectinamide was also synthesized from spectinamide 4 to evaluate the H-bond acceptor character on the C-6 position. All the synthesized analogs were tested for antibacterial activity against a panel of Gram (+) and Gram (-) pathogens, plus Mycobacterium tuberculosis. The molecular contribution of the 2- and 6-hydroxy group and the aryl functionalities of all analogs were examined by measuring inhibition of ribosomal translation and molecular dynamics experiments with MM/GBSA analysis. The results of this work indicate that the 6-hydroxy group, which is the primary target of AMEs, is a required motif for antimicrobial activity in current analogs. Removal of the 6-hydroxy group could be partially rescued by offsetting ribosomal binding contributions made by the aryl side chains found in the spectinamide and amSPCs. This study builds on the knowledge of the structure-activity relationships of spectinomycin analogs and is being used to aid the design of next-generation spectinomycins.

Protein-protein interaction networks are one of the most effective representations of cellular behavior. In order to build these models, high-throughput techniques are required. Next-generation interaction screening (NGIS) protocols that combine yeast two-hybrid (Y2H) with deep sequencing are promising approaches to generate interactome networks in any organism. However, challenges remain to mining reliable information from these screens and thus, limit its broader implementation. Here, we present a computational framework, designated Y2H-SCORES, for analyzing high-throughput Y2H screens. Y2H-SCORES considers key aspects of NGIS experimental design and important characteristics of the resulting data that distinguish it from RNA-seq expression datasets. Three quantitative ranking scores were implemented to identify interacting partners, comprising: 1) significant enrichment under selection for positive interactions, 2) degree of interaction specificity among multi-bait comparisons, and 3) selection of in-frame interactors. Using simulation and an empirical dataset, we provide a quantitative assessment to predict interacting partners under a wide range of experimental scenarios, facilitating independent confirmation by one-to-one bait-prey tests. Simulation of Y2H-NGIS enabled us to identify conditions that maximize detection of true interactors, which can be achieved with protocols such as prey library normalization, maintenance of larger culture volumes and replication of experimental treatments. Y2H-SCORES can be implemented in different yeast-based interaction screenings, with an equivalent or superior performance than existing methods. Proof-of-concept was demonstrated by discovery and validation of novel interactions between the barley nucleotide-binding leucine-rich repeat (NLR) immune receptor MLA6, and fourteen proteins, including those that function in signaling, transcriptional regulation, and intracellular trafficking., Competing Interests: The authors have declared that no competing interests exist.

Increasing rates of drug-resistant Gram-negative (GN) infections, combined with a lack of new GN-effective antibiotic classes, are driving the need for the discovery of new agents. Bacterial metabolism represents an underutilized mechanism of action in current antimicrobial therapies. Therefore, we sought to identify novel antimetabolites that disrupt key metabolic pathways and explore the specific impacts of these agents on bacterial metabolism. This study describes the successful application of this approach to discover a new series of chemical probes, N -(phenyl)thioacetamide-linked 1,2,3-triazoles (TAT), that target cysteine synthase A (CysK), an enzyme unique to bacteria that is positioned at a key juncture between several fundamental pathways. The TAT class was identified using a high-throughput screen against Escherichia coli designed to identify modulators of pathways related to folate biosynthesis. TAT analog synthesis demonstrated a clear structure-activity relationship, and activity was confirmed against GN antifolate-resistant clinical isolates. Spontaneous TAT resistance mutations were tracked to CysK, and mode of action studies led to the identification of a false product formation mechanism between the CysK substrate O -acetyl-l-serine and the TATs. Global transcriptional responses to TAT treatment revealed that these antimetabolites impose substantial disruption of key metabolic networks beyond cysteine biosynthesis. This study highlights the potential of antimetabolite drug discovery as a promising approach to the discovery of novel GN antibiotics and the pharmacological promise of TAT CysK probes.

Introduction: There are no guidelines for opioid use after pediatric urologic surgery, and it is unknown to what extent prescriptions written for these patients may be contributing to the opioid epidemic in the United States. We sought to characterize opioid utilization in a prospective fashion following outpatient pediatric urologic surgery at our institution., Materials and Methods: After obtainingapproval from the Institutional Review Board, we prospectively recruited pediatric patients undergoing outpatient urologic surgery. All patients and families were counseled regarding appropriate use of over-the-counter pain medications as first-line agents, with opioids for breakthrough pain only. All patients received an opioid prescription (ORx), which we attempted to standardize to 10 doses. Parents were provided with a log for keeping track of pain medication administration. Postoperative surveys were sent at various time points after surgery to assess utilization of pain medications at home. We quantified unused opioids prescribed and evaluated factors potentially associated with opioid use., Results: Two hundred and two patients were recruited. All patients were male, with a median age of 2.7 years (interquartile range (IQR) 5.5, range 0.5-17.9 years). One hundred and fifty-four children underwent penile surgery, 22 underwent scrotal surgery, and 27 underwent inguinal surgery. Nearly half of our study patients were black, 33.2% were white, 12.9% were Latino, and 4.0% were Asian. The median number of doses prescribed was 10 (IQR 0, range 4.0-20.8). Postoperative surveys were completed by 80.7% of study patients. The median number of opioid doses used was 0 (IQR 2), whereas the mean was 1.28 (standard deviation (SD) 1.98). None of the factors evaluated (including patient age, surgery type, perioperative pain management techniques, length of surgery, and insurance type) were associated with the amount of opioid used at home after surgery, as utilization was equally low across all groups., Discussion and Conclusions: Ensuring adequate postoperative pain control for children is critical, yet it is also important to minimize excess ORx. We found that the majority of pediatric patients used 0-2 doses of prescription pain medication after discharge following outpatient urologic surgery, representing a small percentage of the total prescribed amount. Low utilization was seen irrespective of patient age, procedure, and perioperative factors. These data can be used to guide perioperative patient and family counseling and to guide future efforts to standardize ORx following outpatient pediatric urologic surgery., (Copyright © 2019 Journal of Pediatric Urology Company. All rights reserved.)

