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Purpose: This study's purpose was to assess caries prevalence by means of teledentistry in 12- to 60-month old children enrolled in Early Head Start inner-city child core centers.Methods: Images of the primary dentition were obtained by trained telehealth assistants using on intraoral camera. Images were entered into a Web-based storage and retrieval program. They were transmitted to a secure, remote-site computer and evaluated by a calibrated pediatric dentist.Results: Of 162 children screened, 93 were caries free and 69 had early childhood caries (ECC). Of these, 28 had severe early childhood caries (S-ECC). The mean dfs score for all 162 children was 1.88. The mean dfs score for the 69 ECC children was 4.42. The mean dfs for the subgroup of 28 S-ECC children was 7.61. Caries scores of S-ECC children were statistically significantly different from caries scores of the entire cohort and from caries scores of the ECC children.Conclusions: This study's results show that: (1) almost half of the preschoolers enrolled in the study were affected by dental caries; (2) only a few children had ever had a dental visit; and (3) teledentistry offers a potentially efficient means of screening high-risk preschool children for signs of early childhood caries. [ABSTRACT FROM AUTHOR]

Abstract Background Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows (“BNP dams”). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and β-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge. Results Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced. Conclusion This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types.

Abstract Background This study sought to map the Insomnia Severity Index (ISI) and symptom variables onto the EQ-5D. Methods A cross-sectional survey was conducted among adult US residents with self-reported sleep problems. Respondents provided demographic, comorbidity, and sleep-related information and had completed the ISI and the EQ-5D profile. Respondents were classified into ISI categories indicating no, threshold, moderate, or severe insomnia. Generalized linear models (GLM) were used to map the ISI's 7 items (Model I), summary scores (Model II), clinical categories (Model III), and insomnia symptoms (Model IV), onto the EQ-5D. We used 50% of the sample for estimation and 50% for prediction. Prediction accuracy was assessed by mean squared errors (MSEs) and mean absolute errors (MAEs). Results Mean (standard deviation) sleep duration for respondents (N = 2,842) was 7.8 (1.9) hours, and mean ISI score was 14.1 (4.8). Mean predicted EQ-5D utility was 0.765 (0.08) from Models I-III, which overlapped with observed utilities 0.765 (0.18). Predicted utility using insomnia symptoms was higher (0.771(0.07)). Based on Model I, predicted utilities increased linearly with improving ISI (0.493 if ISI = 28 vs. 1.00 if ISI = 0, p < 0.01). From Model II, each unit decrease in ISI summary score was associated with a 0.022 (p < 0.001) increase in utility. Predicted utilities were 0.868, 0.809, 0.722, and 0.579, respectively, for the 4 clinical categories, suggesting that lower utility was related to greater insomnia severity. The symptom model (Model IV) indicated a concave sleep-duration function of the EQ-5D; thus, utilities diminished after an optimal amount of sleep. The MSEs/MAEs were substantially lower when predicting EQ-5D > 0.40, and results were comparable in all models. Conclusions Findings suggest that mapping relationships between the EQ-5D and insomnia measures could be established. These relationships may be used to estimate insomnia-related treatment effects on health state utilities.

Abstract Background The purpose of this study was to conduct a scoping review of the literature about the establishment and impact of quality and safety team initiatives in acute care. Methods Studies were identified through electronic searches of Medline, Embase, CINAHL, PsycINFO, ABI Inform, Cochrane databases. Grey literature and bibliographies were also searched. Qualitative or quantitative studies that occurred in acute care, describing how quality and safety teams were established or implemented, the impact of teams, or the barriers and/or facilitators of teams were included. Two reviewers independently extracted data on study design, sample, interventions, and outcomes. Quality assessment of full text articles was done independently by two reviewers. Studies were categorized according to dimensions of quality. Results Of 6,674 articles identified, 99 were included in the study. The heterogeneity of studies and results reported precluded quantitative data analyses. Findings revealed limited information about attributes of successful and unsuccessful team initiatives, barriers and facilitators to team initiatives, unique or combined contribution of selected interventions, or how to effectively establish these teams. Conclusions Not unlike systematic reviews of quality improvement collaboratives, this broad review revealed that while teams reported a number of positive results, there are many methodological issues. This study is unique in utilizing traditional quality assessment and more novel methods of quality assessment and reporting of results (SQUIRE) to appraise studies. Rigorous design, evaluation, and reporting of quality and safety team initiatives are required.

