Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.     

Photochemical oxidation of water and reduction of polyoxometalate anions at interfaces of water with ionic liquids or diethylether

Photoreduction of [P(2)W(18)O(62)](6-), [S(2)Mo(18)O(62)](4-), and [S(2)W(18)O(62)](4-) polyoxometalate anions (POMs) and oxidation of water occurs when water-ionic liquid and water-diethylether interfaces are irradiated with white light (275-750 nm) or sunlight. The ionic liquids (ILs) employed were aprotic ([Bmim]X; Bmim = (1-butyl-3-methylimidazolium, X = BF(4), PF(6)) and protic (DEAS = diethanolamine hydrogen sulphate; DEAP = diethanolamine hydrogen phosphate). Photochemical formation of reduced POMs at both thermodynamically stable and unstable water-IL interfaces led to their initial diffusion into the aqueous phase and subsequent extraction into the IL phase. The mass transport was monitored visually by color change and by steady-state voltammetry at microelectrodes placed near the interface and in the bulk solution phases. However, no diffusion into the organic phase was observed when [P(2)W(18)O(62)](6-) was photo-reduced at the water-diethylether interface. In all cases, water acted as the electron donor to give the overall process: 4POM + 2H(2)O + hν → 4POM(-) + 4H(+) + O(2). However, more highly reduced POM species are likely to be generated as intermediates. The rate of diffusion of photo-generated POM(-) was dependent on the initial concentration of oxidized POM and the viscosity of the IL (or mixed phase system produced in cases in which the interface is thermodynamically unstable). In the water-DEAS system, the evolution of dioxygen was monitored in situ in the aqueous phase by using a Clark-type oxygen sensor. Differences in the structures of bulk and interfacial water are implicated in the activation of water. An analogous series of reactions occurred upon irradiation of solid POM salts in the presence of water vapor.     

A patient with a large pulmonary saddle embolus eluding both clinical gestalt and validated decision rules

We report a patient with chest pain who was classified as having low risk for pulmonary embolism with clinical gestalt and accepted clinical decision rules. An inadvertently ordered D-dimer and abnormal result, however, led to the identification of a large saddle embolus. This case illustrates the fallibility of even well-validated decision aids and that an embolism missed by these tools is not necessarily low risk or indicative of a low clot burden.     

The relationship between clinical outcomes and quality of life in first‐episode mania: a longitudinal analysis

Objectives: Despite growing attention to the relationship between bipolar disorder (BD) and quality of life (QoL), there remains a lack of information about QoL in the early stages of BD, and about the course of QoL in people with BD over time. Here, we report on QoL and symptomatic outcomes over a 1.5‐year period in a Canadian sample of first‐episode mania patients. Methods: Patients (n = 63) with DSM‐IV‐TR BD type I recovering from a recent episode of mania were recruited from a university‐based hospital setting in Vancouver, BC, Canada and assessed at six monthly intervals for 18 months. In addition to symptomatic and cognitive assessments, two self‐report QoL scales [the Quality of Life Enjoyment and Satisfaction Questionnaire (Q‐LES‐Q) and the Medical Outcomes Study Short Form 36 (SF‐36)] were administered. Results: Baseline QoL scores were high, with mean Q‐LES‐Q scores at 70% of the maximum possible score; QoL continued to show a trend towards improvement over time. Multiple hierarchical regressions were used to explore predictors of QoL over time, finding that: (i) length of illness and severity of depressive symptoms at baseline predicted Q‐LES‐Q scores at both baseline and six months; (ii) the number of previous depressive episodes and severity of depression at baseline and 12 months all predicted QoL at 12 months; and (iii) only severity of depressive symptoms at 12 months predicted QoL at 18 months. Conclusions: Our observation that QoL in patients who have recently experienced an episode of mania can be relatively preserved offers hope, both for healthcare providers and for those newly diagnosed. Further, that severity of depressive symptoms even in the early stages of the disease was the consistent predictor of QoL suggests that depressive symptoms need to be aggressively treated to improve QoL.     

Alleviating Social Isolation and Loneliness among Older People

Social isolation and loneliness are often perceived as problems of old age. Although a wide range of services have evolved to combat such ‘negative’ experiences, little is known about the effectiveness, acceptability and accessibility of these interventions. We explored the inter-relationship between older people's and practitioners' perceptions of social isolation and loneliness, and their suggestions for effective interventions. Interviews demonstrated a disparity between definitions of loneliness and social isolation, and perceptions of acceptable interventions. Findings suggest that older people employ a range of coping strategies that are not taken into account when services are planned. Many services treat older people as a homogenous group, giving little consideration to the specific needs of those who are isolated and lonely, or to ways to reach them. Activities often evolve to meet the needs of current participants, rather than of the intended target group, excluding those who are truly isolated and lonely. We conclude that there is inequity between the ‘active lonely’ and those most in need in accessing and using services intended for isolated and lonely older people, because of the lack of need sand evidence-based practice.     

An experimental study exploring the relationship between the size of bacterial inoculum and bacterial adherence to prosthetic mesh

Background

Infection is a major concern with medical implants. Surgical meshes used for the repair of abdominal wall hernias are associated with wound infection rates ranging from 7 to 18 %. Although mesh infection is relatively rare, once a patient shows clinical signs of mesh infection, the surgeon may be required to remove the mesh, resulting in additional surgery, morbidity, and cost. The usual causative organisms associated with cases of mesh infection are Staphylococcus species. The first stage of implant infection is bacterial adherence to the biomaterial. An accurate assessment of adherent bacteria to medical prosthetics is therefore important in order to determine the infection risk associated with surgical implants.

Methods

This experimental study evaluated the relationship between the size of the bacterial inoculum and bacterial adherence to three commonly used hernia prosthetics (polypropylene, polyester, and ePTFE). Tenfold dilutions of S. epidermidis (Evans-ATCC 12228) and S. aureus (Rosenbach-ATCC 25923), created with phosphate-buffered saline, were used to inoculate each of the meshes in 3 ml of tryptone soya broth for 18 h at 37 °C, 95 % air/5 % CO₂. The number of viable bacteria in each dilution was calculated using a spot plate technique. The number of adherent bacteria to the meshes was counted using direct imaging analysis with scanning electron microscopy and expressed as a mean.

Results

One hundred eight mesh samples were analysed. The size of the bacterial inoculum of S. epidermidis significantly influenced the number of adherent bacteria to the mesh, and lower rates of adhesion were observed with smaller inoculums for all three meshes (polypropylene, p = 0.02; ePTFE p = 0.03; polyester p = 0.02). A similar, albeit less profound, pattern of results was observed with S. aureus. Bacterial adherence was observed with inoculum sizes as small as <10 bacteria.

Conclusions

The results demonstrate that even a very low number of bacterial inoculums can result in adherence to hernia biomaterials and that the level of adherence is directly related to the size of the inoculum. These in vitro results provide evidence that the size of the inoculum is important in the colonization of hernia biomaterials and demonstrate the importance of minimising the bacterial inoculum in the clinical setting.