Acyldepsipeptides are a unique class of antibiotics that act via allosterically dysregulated activation of the bacterial caseinolytic protease (ClpP). The ability of ClpP activators to kill nongrowing bacteria represents a new opportunity to combat deep-seated biofilm infections. However, the acyldepsipeptide scaffold is subject to rapid metabolism. Herein, we explore alteration of the potentially metabolically reactive α,β unsaturated acyl chain. Through targeted synthesis, a new class of phenyl urea substituted depsipeptide ClpP activators with improved metabolic stability is described. The ureadepsipeptides are potent activators of Staphylococcus aureus ClpP and show activity against Gram-positive bacteria, including S. aureus biofilms. These studies demonstrate that a phenyl urea motif can successfully mimic the double bond, maintaining potency equivalent to acyldepsipeptides but with decreased metabolic liability. Although removal of the double bond from acyldepsipeptides generally has a significant negative impact on potency, structural studies revealed that the phenyl ureadepsipeptides can retain potency through the formation of a third hydrogen bond between the urea and the key Tyr63 residue in the ClpP activation domain. Ureadepsipeptides represent a new class of ClpP activators with improved drug-like properties, potent antibacterial activity, and the tractability to be further optimized.

Herein we report the antibacterial structure-activity relationships of cyclic hexapeptide wollamide analogs derived from solid-phase library synthesis. Wollamide B, a cyclic hexapeptide natural product, has been previously found to have activity against Mycobacterium bovis. To further evaluate its antimycobacterial/antibacterial potential, 27 peptides including wollamides A/B, and desotamide B, were synthesized and subsequently tested against a panel of clinically significant bacterial pathogens. Biological evaluation revealed that the cyclic scaffold, amide functionality in position I, tryptophan residue in position V, and the original stereochemistry pattern of the core scaffold were key for antituberculosis and/or antibacterial activity. In addition, against M. tuberculosis and Gram-positive bacteria, residues in position II and/or VI greatly impacted antibacterial activity and selectivity. Wollamides A (3) and B (2) along with their corresponding II (l-Leu) analog 10 retained the most promising antituberculosis activity, with the lowest minimum inhibitory concentration (MIC) against virulent M. tuberculosis H37Rv (MIC = 1.56 μg/mL), as well as desirable selectivity indices (>100). Importantly, the antimicrobial activities of wollamides A and B do not result from disruption of the bacterial membrane, warranting further investigation into their mechanism of action.

Root-knot nematodes damage crops around the world by developing complex feeding sites from normal root cells of their hosts. The ability to initiate and maintain this feeding site (composed of individual "giant cells") is essential to their parasitism process. RNA silencing pathways in plants serve a diverse set of functions, from directing growth and development to defending against invading pathogens. Influencing a host's RNA silencing pathways as a pathogenicity strategy has been well-documented for viral plant pathogens, but recently, it has become clear that silencing pathways also play an important role in other plant pathosystems. To determine if RNA silencing pathways play a role in nematode parasitism, we tested the susceptibility of plants that express a viral suppressor of RNA silencing. We observed an increase in susceptibility to nematode parasitism in plants expressing viral suppressors of RNA silencing. Results from studies utilizing a silenced reporter gene suggest that active suppression of RNA silencing pathways may be occurring during nematode parasitism. With these studies, we provide further evidence to the growing body of plant-biotic interaction research that suppression of RNA silencing is important in the successful interaction between a plant-parasitic animal and its host.

Membrane-localized proteins perceive and respond to biotic and abiotic stresses. We performed quantitative proteomics on plasma membrane-enriched samples from Arabidopsis (Arabidopsis thaliana) treated with bacterial flagellin. We identified multiple receptor-like protein kinases changing in abundance, including cysteine (Cys)-rich receptor-like kinases (CRKs) that are up-regulated upon the perception of flagellin. CRKs possess extracellular Cys-rich domains and constitute a gene family consisting of 46 members in Arabidopsis. The single transfer DNA insertion lines CRK28 and CRK29, two CRKs induced in response to flagellin perception, did not exhibit robust alterations in immune responses. In contrast, silencing of multiple bacterial flagellin-induced CRKs resulted in enhanced susceptibility to pathogenic Pseudomonas syringae, indicating functional redundancy in this large gene family. Enhanced expression of CRK28 in Arabidopsis increased disease resistance to P. syringae Expression of CRK28 in Nicotiana benthamiana induced cell death, which required intact extracellular Cys residues and a conserved kinase active site. CRK28-mediated cell death required the common receptor-like protein kinase coreceptor BAK1. CRK28 associated with BAK1 as well as the activated FLAGELLIN-SENSING2 (FLS2) immune receptor complex. CRK28 self-associated as well as associated with the closely related CRK29. These data support a model where Arabidopsis CRKs are synthesized upon pathogen perception, associate with the FLS2 complex, and coordinately act to enhance plant immune responses., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Background: The American Academy of Pediatrics (AAP) Task Force on Circumcision has called for the development of standards of trainee proficiency in regards to evaluation and technique for neonatal clamp circumcision (NCC). At the present time, there is no standardized or general consensus on patient selection for NCC. An improved method to evaluate newborns for NCC is an important first step in this process. Therefore, the authors collaborated to identify criteria useful in the evaluation of newborns for suitability for NCC, and for assessment of success after NCC and have named it "Checklist Assessment for Neonatal Clamp Circumcision Suitability.", Methods: A national multi-institutional collaboration was created to obtain consensus on objective criteria for use in determining patient suitability for NCC, and for assessing post-circumcision success outcomes. Criteria included elements from detailed medical history, bedside physical examination, and post-circumcision follow-up. Patients desiring NCC were enrolled consecutively and prospectively. The Checklist was followed to determine which newborns were suited to NCC, and NCC was done in those cases. The patients' caretakers were given post-circumcision care instructions and a follow-up appointment. Post circumcision, the Checklist was followed to determine if the procedure resulted in a successful circumcision or if there were complications., Results: A total of 193 cases were enrolled prospectively and consecutively from January 2014 through October 2014. The mean age was 15 days (1-30 days). Of those 193 patients, 129 (67%) were deemed suitable for circumcision and underwent NCC. Post-circumcision assessment showed a 100% success rate with no complications. A total of 64 (23%) cases were deemed unsuitable for NCC because at least one checklist criterion was not satisfied, most commonly: penile torsion (n = 25), chordee (n = 19), and penoscrotal webbing (n = 19)., Discussion: Use of the Checklist in the present study has demonstrated a method of patient screening resulting in a 100% success rate with no complications. A high proportion of patients (33%) was identified as unsuited for NCC; however, the patient population consisted of newborn males referred to pediatric urology, and thus does not represent the general population, which is expected to have a lower proportion of unsuited patients. Regardless, the Checklist has the potential to enhance the decision-making process for both urologic and non-urologic care providers., Conclusions: The use of the "Checklist Assessment for Neonatal Clamp Circumcision Suitability" assessment tool improves identification of patients unsuited for NCC and thereby potentially decreases the likelihood of circumcision-related complications., (Copyright © 2016 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