Abstract Background Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Results Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. Conclusions This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques.

Abstract Background Diabetic nephropathy is a serious complication of diabetes mellitus and is associated with considerable morbidity and high mortality. There is increasing evidence to suggest that dysregulation of the epigenome is involved in diabetic nephropathy. We assessed whether epigenetic modification of DNA methylation is associated with diabetic nephropathy in a case-control study of 192 Irish patients with type 1 diabetes mellitus (T1D). Cases had T1D and nephropathy whereas controls had T1D but no evidence of renal disease. Methods We performed DNA methylation profiling in bisulphite converted DNA from cases and controls using the recently developed Illumina Infinium® HumanMethylation27 BeadChip, that enables the direct investigation of 27,578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14,495 genes. Results Singular Value Decomposition (SVD) analysis indicated that significant components of DNA methylation variation correlated with patient age, time to onset of diabetic nephropathy, and sex. Adjusting for confounding factors using multivariate Cox-regression analyses, and with a false discovery rate (FDR) of 0.05, we observed 19 CpG sites that demonstrated correlations with time to development of diabetic nephropathy. Of note, this included one CpG site located 18 bp upstream of the transcription start site of UNC13B, a gene in which the first intronic SNP rs13293564 has recently been reported to be associated with diabetic nephropathy. Conclusion This high throughput platform was able to successfully interrogate the methylation state of individual cytosines and identified 19 prospective CpG sites associated with risk of diabetic nephropathy. These differences in DNA methylation are worthy of further follow-up in replication studies using larger cohorts of diabetic patients with and without nephropathy.

Abstract Background The Spemann/Mangold organizer is a transient tissue critical for patterning the gastrula stage vertebrate embryo and formation of the three germ layers. Despite its important role during development, there are still relatively few genes with specific expression in the organizer and its derivatives. Foxa2 is a forkhead transcription factor that is absolutely required for formation of the mammalian equivalent of the organizer, the node, the axial mesoderm and the definitive endoderm (DE). However, the targets of Foxa2 during embryogenesis, and the molecular impact of organizer loss on the gastrula embryo, have not been well defined. Results To identify genes specific to the Spemann/Mangold organizer, we performed a microarray-based screen that compared wild-type and Foxa2 mutant embryos at late gastrulation stage (E7.5). We could detect genes that were consistently down-regulated in replicate pools of mutant embryos versus wild-type, and these included a number of known node and DE markers. We selected 314 genes without previously published data at E7.5 and screened for expression by whole mount in situ hybridization. We identified 10 novel expression patterns in the node and 5 in the definitive endoderm. We also found significant reduction of markers expressed in secondary tissues that require interaction with the organizer and its derivatives, such as cardiac mesoderm, vasculature, primitive streak, and anterior neuroectoderm. Conclusion The genes identified in this screen represent novel Spemann/Mangold organizer genes as well as potential Foxa2 targets. Further investigation will be needed to define these genes as novel developmental regulatory factors involved in organizer formation and function. We have placed these genes in a Foxa2-dependent genetic regulatory network and we hypothesize how Foxa2 may regulate a molecular program of Spemann/Mangold organizer development. We have also shown how early loss of the organizer and its inductive properties in an otherwise normal embryo, impacts on the molecular profile of surrounding tissues.

