Thyroglossal Duct Cyst Infection Caused by Neisseria gonorrhoeae: An Unusual Complication of Pharyngeal Gonorrhea.

ABSTRACT: Neisseria gonorrhoeae is a human obligate pathogen whose clinical expression of disease ranges from localized genital infection to involvement of extragenital sites such as the conjunctiva and throat. We describe the second case of a thyroglossal duct abscess due to N. gonorrhoeae, an uncommon complication of pharyngeal gonococcal infection. The fortuitous occurrence in the same individual of these 2 conditions that both exhibit an occult clinical presentation likely accounts for rarity of this infection. We discuss the pertinent gonococcal and host factors that underlie the clinical manifestations of this infection. A particular focus is the fundamental role that the binding of the gonococcal opacity-associated protein to the ubiquitous human carcinoembryonic cell adhesion molecule plays in the pathogenesis of pharyngeal gonorrhea.

Competitive Specific Anchorage of Molecules onto Surfaces: Quantitative Control of Grafting Densities and Contamination by Free Anchors.

The formation of surfaces decorated with biomacromolecules such as proteins, glycans, or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes and interaction assays. To this effect, ligand molecules are often functionalized with an anchor that specifically binds to a surface with a high density of binding sites, providing control over the presentation of the molecules. Here, we present a method to robustly and quantitatively control the surface density of one or several types of anchor-bearing molecules by tuning the relative concentrations of target molecules and free anchors in the incubation solution. We provide a theoretical background that relates incubation concentrations to the final surface density of the molecules of interest and present effective guidelines toward optimizing incubation conditions for the quantitative control of surface densities. Focusing on the biotin anchor, a commonly used anchor for interaction studies, as a salient example, we experimentally demonstrate surface density control over a wide range of densities and target molecule sizes. Conversely, we show how the method can be adapted to quality control the purity of end-grafted biopolymers such as biotinylated glycosaminoglycans by quantifying the amount of residual free biotin reactant in the sample solution.

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors.

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation due to a lack of mechanistic understanding. Specifically, CXCL4 (Platelet Factor 4, PF4) has no established receptor that explains its function. Here, we use biophysical, in vitro, and in vivo techniques to determine the mechanism underlying CXCL4-mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars on proteoglycans within the endothelial extracellular matrix, resulting in increased adhesion of leukocytes to the vasculature, increased vascular permeability, and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulfation confers selectivity onto chemokine localization. These findings present mechanistic insights into chemokine biology and provide future therapeutic targets.

A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces.

Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (- Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ~ 2 kDa tetrasaccharides to ~ 1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/ - Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.

Cholesterol is required for transcriptional repression by BASP1.

Lipids are present within the cell nucleus, where they engage with factors involved in gene regulation. Cholesterol associates with chromatin in vivo and stimulates nucleosome packing in vitro, but its effects on specific transcriptional responses are not clear. Here, we show that the lipidated Wilms tumor 1 (WT1) transcriptional corepressor, brain acid soluble protein 1 (BASP1), interacts with cholesterol in the cell nucleus through a conserved cholesterol interaction motif. We demonstrate that BASP1 directly recruits cholesterol to the promoter region of WT1 target genes. Mutation of BASP1 to ablate its interaction with cholesterol or the treatment of cells with drugs that block cholesterol biosynthesis inhibits the transcriptional repressor function of BASP1. We find that the BASP1-cholesterol interaction is required for BASP1-dependent chromatin remodeling and the direction of transcription programs that control cell differentiation. Our study uncovers a mechanism for gene-specific targeting of cholesterol where it is required to mediate transcriptional repression.

Process evaluation of specialist nurse implementation of a soft opt-out organ donation system in Wales.

BACKGROUND: Wales introduced a soft opt-out organ donation system on 1st December 2015 with the aim of improving consent rates. In the first 18 months consent rates improved but the difference could not solely be attributed to the soft opt-out system when compared with similar improvements in consent rates in other UK nations. METHODS: We conducted an 18 month post-intervention qualitative process evaluation involving 88 family members of 60/211 potential organ donor cases, and 19 professionals. Views and experiences of Specialist Nurses in Organ Donation who implemented the new system and family members who were involved in decision making were collected to see how their respective behaviours impacted on implementation. Data collection included interviews, focus groups and qualitative questionnaire data. RESULTS: Implementation was considered a success by Specialist Nurses in Organ Donation. The bespoke retraining programme and responsive approach to addressing initial implementation issues were identified as examples of best practice. Specialist Nurses in Organ Donation were valued by family members. Six implementation issues had an impact on consent rates - the media campaign had gaps, the system was more complex, challenges in changing professional behaviours, inability to obtain the required standard of evidence from family members to overturn a donation decision, increased complexity of consent processes, and additional health systems issues. CONCLUSION: This is the first comprehensive process evaluation of implementing a soft opt-out system of organ donation. Specific elements of good implementation practice (such as investment in the retraining programme and the responsiveness of Specialist Nurses in Organ Donation and managers to feedback) were identified. The key message is that despite retraining, nursing practice did not radically change overnight to accommodate the new soft opt-out system. Policy makers and health service managers should not assume that nurses simply need more time to implement the soft-out as intended. Additional responsive modification of processes, ongoing training and support is required to help with implementation as originally intended. Scotland, England and the Netherlands are introducing soft opt-out systems. There is an opportunity to learn from initial implementation in Wales, by acknowledging gaps, good practice and opportunities to further improve processes and nursing practices.