To establish infection, pathogens deliver effectors into host cells to target immune signaling components, including elements of mitogen-activated protein kinase (MPK) cascades. The virulence function of AvrRpt2, one of the first identified Pseudomonas syringae effectors, involves cleavage of the plant defense regulator, RPM1-INTERACTING PROTEIN4 (RIN4), and interference with plant auxin signaling. We show now that AvrRpt2 specifically suppresses the flagellin-induced phosphorylation of Arabidopsis (Arabidopsis thaliana) MPK4 and MPK11 but not MPK3 or MPK6. This inhibition requires the proteolytic activity of AvrRpt2, is associated with reduced expression of some plant defense genes, and correlates with enhanced pathogen infection in AvrRpt2-expressing transgenic plants. Diverse AvrRpt2-like homologs can be found in some phytopathogens, plant-associated and soil bacteria. Employing these putative bacterial AvrRpt2 homologs and inactive AvrRpt2 variants, we can uncouple the inhibition of MPK4/MPK11 activation from the cleavage of RIN4 and related members from the so-called nitrate-induced family as well as from auxin signaling. Thus, this selective suppression of specific mitogen-activated protein kinases is independent of the previously known AvrRpt2 targets and potentially represents a novel virulence function of AvrRpt2., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Introduction and Objective: While open ureteral reimplantation remains the gold standard for surgical treatment of vesicoureteral reflux (VUR), minimally invasive approaches offer potential benefits. This study evaluated the outcomes of children undergoing complex robot-assisted laparoscopic ureteral reimplantation (RALUR) for failed previous anti-reflux surgery, complex anatomy, or ureterovesical junction obstruction (UVJO), and compared them with patients undergoing open extravesical repair., Study Design: Children undergoing complex RALUR or open extravesical ureteral reimplantation (OUR) were identified. Reimplantation was classified as complex if ureters: 1) had previous anti-reflux surgery, 2) required tapering and/or dismembering, or 3) had associated duplication or diverticulum., Results: Seventeen children underwent complex RALUR during a 24-month period, compared with 41 OUR. The mean follow-up was 16.6 ± 6.5 months. The RALUR children were significantly older (9.3 ± 3.7 years) than the OUR patients (3.1 ± 2.7 years; P < 0.001). All RALUR patients were discharged on postoperative day one, while 24.4% of children in the open group required longer hospitalization (mean 1.3 ± 0.7 days; P = 0.03). Adjusting for age, there was no significant difference in inpatient analgesic usage between the two cohorts. Three OUR patients (7.3%) developed postoperative febrile urinary tract infection compared with a single child (5.9%) undergoing RALUR (P = 1.00). There was no significant difference in complication rate between the two groups (12.2% OUR versus 11.8% RALUR; P = 1.00). A postoperative cystogram was performed in the majority of RALUR patients, with no persistent VUR detected, and one child (6.7%) was diagnosed with contralateral reflux., Discussion: Reported VUR resolution rates following robot-assisted ureteral reimplantation are varied. In the present series, children undergoing RALUR following failed previous anti-reflux surgery, with complex anatomy, or UVJO experienced a shorter length of stay but had similar analgesic requirements to those undergoing open repair. Radiographic, clinical success rates and complication risk were comparable. This study had several limitations, aside from lack of randomization. Analgesic use was limited to an inpatient setting, and pain scores were not assessed. Not all children underwent a postoperative VCUG, so the true radiographic success rate is unknown. A larger patient cohort with longer follow-up is necessary to determine predictors of radiographic and clinical failure., Conclusion: Older children with a previous history of anti-reflux surgery were more likely to undergo RALUR. These children had success and complication rates comparable to younger patients following complex open extravesical reimplantation, which underscores the expanding role of robot-assisted lower urinary tract reconstructive surgery in the pediatric population., (Copyright © 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Introduction and Objective: The Glans-Urethral Meatus-Shaft (GMS) score is a concise and reproducible way to describe hypospadias severity. We classified boys undergoing primary hypospadias repair to determine the correlation between GMS score and postoperative complications., Study Design: Between February 2011 and August 2013, patients undergoing primary hypospadias repair were prospectively scored using the GMS classification. GMS scoring included a 1-4 scale for each component: G - glans size/urethral plate quality, M - meatal location, and S - degree of shaft curvature, with more unfavorable characteristics assigned higher scores [Figure]. Demographics, repair type, and complications (urethrocutaneous fistula, meatal stenosis, glans dehiscence, phimosis, recurrent chordee and stricture) were assessed. Total and individual component scores were tested in uni- and multivariate analysis., Results: Two-hundred and sixty-two boys (mean age 12.3 ± 13.7 months) undergoing primary hypospadias repair had a GMS score assigned. Mean GMS score was 7 ± 2.5 (G 2.1 ± 0.9, M 2.4 ± 1, S 2.4 ± 1). Mean clinical follow-up was 17.7 ± 9.3 months. Thirty-seven children (14.1%) had 45 complications. A significant relationship between the total GMS score and presence of any complication (p < 0.001) was observed; for every unit increase in GMS score the odds of any postoperative complication increased 1.44 times (95% CI, 1.24-1.68). Urethrocutaneuous fistula was the most common complication, occurring in 21 of 239 (8.8%) of single-stage repairs. Patients with mild hypospadias (GMS 3-6) had a 2.4% fistula rate vs. 11.1% for moderate (GMS 7-9) and 22.6% for severe (GMS 10-12) hypospadias (p < 0.001). Degree of chordee was an independent predictor of fistula on multivariate analysis; S4 (>60° ventral curvature) patients were 27 times more likely to develop a fistula than S1 (no curvature) boys (95% CI, 3.2-229)., Discussion: The GMS score is based on anatomic features (i.e. glans size/urethral plate quality, location of meatus, and degree of chordee) felt to most likely