Abstract Background The orphan GPCR MrgE is one of an extended family of GPCRs that are expressed in dorsal root ganglia (DRG). Based on these expression patterns it has been suggested that GPCRs like MrgE may play a role in nociception however, to date, no direct supporting evidence has emerged. We generated mutant mice lacking MrgE and examined the effects of deletion of this gene in three pain behavioural models. The effect of MrgE gene deletion on expression of Mrgs and genes involved in sensory neurone function was also investigated. Results The absence of MrgE had no effect on the development of pain responses to a noxious chemical stimulus or an acute thermal stimulus. However, in contrast, the development but not the maintenance of neuropathic pain was affected by deletion of MrgE. The expression of Mrg genes was not significantly affected in the MrgE knockout (KO) mice with the sole exception of MrgF. In addition, the expression of 77 of 84 genes involved in sensory neuron development and function was also unaffected by deletion of MrgE. Of the 7 genes affected by MrgE deletion, 4 have previously been implicated in nociception. Conclusion The data suggests that MrgE may play a role in selective pain behavioural responses in mice.

Abstract Background Extraordinary infection control measures limited access to medical care in the Greater Toronto Area during the 2003 Severe Acute Respiratory Syndrome (SARS) outbreak. The objective of this study was to determine if the period of these infection control measures was associated with changes in overall population mortality due to causes other than SARS. Methods Observational study of death registry data, using Poisson regression and interrupted time-series analysis to examine all-cause mortality rates (excluding deaths due to SARS) before, during, and after the SARS outbreak. The population of Ontario was grouped into the Greater Toronto Area (N = 2.9 million) and the rest of Ontario (N = 9.3 million) based upon the level of restrictions on delivery of clinical services during the SARS outbreak. Results There was no significant change in mortality in the Greater Toronto Area before, during, and after the period of the SARS outbreak in 2003 compared to the corresponding time periods in 2002 and 2001. The rate ratio for all-cause mortality during the SARS outbreak was 0.99 [95% Confidence Interval (CI) 0.93–1.06] compared to 2002 and 0.96 [95% CI 0.90–1.03] compared to 2001. An interrupted time series analysis found no significant change in mortality rates in the Greater Toronto Area associated with the period of the SARS outbreak. Conclusion Limitations on access to medical services during the 2003 SARS outbreak in Toronto had no observable impact on short-term population mortality. Effects on morbidity and long-term mortality were not assessed. Efforts to contain future infectious disease outbreaks due to influenza or other agents must consider effects on access to essential health care services.

Abstract Background It is uncertain whether patients with renal vascular disease will have renal or mortality benefit from re-establishing renal blood flow with renal revascularization procedures. The RAVE study will compare renal revascularization to medical management for people with atherosclerotic renal vascular disease (ARVD) and the indication for revascularization. Patients will be assessed for the standard nephrology research outcomes of progression to doubling of creatinine, need for dialysis, and death, as well as other cardiovascular outcomes. We will also establish whether the use of a new inexpensive, simple and available ultrasound test, the renal resistance index (RRI), can identify patients with renal vascular disease who will not benefit from renal revascularization procedures1. Methods/design This single center randomized, parallel group, pilot study comparing renal revascularization with medical therapy alone will help establish an infrastructure and test the feasibility of answering this important question in clinical nephrology. The main outcome will be a composite of death, dialysis and doubling of creatinine. Knowledge from this study will be used to better understand the natural history of patients diagnosed with renal vascular disease in anticipation of a Canadian multicenter trial. Data collected from this study will also inform the Canadian Hypertension Education Program (CHEP) Clinical Practice Guidelines for the management of Renal and Renal Vascular Disease. The expectation is that this program for ARVD, will enable community based programs to implement a comprehensive guidelines based diagnostic and treatment program, help create an evidence based approach for the management of patients with this condition, and possibly reduce or halt the progression of kidney disease in these patients. Discussion Results from this study will determine the feasibility of a multicentered study for the management of renovascular disease.