Designing a co-productive study to overcome known methodological challenges in organ donation research with bereaved family members.

BACKGROUND: Co-production of research into public health services has yet to demonstrate tangible benefits. Few studies have reported the impact of co-production on research outcomes. The previous studies of organ donation have identified challenges in engaging with public organizations responsible, gaining ethical approval for sensitive studies with the recently bereaved and difficulty in recruiting bereaved family members who were approached about organ donation. OBJECTIVE: To address these challenges, we designed the first large co-productive observational study to evaluate implementation of a new system of organ donation in Wales. This paper outlines the co-productive strategies that were designed to overcome known methodological challenges and reports what impact they had on resolving these challenges. DESIGN: Two-year co-produced study with multiple stakeholders with the specific intention of maximizing engagement with the National Health Service arm in Wales responsible for organ donation, and recruitment of bereaved family members whose perspectives are essential but commonly absent from studies. SETTING AND PARTICIPANTS: NHS Blood and Transplant, Welsh Government and multiple patient and public representatives who served as co-productive partners with the research team. RESULTS: Co-productive strategies enabled a smooth passage through four different ethics processes within the 10-week time frame, family member recruitment targets to be surpassed, sharing of routinely collected data on 100% of potential organ donor cases and development of further research capacity and capability in a critically under researched area. DISCUSSION AND CONCLUSION: Although expensive and time consuming, co-production was effective and added value to research processes and study outcomes.

Short-term impact of introducing a soft opt-out organ donation system in Wales: before and after study.

OBJECTIVES: To determine the short-term impact of a soft opt-out organ donation system on consent rates and donor numbers. DESIGN: Before and after observational study using bespoke routinely collected data. SETTING: National Health Service Blood and Transplant. PARTICIPANTS: 205 potential organ donor cases in Wales. INTERVENTIONS: The Act and implementation strategy. PRIMARY AND SECONDARY OUTCOMES: Consent rates at 18 months post implementation compared with 3 previous years, and organ donor numbers 21 months before and after implementation. Changes in organ donor register activity post implementation for 18 months. RESULTS: The consent rate for all modes of consent was 61.0% (125/205), showing a recovery from the dip to 45.8% in 2014/2015. 22.4% (46/205) were deemed consented donors: consent rate 60.8% (28/46). Compared with the 3 years before the switch there was a significant difference in Welsh consent rates (χ2 p value=0.009). Over the same time period, rest of the UK consent rates also significantly increased from 58.6% (5256/8969) to 63.1% (2913/4614) (χ2 p value<0.0001), therefore the Wales increase cannot be attributed to the Welsh legislation change. Deceased donors did not increase: 101 compared with 104. Organ donation registration increased from 34% to 38% with 6% registering to opt-out. CONCLUSION: This is the first rigorous initial evaluation with bespoke data collected on all cases. The longer-term impact on consent rates and donor numbers is unclear. Concerns about a potential backlash and mass opting out were not realised. The move to a soft opt-out system has not resulted in a step change in organ donation behaviour, but can be seen as the first step of a longer journey. Policymakers should not assume that soft opt-out systems by themselves simply need more time to have a meaningful effect. Ongoing interventions to further enhance implementation and the public's understanding of organ donation are needed to reach the 2020 target of 80% consent rates. Further longitudinal monitoring is required.

Family attitudes, actions, decisions and experiences following implementation of deemed consent and the Human Transplantation (Wales) Act 2013: mixed-method study protocol.

INTRODUCTION: The Human Transplantation (Wales) Act 2013 (the Act) introduced a 'soft opt-out' system of organ donation on 1 December 2015. Citizens are encouraged to make their organ donation decision known during their lifetime. In order to work, the Act and media campaign need to create a context, whereby organ donation becomes the norm, and create a mechanism for people to behave as intended (formally register their decision; consider appointing a representative; convey their donation decision to their families and friends or do nothing-deemed consent). In addition, family members/appointed representatives need to be able to put their own views aside to support the decision of their loved one. The aim of this study is to evaluate initial implementation, outcomes and impact on families and appointed representatives who were approached about organ donation during the first 18 months. METHODS AND ANALYSIS: Prospective mixed-method coproductive study undertaken with National Health Service Blood and Transplant (NHSBT), and multiple patient/public representatives. The study is designed to collect information on all cases who meet specified criteria (≥18 years, deceased person voluntarily resident in Wales and died in Wales or England) whose family were approached between 1 December 2015 and 31 June 2017). Data for analysis include: NHSBT routinely collected anonymised audit data on all cases; Specialist Nurse in Organ Donation (SNOD) completed anonymised form for all cases documenting their perception of the families' understanding of the Act, media campaign and outcome of the donation approach; questionnaires and depth interviews with any family member or appointed representative (minimum 50 cases). Additional focus groups and interviews with SNODs. Anonymised donation outcomes and registration activity reports for Wales provide additional context. ETHICS AND DISSEMINATION: Approved by NHSBT Research, Innovation and Technology Advisory Group on 23 October 2015; Wales Research Ethics Committee 5 (IRAS190066; Rec Reference 15/WA/0414) on 25 November 2015 and NHSBT R&D Committee (NHSBT ID: AP-15-02) on 24 November 2015. REGISTRATION: The protocol is registered on the Health and Care Research Wales Clinical Research Portfolio. Study ID number 34396, www.ukctg.nihr.ac.uk.