impact functional and cosmetic outcomes following hypospadias repair. We demonstrated a statistically significant increase in the likelihood of any postoperative complication with every unit increase in total GMS score. The concept that factors aside from meatal location affect hypospadias repair and outcomes is not novel, and degree of ventral curvature and urethral plate quality are often cited as important factors. In our series, boys with greater than 60° of ventral curvature undergoing a single-stage repair were 27 times more likely to develop a fistula than those without chordee on multivariate analysis, making severe curvature an independent predictor of urethrocutaneous fistula formation. That meatal location did not retain significance on multivariate analysis highlights the importance of considering the entire hypospadias complex when determining severity, rather than just evaluating the position of the meatus. Our study has several limitations that warrant consideration. While GMS scores were assigned prospectively, the data was collected retrospectively, subjecting it to flaws inherent with such study design. Furthermore, type of repair is influenced by surgeon preference and subjective assessment of hypospadias characteristics not incorporated in our scoring system (i.e. tissue quality, urethral hypoplasia, penoscrotal transposition). Despite these limitations, our study demonstrates a strong correlation between the GMS classification and surgical complications, furthering supporting its potential as a tool to standardize hypospadias severity and gauge postoperative complications., Conclusion: The Glans-Urethral Meatus-Shaft (GMS) classification provides a means by which hypospadias severity and reporting can be standardized, which may improve inter-study comparison of reconstructive outcomes. There is a strong correlation between complication risk and total GMS score. Degree of chordee (S score) is independently predictive of fistula rate., (Copyright © 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Objective: Reducing readmissions has become a focal point to increase quality of care while reducing costs. We report all-cause unplanned return visits following urologic surgery in children at our institution., Materials and Methods: Children undergoing urology procedures with returns within 30 days of surgery were identified. Patient demographics, insurance status, type of surgery, and reason for return were assessed., Results: Four thousand and ninety-seven pediatric urology surgeries were performed at our institution during 2012, with 106 documented unplanned returns (2.59%). Mean time from discharge to return was 5.9 ± 4.9 days (range, 0.3-24.8 days). Returns were classified by chief complaint, including pain (32), infection (30), volume status (14), bleeding (11), catheter concern (8), and other (11). Circumcision, hypospadias repair, and inguinal/scrotal procedures led to the majority of return visits, accounting for 21.7%, 20.7%, and 18.9% of returns, respectively. Twenty-two returns (20.75%) resulted in hospital readmission and five (4.72%) required a secondary procedure. Overall readmission rate was 0.54%, with a reoperation rate of 0.12%., Conclusions: The rate of unplanned postoperative returns in the pediatric population undergoing urologic surgery is low, further strengthening the argument that readmission rates in children are not necessarily a productive focal point for financial savings or quality control., (Copyright © 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Objective: Absorbable staples facilitate detubularization and reconfiguration of the bowel when performing augmentation colocystoplasty. We compared the outcomes of stapled sigmoid augmentation with standard sutured colocystoplasty., Materials and Methods: Between 2003 and 2011, 108 children underwent bladder augmentation at our institution. Colocystoplasty was used in 30 patients (27.8%). Medical charts of children who underwent stapled (n = 8) or sutured (n = 22) sigmoid augmentation were compared with regard to patient demographics and surgical complications, including anastomotic leak and urolithiasis., Results: Eight children with underwent stapled sigmoid colocystoplasty. Average age at surgery was 8 years (range 4-17 years). Time to detubularize and refashion the bowel segment prior to augmentation was consistently under 5 min. Average length of follow-up was 44 months (range 12-80 months). One patient experienced anastomotic leak. Two of eight children (25%) in the stapled anastomosis cohort developed bladder stones. Twenty-two patients underwent standard sigmoid augmentation during the same time period (average age 8.2 years; range 4-16 years). One of 22 (4.5%) experienced anastomotic leak. Seven of 22 (31.8%) developed cystolithiasis., Conclusions: Complications from stapled sigmoid anastomosis are similar to those from standard colocystoplasty. Use of absorbable staples decreases operating time by avoiding bowel spatulation and suturing, and should be considered in pediatric patients undergoing colocystoplasty., (Copyright © 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Objective: An agreed upon method for describing the severity of hypospadias has not been established. Herein we assess the inter-observer reliability of the GMS hypospadias score and correlate it with the risk of a post-operative complication., Methods: A 3-component method for grading the severity of hypospadias was developed (GMS). Eighty-five consecutive patients presenting for hypospadias repair were graded independently by at least 2 surgeons using the GMS criteria. Scores were compared statistically to determine agreement between the observers. The outcomes of these patients were then reviewed to determine how the GMS score correlates to the risk of a surgical complication., Results: The G, M, and S scores had excellent agreement between observers. The GMS total score was exactly the same or differed by one point in 79/85 (93%) of patients. The complication rate was 5.6% for patients with a GMS score of 6 or less, but was 25.0% for patients with a GMS score greater than 6., Conclusions: The GMS score provides a concise method for describing the severity of hypospadias and appears to have high inter-observer reliability. The GMS score also appears to correlate with the risk of a surgical complication., (Copyright © 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