Abstract Background Cocaine and amphetamine regulated transcript (CART) is an anorectic neuropeptide located principally in hypothalamus. CART has been shown to be involved in control of feeding behavior, but a direct relationship with obesity has not been established. The aim of this study was to evaluate the effect of polymorphisms within the CART gene with regards to a possible association with obesity in a Caucasian population. Results Screening of the entire gene as well as a 3.7 kb region of 5' upstream sequence revealed 31 SNPs and 3 rare variants ; 14 of which were subsequently genotyped in 292 French morbidly obese subjects and 368 controls. Haplotype analysis suggested an association with obesity which was found to be mainly due to SNP-3608T>C (rs7379701) (p = 0.009). Genotyping additional cases and controls also of European Caucasian origin supported further this possible association between the CART SNP -3608T>C T allele and obesity (global p-value = 0.0005). Functional studies also suggested that the SNP -3608T>C could modulate nuclear protein binding. Conclusion CART SNP -3608T>C may possibly contribute to the genetic risk for obesity in the Caucasian population. However confirmation of the importance of the role of the CART gene in energy homeostasis and obesity will require investigation and replication in further populations.

Polysorbates are the predominant surfactants used to stabilize protein formulations. Unfortunately, polysorbates can undergo hydrolytic degradation, which releases fatty acids that can accumulate to form visible particles. The detection and quantitation of these fatty acid degradation products are critical for assessing the extent of polysorbate degradation and the associated risks of particle formation. We previously developed a user-friendly mass spectrometric method called Fatty Acids by Mass Spectrometry (FAMS) to quantify the free fatty acids. The FAMS method was validated according to ICH Q2 (R1) guidelines and is suitable for a wide range of products, buffers and protein concentrations. The end-to-end workflow can be automated from sample preparation to data analysis. To broaden method accessibility, the QDa detector selected for fatty acid measurement does not require specific mass spectrometry experience. We provide here a detailed procedure for both manual and automated sample preparation for high-throughput analysis. In addition, we highlight in this protocol the critical operational details, procedural watchouts and troubleshooting tips to support the successful execution of this method in another laboratory.

Palliative care has been defined as 'the active holistic care of individuals across all ages with serious health-related suffering due to severe illness, and especially of those near the end of life'. Unfortunately, palliative care and especially paediatric palliative care remain a neglected area of medicine and are widely misunderstood, with few healthcare providers having any formal training in South Africa. To relieve health-related suffering, healthcare providers must understand that the field is not limited to end-of-life care for the terminally ill, and holistic care (physical, emotional, social and spiritual) should commence at the time of diagnosis of a serious illness. It is imperative that all healthcare providers develop the knowledge and skills to provide this essential care across all levels of care and disciplines. The article aims to raise awareness and show how to practically implement palliative care through case studies.

Surfactant degradation in biopharmaceuticals has recently gained significant attention in the pharmaceutical industry. Specifically, hydrolytic degradation of polysorbates, leading to the release of free fatty acids potentially forming visible particles, is a key theme in technical development. To address this emerging topic, we present the development of a fully automated liquid-chromatography single quad mass detector method for the quantification of free fatty acids in biopharmaceuticals. For the first time, we have quantified the longer chain fatty acid degradation products of polysorbate, palmitic and stearic acid, allowing reliable detection and early critical insights for process improvements. This high-throughput method was validated underlining its robust performance in an interlaboratory trial as well as high flexibility allowing different robotic platforms and preparation techniques. The combination of automated sample preparation, separation by liquid chromatography and single quad mass detection makes the validated fatty acid mass spectrometry assay ready for routine use in a regulated environment., 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 © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The birth plan was introduced in the 1980s to facilitate communication between maternity care providers and women and increase agency for childbearing women in the face of medicalised birth. Forty years on, the birth plan is a heterogeneous document with uncertainty surrounding its purpose, process, and impact. The aim of this review was to synthesise the evidence and improve understanding of the purpose, process and impact of the birth plan on childbearing women's experiences and outcomes., Methods: This systematic review followed the PRISMA guidelines. A comprehensive search strategy was designed and applied to electronic databases CINAHL, MEDLINE, PsychINFO, Cochrane Library, Scopus, and ClinicalTrials.gov. Articles were appraised using the Crowe Critical Appraisal Tool and a five-step integrative approach to analysis followed., Findings: Eleven articles were identified, all quantitative in nature. It is clear that the general purpose of birth plans is communication, with decision making a key factor. Even though the processes of birth planning were varied, having a birth plan was associated with generally positive birth outcomes., Conclusions: Despite the heterogeneity of birth plans, birth plans were associated with positive outcomes for childbearing women when developed in collaboration with care providers. The act of collaboratively creating a birth plan may improve obstetric outcomes, aid realistic expectations, and improve satisfaction and the sense of control., Competing Interests: Declaration of Competing Interest None declared, (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Microbial community compositions and functional profiles were analyzed in microcosms established using aquifer materials from a former automobile factory site, where 1,4-dioxane was identified as the primary contaminant of concern. Propane or oxygen biostimulation resulted in limited 1,4-dioxane degradation, which was markedly enhanced with the addition of nutrients, resulting in abundant Mycobacterium and Methyloversatilis taxa and high expressions of propane monooxygenase gene, prmA. In bioaugmented treatments, Pseudonocardia dioxanivorans CB1190 or Rhodococcus ruber ENV425 strains dominated immediately after augmentation and degraded 1,4-dioxane rapidly which was consistent with increased representation of xenobiotic and lipid metabolism-related functions. Although the bioaugmented microbes decreased due to insufficient growth substrates and microbial competition, they did continue to degrade 1,4-dioxane, presumably by indigenous propanotrophic and heterotrophic bacteria, inducing similar community structures across bioaugmentation conditions. In various treatments, functional redundancy acted as buffer capacity to ensure a stable microbiome, drove the restoration of the structure and microbial functions to original levels, and induced the decoupling between basic metabolic functions and taxonomy. The results of this study provided valuable information for design and decision-making for ex-situ bioreactors and in-situ bioremediation applications. A metagenomics-based understanding of the treatment process will enable efficient and accurate adjustments when encountering unexpected issues in bioremediation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Polysorbates and Poloxamer 188 constitute the most common surfactants used in biopharmaceutical formulations owing to their excellent protein-stabilizing properties and good safety profiles. In recent years, however, a vast number of reports concerning potential risk factors closely related with their applications, such as the accumulation of degradation products, their inherent heterogeneity and adsorption effects of proteins at silicon/oil interfaces have drawn the focus to potential alternatives. Apart from tedious efforts to evaluate new excipient candidates, the use of mixed formulations leveraging combinations of well-established surfactants appears to be a promising approach to eliminate or, at least, minimize and postpone adverse effects associated with the single compounds. Due to the similar molecular properties of non-ionic surfactants, however, baseline separation of these mixtures, which is mandatory for their reliable quantification, poses a great challenge to analytical scientists. For this purpose, the present work describes the development of a robust mixed-mode liquid chromatography method coupled to evaporative light scattering detection (mixed-mode LC-ELSD) for simultaneous determination of the (intact) Polysorbate 20 and Poloxamer 188 content in biopharmaceutical formulations containing monoclonal antibodies. Extensive qualification and validation studies, comprising the evaluation of method specificity, robustness, linearity, accuracy and precision according to ICH guidelines, demonstrated its suitability for quality control studies. A case study on the storage stability of a formulated antibody was conducted to underline the method's practical utility. Finally, the versatility of the developed approach was successfully tested by quantifying Polysorbate 20-related surfactants, such as Polysorbate 80 and super-refined Polysorbate., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hydrolysis of the non-ionic surfactant polysorbate upon long-term storage poses significant challenges to development of biopharmaceutical liquid formulations. Low concentrations of intact surfactant may compromise its protective properties and thus affect protein stability. In addition, accumulation of polysorbate hydrolysis products is increasingly put into context with the formation of visible and subvisible particulates based on the low solubility of the main degradation products. Despite of this potential negative impact on product quality, quantification of the released free fatty acids is performed commonly in an indirect and consequently insensitive manner by determining the remaining PS content or by cumbersome methods, which are unsuitable for routine testing in quality control laboratories. For this purpose, this study describes the development and qualification of a label-free, reliable liquid-chromatography single quad mass detector (LC-QDa)-based method capable of resolving slight changes in the free fatty acid profile which can be readily integrated into quality control facilities. The practical utility of the herein described method is outlined by a case study on the real-time storage stability of a formulated monoclonal antibody., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rhodobacter sphaeroides has emerged as a model system for studies of the complex chemotaxis pathways that are a hallmark of many non-enteric bacteria. The genome of R. sphaeroides encodes two sets of flagellar genes, fla1 and fla2, that are controlled by three different operons. Each operon encodes homologues of most of the proteins required for the well-studied E. coli chemotaxis pathway. R. sphaeroides has six homologues of the response regulator CheY that are localized to and are regulated by different clusters of chemosensory proteins in the cell and have different effects on chemotaxis. CheY6 is the major CheY stopping the fla1 flagellar motor and associated with a cytoplasmically localised chemosensory pathway. CheY3 and CheY4 are associated with a membrane localised polar chemosensory cluster, and can bind to but not stop the motor. CheY6 and either CheY3 or CheY4 are required for chemotaxis. We are using NMR spectroscopy to characterise and compare the structure and dynamics of CheY3 and CheY6 in solution. We are interested in defining the conformational changes that occur upon activation of these two proteins and to identify differences in their properties that can explain the different functions they play in chemotaxis in R. sphaeroides. Here we present the (1)H, (13)C and (15)N assignments for CheY3 in its active, inactive and Mg(2+)-free apo form. These assignments provide the starting point for detailed investigations of the structure and function of CheY3.