The plasma membrane (PM) regulates diverse processes essential to plant growth, development, and survival in an ever-changing environment. In addition to maintaining normal cellular homeostasis and plant nutrient status, PM proteins perceive and respond to a myriad of environmental cues. Here we review recent advances in the analysis of the plant PM proteome with a focus on the model plant Arabidopsis thaliana. Due to membrane heterogeneity, hydrophobicity, and low relative abundance, analysis of the PM proteome has been a special challenge. Various experimental techniques to enrich PM proteins and different protein and peptide separation strategies have facilitated the identification of thousands of integral and membrane-associated proteins. Numerous classes of proteins are present at the PM with diverse biological functions. PM microdomains have attracted much attention. However, it still remains a challenge to characterize these cell membrane compartments. Dynamic changes in the PM proteome in response to different biotic and abiotic stimuli are highlighted. Future prospects for PM proteomics research are also discussed.

A key virulence strategy of bacterial pathogens is the delivery of multiple pathogen effector proteins into host cells during infection. The Hrp outer protein Q (HopQ1) effector from Pseudomonas syringae pv tomato (Pto) strain DC3000 is conserved across multiple bacterial plant pathogens. Here, we investigated the virulence function and host targets of HopQ1 in tomato (Solanum lycopersicum). Transgenic tomato lines expressing dexamethasone-inducible HopQ1 exhibited enhanced disease susceptibility to virulent Pto DC3000, the Pto ΔhrcC mutant, and decreased expression of a pathogen-associated molecular pattern-triggered marker gene after bacterial inoculation. HopQ1-interacting proteins were coimmunoprecipitated and identified by mass spectrometry. HopQ1 can associate with multiple tomato 14-3-3 proteins, including TFT1 and TFT5. HopQ1 is phosphorylated in tomato, and four phosphorylated peptides were identified by mass spectrometry. HopQ1 possesses a conserved mode I 14-3-3 binding motif whose serine-51 residue is phosphorylated in tomato and regulates its association with TFT1 and TFT5. Confocal microscopy and fractionation reveal that HopQ1 exhibits nucleocytoplasmic localization, while HopQ1 dephosphorylation mimics exhibit more pronounced nuclear localization. HopQ1 delivered from Pto DC3000 was found to promote bacterial virulence in the tomato genotype Rio Grande 76R. However, the HopQ1(S51A) mutant delivered from Pto DC3000 was unable to promote pathogen virulence. Taken together, our data demonstrate that HopQ1 enhances bacterial virulence and associates with tomato 14-3-3 proteins in a phosphorylation-dependent manner that influences HopQ1's subcellular localization and virulence-promoting activities in planta.

Objective: The objective of this study was to evaluate risk factors for new contralateral vesicoureteral reflux (NCVUR) and to investigate whether assessment of the non-refluxing contralateral ureter (NRCU) by hydrodistention and selective treatment can reduce the incidence of NCVUR., Materials and Methods: From 2001 to 2007, 339 of 841 patients (40%) were treated for unilateral VUR by endoscopic injection. While in the first 267 patients the NRCU was only assessed by hydrodistention but not injected (observation group), NRCUs of the subsequent 72 patients were prophylactically treated if deemed at high risk for NCVUR (H2 or H3) (prophylaxis group)., Results: NCVUR occurred in 30 of 267 patients (11.2%) whose NRCUs were observed. No statistically significant risk factors for NCVUR were found in this group. In the subsequent 72 patients, whose H2 and H3 ureters were selectively injected (N = 56), no cases of NCVUR were seen., Conclusions: Prophylactic endoscopic treatment of NRCU H2 and H3 ureters successfully prevented the occurrence of NCVUR., (Copyright © 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Objective: Criteria for success following endoscopic vesicoureteral reflux (VUR) surgery vary greatly. We sought to define outcomes based on radiographic and long-term clinical follow up., Methods: We reviewed the charts and interviewed parents of children who underwent endoscopic treatment for primary VUR (grades I-IV). All patients had a postoperative voiding cystourethrogram (VCUG) at mean of 3 months (1-21 months) and all cases of postoperative febrile urinary tract infection (FUTI) prompted repeat VCUG. Radiographic success was defined as no VUR on postoperative VCUG and clinical success as no FUTIs during follow up of 12-36 months. To demonstrate how criteria for success can affect outcomes, we calculated the success rates using different definitions., Results: In 2004-2008, 336 patients (296 female and 40 male, mean age 4 years) were treated with dextranomer/hyaluronic acid via the Double-HIT method. Initial radiographic success was 90% (302/336). Of these, 19 (6%) developed FUTIs, 12 (4%) of whom had recurrent VUR, and 5 (2%) went on to open surgery. Of the radiographic failures, 18% were observed with no further treatment. Success defined clinically was 94% (281/300), and as 'radiographic cure and no clinical evidence of FUTIs' it was 82% (275/336)., Conclusions: It is important to agree on a universal definition of success for VUR interventions to compare across studies and across therapies. Clinical success is more meaningful to the patient, and initial radiographic success could be followed by UTI necessitating further intervention. We question the need for routine postoperative VCUG., (Copyright © 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