Repulsive guidance molecules (RGMs) control crucial processes including cell motility, adhesion, immune-cell regulation and systemic iron metabolism. RGMs signal via the neogenin (NEO1) and the bone morphogenetic protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a new protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the crystal structure of the ternary BMP2-RGM-NEO1 complex, which, along with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM-NEO1 complex. Our results show how RGM acts as the central hub that links BMP and NEO1 and physically connects these fundamental signaling pathways.

Repulsive guidance molecule family members (RGMs) control fundamental and diverse cellular processes, including motility and adhesion, immune cell regulation, and systemic iron metabolism. However, it is not known how RGMs initiate signaling through their common cell-surface receptor, neogenin (NEO1). Here, we present crystal structures of the NEO1 RGM-binding region and its complex with human RGMB (also called dragon). The RGMB structure reveals a previously unknown protein fold and a functionally important autocatalytic cleavage mechanism and provides a framework to explain numerous disease-linked mutations in RGMs. In the complex, two RGMB ectodomains conformationally stabilize the juxtamembrane regions of two NEO1 receptors in a pH-dependent manner. We demonstrate that all RGM-NEO1 complexes share this architecture, which therefore represents the core of multiple signaling pathways.

Plexins are cell surface receptors for the semaphorin family of cell guidance cues. The cytoplasmic region comprises a Ras GTPase-activating protein (GAP) domain and a RhoGTPase binding domain. Concomitant binding of extracellular semaphorin and intracellular RhoGTPase triggers GAP activity and signal transduction. The mechanism of this intricate regulation remains elusive. We present two crystal structures of the human Plexin-B1 cytoplasmic region in complex with a constitutively active RhoGTPase, Rac1. The structure of truncated Plexin-B1-Rac1 complex provides no mechanism for coupling RhoGTPase and Ras binding sites. On inclusion of the juxtamembrane helix, a trimeric structure of Plexin-B1-Rac1 complexes is stabilised by a second, novel, RhoGTPase binding site adjacent to the Ras site. Site-directed mutagenesis combined with cellular and biophysical assays demonstrate that this new binding site is essential for signalling. Our findings are consistent with a model in which extracellular and intracellular plexin clustering events combine into a single signalling output., Competing Interests: The authors have declared that no competing interests exist.