The plant plasma membrane is a crucial mediator of the interaction between plants and microbes. Understanding how the plasma membrane proteome responds to diverse immune signaling events will lead to a greater understanding of plant immunity and uncover novel targets for crop improvement. Here we report the results from a large scale quantitative proteomics study of plasma membrane-enriched fractions upon activation of the Arabidopsis thaliana immune receptor RPS2. More than 2300 proteins were identified in total, with 1353 proteins reproducibly identified across multiple replications. Label-free spectral counting was employed to quantify the relative protein abundance between different treatment samples. Over 20% of up-regulated proteins have known roles in plant immune responses. Significantly changing proteins include those involved in calcium and lipid signaling, membrane transport, primary and secondary metabolism, protein phosphorylation, redox homeostasis, and vesicle trafficking. A subset of differentially regulated proteins was independently validated during bacterial infection. This study presents the largest quantitative proteomics data set of plant immunity to date and provides a framework for understanding global plasma membrane proteome dynamics during plant immune responses.

Plant disease resistance proteins commonly belong to the nucleotide binding-leucine rich repeat (NB-LRR) protein family. These specialized immune proteins mediate recognition of diverse pathogen-derived effector proteins and initiate potent defense responses. NB-LRRs exhibit a multidomain architecture and each domain appears to have discrete functions depending on the stage of NB-LRR signaling. Novel proteins that were found to interact with the core HSP90 chaperone complex regulate accumulation and activation of NB-LRR immune receptors. Recent studies have also advanced our understanding of how accessory proteins contribute to NB-LRR activation. The dynamic nature of NB-LRR localization to different subcellular compartments before and after activation suggests that NB-LRRs may activate immune responses in multiple parts of the cell. In this review we highlight recent advances in understanding NB-LRR function., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Plasma membrane (PM) H+-ATPases are the primary pumps responsible for the establishment of cellular membrane potential in plants. In addition to regulating basic aspects of plant cell function, these enzymes contribute to signaling events in response to diverse environmental stimuli. Here, we focus on the roles of the PM H+-ATPase during plant-pathogen interactions. PM H+-ATPases are dynamically regulated during plant immune responses and recent quantitative proteomics studies suggest complex spatial and temporal modulation of PM H+-ATPase activity during early pathogen recognition events. Additional data indicate that PM H+-ATPases cooperate with the plant immune signaling protein RIN4 to regulate stomatal apertures during bacterial invasion of leaf tissue. Furthermore, pathogens have evolved mechanisms to manipulate PM H+-ATPase activity during infection. Thus, these ubiquitous plant enzymes contribute to plant immune responses and are targeted by pathogens to increase plant susceptibility.

Plants have evolved sophisticated surveillance systems to recognize pathogen effectors delivered into host cells. RPM1 is an NB-LRR immune receptor that recognizes the Pseudomonas syringae effectors AvrB and AvrRpm1. Both effectors associate with and affect the phosphorylation of RIN4, an immune regulator. Although the kinase and the specific mechanisms involved are unclear, it has been hypothesized that RPM1 recognizes phosphorylated RIN4. Here, we identify RIPK as a RIN4-interacting receptor-like protein kinase that phosphorylates RIN4. In response to bacterial effectors, RIPK phosphorylates RIN4 at amino acid residues T21, S160, and T166. RIN4 phosphomimetic mutants display constitutive activation of RPM1-mediated defense responses and RIN4 phosphorylation is induced by AvrB and AvrRpm1 during P. syringae infection. RIPK knockout lines exhibit reduced RIN4 phosphorylation and blunted RPM1-mediated defense responses. Taken together, our results demonstrate that the RIPK kinase associates with and modifies an effector-targeted protein complex to initiate host immunity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Hypospadias is a common congenital anomalies, yet its molecular basis remains unknown. Recent studies have linked perturbations in the Hedgehog signaling pathway to hypospadias. However, the expression of Sonic hedgehog (Shh) has not been reported during genital development. Immunohistochemical staining for Shh and its receptors was applied to 10 human fetal penises ranging from 12 to 29 weeks gestation. The intensity of Shh staining was greatest in the urethral epithelium at 14 weeks gestation, correlating with the time of urethral tubularization. Results suggest a role for Shh in human male genital development.

Protein complex purification represents a powerful approach to identify novel players in plant innate immunity. However, the identification of interacting protein partners within a natural context has been a challenge for researchers. In this chapter, we describe a method of immunoaffinity chromatography using purified, antibodies to isolate native protein complexes from wild-type tissue. We detail the antibody purification and immobilization steps in addition to the co-immunoprecipitation protocol. In addition, a method to prepare protein samples for mass spectroscopy analysis is described. This straightforward protocol has been used to isolate and identify novel components of Arabidopsis immunity-associated protein complexes.