Objective: • To determine if hyaluronic acid (HA) can be incorporated into porcine small intestinal submucosa (SIS) through poly (lactide-co-glycolide-acid) (PLGA) nanoparticles to improve the consistency of the naturally derived biomaterial and promote bladder tissue regeneration., Methods: • Beagle dogs were subjected to 40% partial cystectomy followed by bladder augmentation with commercial SIS or HA-PLGA-modified SIS. • Urodynamic testing was performed before and after augmentation to assess bladder volume. • A scoring system was created to evaluate gross and histological presentations of regenerative bladders., Results: • All dogs showed full-thickness bladder regeneration. • Histological assessment showed improved smooth muscle regeneration in the HA-PLGA-modified SIS group. • For both groups of dogs, urodynamics and graft measurements showed an approximate 40% reduction in bladder capacity and graft size from pre-augmentation to post-regeneration measurements. • Application of the scoring system and statistical analysis failed to show a significant difference between the groups., Conclusions: • SIS can be modified through the addition of HA-PLGA nanoparticles. The modified grafts showed evidence of improved smooth muscle regeneration on histological assessment, although this difference was not evident on a novel grading scale. • The volume loss and graft shrinkage experienced are consistent with previous models of SIS bladder regeneration at the 10-week time point. • Additional research into the delivery of HA and the long-term benefits of HA on bladder regeneration is needed to determine the full benefit of HA-PLGA-modified SIS. In addition, a more objective biochemical characterization will be needed to evaluate the quality of regeneration., (© 2010 THE AUTHORS. BJU INTERNATIONAL © 2010 BJU INTERNATIONAL.)

Borderline personality disorder (BPD) is a complex psychiatric disorder that involves the core feature of affect dysregulation. Prior neuroimaging studies have indicated that BPD patients have (1) excessive amygdala activation to negative emotion and (2) diminished frontal regulation. This study examined amygdala functional connectivity in 12 women with BPD and 12 matched healthy comparison volunteers. We explored how connectivity patterns would change in the context of processing neutral, overt fear, or masked fear face expressions. Each participant underwent three 5-min fMRI scans in which they primarily viewed: (1) neutral, (2) overt fear, and (3) masked fear faces. In comparison to their healthy counterparts, young women with BPD showed (1) lower connectivity between bilateral amygdala and mid-cingulate cortex during the neutral scan; (2) higher connectivity between bilateral amygdala and rostral anterior cingulate cortex during the overt fear scan; and (3) higher right amygdala connectivity with bilateral thalamus and right caudate during the masked fear scan. Exploratory analyses revealed interesting correlations between amygdala connectivity in these conditions with multiple clinical measures. Results from the neutral scan add to the few prior connectivity studies in BPD that have been suggestive of lower fronto-limbic connectivity in BPD. However, the connectivity findings during fear processing are novel, and map onto basic research models for amygdala connectivity, that is, connections to frontal areas for overt fear processing versus connections to thalamus for automatic fear processing. Further, results suggest that BPD subjects tap into both pathways more strongly than healthy comparisons.

Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. Here we present crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB1(1-2)-SEMA4D(ecto) and murine PlxnA2(1-4)-Sema6A(ecto)), plus unliganded structures of PlxnA2(1-4) and Sema6A(ecto). These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, our data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed β-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin-plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity.

Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 A crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA(3), in complex with its cognate RR, CheY(6). A methionine finger on CheY(6) that nestles in a hydrophobic pocket in CheA(3) was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA(3), CheY(6), and CheB(2). Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA(3)-P to CheY(6). Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA(3)-P. The structure presented here has allowed us to identify specificity determinants for the CheA-CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction., Competing Interests: The authors have declared that no competing interests exist.

Objective: To determine if porcine small intestinal submucosa (SIS)-regenerated urothelium expresses markers of urothelial differentiation, uroplakin and zona occludens-1 (ZO-1), and whether their expression correlates with the histological appearance of the urothelium., Materials and Methods: In all, 15 rats underwent partial cystectomy and bladder replacement with SIS. Regenerated bladders were harvested at either 2, 7, 14, 28, or 56 days after SIS grafting. Histological examination with haematoxylin and eosin staining was conducted to assess tissue regeneration. Immunohistochemistry was performed with uroplakin and ZO-1 antibodies., Results: By 14 days after SIS grafting, the urothelial layer was completely confluent over the SIS. Expression of uroplakin and ZO-1, evident at 2 days after SIS grafting, progressed from a cytoplasmic pattern of expression to a mature pattern of cytoplasmic and membrane expression by 56 days after SIS grafting., Conclusion: In vivo tissue regeneration produces histologically and phenotypically mature urothelium within 2 weeks of SIS implantation. Regeneration of functional urothelium is probably essential for the subsequent development of the remaining bladder.

F425-B4e8 (B4e8) is a monoclonal antibody isolated from a human immunodeficiency virus type 1 (HIV-1)-infected individual that recognizes the V3 variable loop on the gp120 subunit of the viral envelope spike. B4e8 neutralizes a subset of HIV-1 primary isolates from subtypes B, C and D, which places this antibody among the very few human anti-V3 antibodies with notable cross-neutralizing activity. Here, the crystal structure of the B4e8 Fab' fragment in complex with a 24-mer V3 peptide (RP142) at 2.8 A resolution is described. The complex structure reveals that the antibody recognizes a novel V3 loop conformation, featuring a five-residue alpha-turn around the conserved GPGRA apex of the beta-hairpin loop. In agreement with previous mutagenesis analyses, the Fab' interacts primarily with V3 through side-chain contacts with just two residues, Ile(P309) and Arg(P315), while the remaining contacts are to the main chain. The structure helps explain how B4e8 can tolerate a certain degree of sequence variation within V3 and, hence, is able to neutralize an appreciable number of different HIV-1 isolates.

Background: The orphan GPCR MrgE is one of an extended family of GPCRs that are expressed in dorsal root ganglia (DRG). Based on these expression patterns it has been suggested that GPCRs like MrgE may play a role in nociception however, to date, no direct supporting evidence has emerged. We generated mutant mice lacking MrgE and examined the effects of deletion of this gene in three pain behavioural models. The effect of MrgE gene deletion on expression of Mrgs and genes involved in sensory neurone function was also investigated., Results: The absence of MrgE had no effect on the development of pain responses to a noxious chemical stimulus or an acute thermal stimulus. However, in contrast, the development but not the maintenance of neuropathic pain was affected by deletion of MrgE. The expression of Mrg genes was not significantly affected in the MrgE knockout (KO) mice with the sole exception of MrgF. In addition, the expression of 77 of 84 genes involved in sensory neuron development and function was also unaffected by deletion of MrgE. Of the 7 genes affected by MrgE deletion, 4 have previously been implicated in nociception., Conclusion: The data suggests that MrgE may play a role in selective pain behavioural responses in mice.

The results of a laboratory investigation into the effects of modified atmospheres (MA) on the eggs of mite pests of grain and cheese are presented. Four species of astigmatid mite were tested; Acarus farris (Oudemans). A. siro L., Lepidoglyphus destructor (Schrank) and Tyrophagus longior (Gervais). All are found in many habitats including grain and cheese stores. Three low oxygen (O2) MA mixtures were used, based on carbon dioxide (CO2), nitrogen (N2) or simulated burner gas (0.5 or 2% O2, 10% CO2, balance N2) plus 60% CO2 in air (8% O2). The mites were exposed at 15 degrees C and 80% r.h., a combination of conditions that occurs at the surface of stored grain during the autumn which promotes mite population growth. The exposure periods required to prevent egg hatch for each species in every mixture are given. Tyrophagus longior was the most tolerant species, followed by A. siro and A. farris, with L. destructor the most susceptible. Burner gas was the most effective mixture overall with 0.5% O2 but with an increase in the O2 level to 2% for all the mixtures, CO2 became the more effective control agent. With 60% CO2 in air some loss of efficacy was observed against the three most tolerant species and even more so for L. destructor. Sublethal exposures to MAs for at least 4 days in L. destructor, 6 days in A. farris and A. siro and 8 days for T. longior caused a delay in egg hatch.

Pseudoporphyria cutanea tarda occurred in two children undergoing peritoneal dialysis and receiving erythropoietin therapy. The mechanism whereby erythropoietin might lead to photosensitization is unknown, but physicians should be aware of this possible association.