Purpose: We previously reported our success with sutureless circumcision using 2-octyl cyanoacrylate in 267 patients. We have since modified our technique by making incisions with electrocautery. We report our results with this novel technique. We also performed a cost analysis., Materials and Methods: We compiled data on all patients 6 months to 12 years old who underwent primary circumcision and circumcision revision in a 39-month period, as done by 3 surgeons. Study exclusion criteria were complexity beyond phimosis and Gomco clamp use. The technique included 1) a circumferential inner incision using electrocautery on cutting current, 2) a circumferential outer incision using electrocautery, 3) foreskin removal, 4) hemostasis with electrocautery, 5) skin edge approximation with 2-octyl cyanoacrylate or 6-zero suture and 6) antibiotic ointment application. We also determined the cost of all procedures based on anesthesia and operating room facility fees, and material costs., Results: Between July 1, 2006 and October 1, 2009 we performed 493 primary circumcisions and 248 revisions using 2-octyl cyanoacrylate, and 152 primary circumcisions and 115 revisions using 6-zero sutures. Mean operative time for primary circumcision and revision using 2-octyl cyanoacrylate was 8 minutes (range 6 to 18), and for sutured primary circumcision and revision it was 27 minutes (range 18 to 48). At a mean 18-month followup (range 1 to 39) 3 patients treated with 2-octyl cyanoacrylate and 2 treated with sutures were rehospitalized for bleeding. When done with electrocautery, the cost of the 2-octyl cyanoacrylate technique was $743.55 less than the sutured technique as long as the 2-octyl cyanoacrylate procedures required less than 15 minutes and the sutured procedures required more than 15 minutes., Conclusions: Combined electrocautery and 2-octyl cyanoacrylate for circumcision is a safe, efficient, financially beneficial, cosmetically appealing alternative to traditional circumcision done with scalpel and sutures., (Copyright © 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Gross, intractable hematuria is rare in children. Although the role of epsilon aminocaproic acid in the management of refractory hematuria is well established in the adult population, few data exist about its use in children for this indication. We present our initial experience with epsilon aminocaproic acid for the treatment of intractable hematuria after more conservative measures failed, and propose an algorithm for administration of epsilon aminocaproic acid in children., Materials and Methods: We reviewed the charts of all patients treated with epsilon aminocaproic acid for intractable gross hematuria at our institution during a period of 36 months. All patients underwent hematological evaluation and any underlying bleeding dyscrasias were addressed. All patients also underwent renal and bladder ultrasound, retrograde pyelogram and ureteroscopy. Demographic information, medical and surgical histories, and epsilon aminocaproic acid dosing and outcomes were recorded., Results: Three boys and 1 girl 11 to 17 years old were treated with epsilon aminocaproic acid. Three patients had sickle trait (1 with nutcracker phenomenon) and 1 had hemophilia A. Three patients required packed red blood cell transfusions to maintain hematocrit. Three renal angiograms were performed, all of which were nondiagnostic. Duration of hematuria ranged from 1 to 52 weeks before administration of epsilon aminocaproic acid. Endoscopic evaluation demonstrated hematuria localized to 1 ureteral orifice in all 4 patients. All patients received 100 mg/kg epsilon aminocaproic acid orally every 6 hours, which uniformly led to cessation of hematuria., Conclusions: Epsilon aminocaproic acid is useful for the management of gross refractory hematuria when more conservative measures fail. Because of its potential side effects, it should be used cautiously., (2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Pathogen recognition by the plant innate immune system invokes a sophisticated signal transduction network that culminates in disease resistance. The Arabidopsis protein RIN4 is a well-known regulator of plant immunity. However, the molecular mechanisms by which RIN4 controls multiple immune responses have remained elusive. in our recently published study, we purified components of the RIN4 protein complex from A. thaliana and identified several novel RIN4-associated proteins.1 we found that one class of RIN4-associated proteins, the plasma membrane H(+)-ATPases AHA1 and AHA2, play a crucial role in resisting pathogen invasion. Plants use RIN4 to regulate H(+)-ATPase activity during immune responses, thereby controlling stomatal apertures during pathogen attack. Stomata were previously identified as active regulators of plant immune responses during pathogen invasion, but how the plant innate immune system coordinates this response was unknown.2,3 Our investigations have revealed a novel function of rin4 during pathogenesis. Here, we discuss the rin4-AHA1/2 interaction and highlight additional RIN4-associated proteins (RAPs) as well as speculate on their potential roles in plant innate immunity.

Purpose: We evaluated the usefulness and interobserver concordance of a novel grading system for dynamic ureteral hydrodistention., Materials and Methods: Between May 1, 2002 and July 1, 2008 the hydrodistention grade in 697 ureters was prospectively assigned and recorded, including H0-no hydrodistention, H1-ureteral orifice open but tunnel not evident, H2-tunnel seen only and H3-extravesical ureter visualized. Specifically 489 refluxing ureters (vesicoureteral reflux group) were compared to 100 normal control ureters (normal control group). Additionally, the posttreatment hydrodistention grade in 56 ureters in which surgery for reflux failed was compared to that in 52 ureters with successful surgery. Hydrodistention grades assigned to an additional 77 ureters by 3 blinded observers were compared to assess the interobserver concordance of this system., Results: Vesicoureteral reflux and hydrodistention grades correlated significantly (p <0.001). Ureters with a higher reflux grade also showed a higher hydrodistention grade. The normal control group (mean +/- SEM hydrodistention grade 0.62 +/- 0.07) showed a statistically lower hydrodistention grade than the reflux groups (overall mean hydrodistention grade 2.26 +/- 0.01). Mean posttreatment hydrodistention grade in the failed reflux surgery group was statistically higher than that in the mean successful reflux surgery group (2.03 +/- 0.09 vs 1.33 +/- 0.08). By defining the degree of hydrodistention as normal (H0-H1) and abnormal (H2-H3) the concordance between observers was 95% and 96%., Conclusions: The dynamic hydrodistention classification is a reliable method of evaluating the presence or absence of vesicoureteral reflux as it correlates significantly with radiographic reflux grade. It has high interobserver concordance.

Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4-mediated immune signal transduction, we purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H(+)-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines exhibit differential PM H(+)-ATPase activity. PM H(+)-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H(+)-ATPase activity and, importantly, its stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H(+)-ATPase to regulate stomatal apertures, inhibiting the entry of bacterial pathogens into the plant leaf during infection., Competing Interests: The authors have declared that no competing interests exist.

The efficiency of beta-glucuronidase (GUS) expression was evaluated with five viral promoters to identify the most suitable promoter or promoters for use in soybean hairy roots, including applications to study the symbiotic interaction with Bradyrhizobium japonicum. Levels of GUS activity were fluorimetrically and histochemically assayed when the GUS (uidA) gene was driven by the Cauliflower mosaic virus (CaMV) 35S promoter and enhanced 35S (E35S) promoter, the Cassava vein mosaic virus (CsVMV) promoter, the Figwort mosaic virus (FMV) promoter, and the Strawberry vein banding virus (SVBV2) promoter. We demonstrate that GUS activity was highest when driven by the FMV promoter and that the promoter activity of 35S and SVBV2 was significantly lower than that of the CsVMV and E35S promoters when tested in soybean hairy roots. In mature soybean root nodules, strong GUS activity was evident when the FMV, 35S, and CsVMV promoters were used. These results indicate that the FMV promoter facilitates the strong expression of target genes in soybean hairy roots and root nodules.

Purpose: Endoscopic implantation of dextranomer/hyaluronic acid has proved to be an effective minimally invasive technique for correcting vesicoureteral reflux in children. There is some evidence suggesting that in addition to being less invasive, successful dextranomer/hyaluronic acid implantation compared to successful antireflux surgery is associated with fewer febrile and nonfebrile urinary tract infections. We review the clinical outcomes of 2 groups of children cured of reflux with open surgery and dextranomer/hyaluronic acid implantation to determine if a difference in clinical outcomes exists., Materials and Methods: We reviewed the charts of 43 patients who underwent dextranomer/hyaluronic acid implantation and 33 who underwent open surgery for vesicoureteral reflux. Data collected included age, gender, preoperative and postoperative grades of reflux, and urinalysis and urine culture results. Urinary tract infection was defined as any culture that grew more than 10(5) colonies of a single organism, with symptoms typical of cystitis (urgency, frequency, dysuria). A febrile urinary tract infection was defined as an infection accompanied by a temperature greater than 101.5F. Any hospitalizations for febrile episodes were also recorded., Results: The incidence of urinary tract infection after successful open surgery (38%) was significantly higher than that observed following successful dextranomer/hyaluronic acid treatment (15%, p = 0.03). Febrile urinary tract infections occurred in 24% of the children who underwent open surgery and in 5% of those who underwent dextranomer/hyaluronic acid implantation (p = 0.02). Hospital readmissions occurred only in the group undergoing open surgery., Conclusions: Children successfully cured of vesicoureteral reflux with dextranomer/hyaluronic acid implantation have a lower incidence of febrile and nonfebrile urinary tract infections compared to those cured with open surgery. These findings suggest that dextranomer/hyaluronic acid implantation, when successful, may result in more favorable clinical outcomes.

Introduction: 2-Octyl cyanoacrylate (2-OCA; Dermabond) is a synthetic tissue adhesive that has proven utility in the closure of minor surgical incisions. Recently, we began using this adhesive for skin closure after circumcision and herein report our experience., Technical Considerations: A total of 267 patients (mean age 4 years) underwent circumcision (n = 208) or circumcision revision (n = 59) using 2-OCA for skin closure. The circumcision was performed using a sleeve technique in 219 patients and a Gomco clamp in 48. After meticulous hemostasis, the incision was cleansed, and the skin edges were aligned. Two thin layers of 2-OCA were applied to the incision and allowed to dry. Antibiotic ointment was then applied to the glans and penis. The technique was not used if poor skin edge apposition or bleeding was noted after circumcision. The skin closure time using 2-OCA averaged 3 minutes for the first 150 patients and less than 90 seconds for the last 120 patients, reflecting a learning curve associated with application. At a mean follow-up of 13 months (range 2 to 18), three postoperative complications had occurred. The cosmetic outcomes have been subjectively better than those with standard closure using interrupted 5-0 or 6-0 sutures, with no risk of suture tracks or sinuses. Parental satisfaction has been consistently great., Conclusions: Sutureless circumcision closure using 2-OCA is a safe, fast, and cosmetically appealing alternative to standard interrupted suture reapproximation. These advantages have made it our technique of choice. A direct economic and outcome comparison to sutured repairs is ongoing.

Purpose: Severe chordee associated with proximal hypospadias is a challenging problem. Occasionally division of the urethral plate and corporeal body grafting are necessary to achieve a straight phallus. We report our experience with small intestinal submucosa for corporeal body grafting to correct severe chordee associated with proximal hypospadias., Materials and Methods: We reviewed the charts of all 28 patients who underwent small intestinal submucosa corporeal body grafting to correct severe chordee during stage 1 of planned 2-stage proximal hypospadias repair from January 2001 to June 2006. After mobilization and division of the urethral plate ventral horizontal corporotomies were performed at the site of maximum curvature. Four and 1 ply small intestinal submucosa in 21 and 7 cases, respectively, was then fashioned to size and secured into the defect. Preoperative, operative and postoperative clinic visit records were reviewed to assess outcomes., Results: Preoperatively the meatal position was penoscrotal in 18 patients, scrotal in 5 and perineal in 5. In all patients, who had a mean age of 16 months, the urethral plate was divided and the corporeal bodies were grafted during stage 1 surgery. Vascularized preputial flaps were rotated ventrally for subsequent urethroplasty in 25 patients and buccal mucosa was used for urethroplasty in the remaining 3. A total of 26 patients underwent stage 2 repair with a mean followup of 38 months (range 6 to 61). Overall there were 6 complications, including epidermal retention cysts in 2 cases, partial urethral dehiscence in 2 and urethral diverticulum requiring reduction urethroplasty in 1, while a perimeatal granuloma developed in 1 following stage 1 repair, which required excision. Except the possibility of this latter patient there were no complications related to the small intestinal submucosa graft and a straight phallus without fibrosis was achieved in all 26., Conclusions: Our experience suggests that small intestinal submucosa is a suitable material for corporeal body grafting in 2-stage hypospadias repairs when there is associated severe chordee. In rare instances in which other maneuvers are inadequate to correct chordee small intestinal submucosa grafting is a reasonable option and a low complication rate can be anticipated.