Concentric lamellae - novel microanatomical structures in the articular calcified cartilage of mice.

The structure, ultrastructure and function of hyaline articular cartilage (HAC) and subchondral bone (SCB), and their involvement in the pathogenesis of osteoarthritis (OA) have been extensively researched. However, much less attention has been focused on the intervening tissue, articular calcified cartilage (ACC) and its role in the initiation and progression of OA. Using both light microscopy (LM) and transmission electron microscopy (TEM), a study of ACC in wild type (WT) mice, and mice with genetic osteoarthropathies (AKU) was undertaken to further understand the role played by ACC in the early stages of OA.Tibio-femoral joints were obtained from BALB/c WT and BALB/c AKU mice aged between 7 and 69 weeks. One joint was processed for routine histological analysis. The tip of the medial femoral condyle (MFC), which contained HAC, ACC, and SCB, was dissected from the contra-lateral joint and processed for TEM.In WT and AKU mice novel microanatomical structures, designated concentric lamellae, were identified surrounding chondrocytes in the ACC. The lamellae appeared to be laid down in association with advancement of the tidemark indicating they may be formed during calcification of cartilage matrix. The lamellae were associated with hypertrophic chondrocytes throughout the ACC.Novel microanatomical structures, termed concentric lamellae, which were present around hypertrophic chondrocytes in the ACC are described for the first time. Their apparent association with mineralisation, advancement of the tidemark, and greater abundance in a model of osteoarthropathy indicate their formation could be important in the pathogenesis of OA and AKU

Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; and fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of microtubules within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle

Collaborative conversation as a method for exploring multiple perspectives on 'community' and forms of knowledge in the Congruence Engine

This article explores collaborative conversation as a method to surface multiple perspectives on community engagement and forms of knowledge creation in the Congruence Engine project. Our exchanges naturally converged around four main areas: the multiple meanings of the term ‘community’ and the nature of these relationships; the modes and spaces for engagement; the different nature of knowledge emerging from these interactions; and, finally, a series of practical issues and challenges that can act as potential barriers. The article also reflects on the opportunities of dialogic writing to enable participatory, inclusive and polyvocal approaches in the development of a national collection

Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate

This is the final version of the article. Available from the publisher via the DOI in this record.Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.This work was funded by the United Kingdom Ministry of Defence. The mass spectral data described here were acquired on an Orbitrap Fusion mass spectrometer funded by National Institutes of Health grant 1S10OD010645-01A1

The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-alpha(1)-antitrypsin

α1-Antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming polymers that become trapped in inclusions of endoplasmic reticulum (ER). It is unclear whether these inclusions are connected to the main ER network in Z-α1-antitrypsin-expressing cells. Using live cell imaging, we found that despite inclusions containing an immobile matrix of polymeric α1-antitrypsin, small ER resident proteins can diffuse freely within them. Inclusions have many features to suggest they represent fragmented ER, and some are physically separated from the tubular ER network, yet we observed cargo to be transported between them in a cytosol-dependent fashion that is sensitive to N-ethylmaleimide and dependent on Sar1 and sec22B. We conclude that protein recycling occurs between ER inclusions despite their physical separation.—Dickens, J. A., Ordóñez, A., Chambers, J. E., Beckett, A. J., Patel, V., Malzer, E., Dominicus, C. S., Bradley, J., Peden, A. A., Prior, I. A., Lomas, D. A., Marciniak, S. J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin

Serial block-face scanning electron microscopy applied to study the trafficking of 8D3-coated gold nanoparticles at the blood-brain barrier

Due to the physical and physiological properties of the blood-brain barrier (BBB), the transport of neurotherapeutics from blood to brain is still a pharmaceutical challenge. We previously conducted a series of experiments to explore the potential of the anti-transferrin receptor 8D3 monoclonal antibody (mAb) to transport neurotherapeutics across the BBB. In that study, gold nanoparticles (AuNPs) were coated with the 8D3 antibody and administered intravenously to mice. Transmission electron microscopy was used and a two-dimensional (2D) image analysis was performed to detect the AuNPs in the brain capillary endothelial cells (BCECs) and brain parenchyma. In the present work, we determined that serial block-face scanning electron microscopy (SBF-SEM) is a useful tool to study the transcytosis of these AuNPs across the BBB in three dimensions and we, therefore, applied it to gain more knowledge of their transcellular trafficking. The resulting 3D reconstructions provided additional information on the endocytic vesicles containing AuNPs and the endosomal processing that occurs inside BCECs. The passage from 2D to 3D analysis reinforced the trafficking model proposed in the 2D study, and revealed that the vesicles containing AuNPs are significantly larger and more complex than described in our 2D study. We also discuss tradeoffs of using this technique for our application, and conclude that together with other volume electron microscopy imaging techniques, SBF-SEM is a powerful approach that is worth of considering for studies of drug transport across the BBB

Modular approach for bimodal antibacterial surfaces combining photo-switchable activity and sustained biocidal release

Photo-responsive antibacterial surfaces combining both on-demand photo-switchable activity and sustained biocidal release were prepared using sequential chemical grafting of nano-objects with different geometries and functions. The multi-layered coating developed incorporates a monolayer of near-infrared active silica-coated gold nanostars (GNS) decorated by silver nanoparticles (AgNP). This modular approach also enables us to unravel static and photo-activated contributions to the overall antibacterial performance of the surfaces, demonstrating a remarkable synergy between these two mechanisms. Complementary microbiological and imaging evaluations on both planktonic and surface-attached bacteria provided new insights on these distinct but cooperative effects

Human 13N-ammonia PET studies: the importance of measuring 13N-ammonia metabolites in blood

Dynamic 13N-ammonia PET is used to assess ammonia metabolism in brain, liver and muscle based on kinetic modeling of metabolic pathways, using arterial blood 13N-ammonia as input function. Rosenspire et al. (1990) introduced a solid phase extraction procedure for fractionation of 13N-content in blood into 13N-ammonia, 13N-urea, 13N-glutamine and 13N-glutamate. Due to a radioactive half-life for 13N of 10 min, the procedure is not suitable for blood samples taken beyond 5–7 min after tracer injection. By modifying Rosenspire’s method, we established a method enabling analysis of up to 10 blood samples in the course of 30 min. The modified procedure was validated by HPLC and by 30-min reproducibility studies in humans examined by duplicate 13N-ammonia injections with a 60-min interval. Blood data from a 13N-ammonia brain PET study (from Keiding et al. 2006) showed: (1) time courses of 13N-ammonia fractions could be described adequately by double exponential functions; (2) metabolic conversion of 13N-ammonia to 13N-metabolites were in the order: healthy subjects > cirrhotic patients without HE > cirrhotic patients with HE; (3) kinetics of initial tracer distribution in tissue can be assessed by using total 13N-concentration in blood as input function, whereas assessment of metabolic processes requires 13N-ammonia measurements

Second primary malignancies after treatment for malignant lymphoma

To determine the incidence and possible causes of second primary malignancies after treatment for Hodgkin's and Non-Hodgkin's lymphoma (HL and NHL). A cohort of 3764 consecutive patients diagnosed with HL or NHL between January 1970 and July 2001 was identified using the Sheffield Lymphoma Group database. A search was undertaken for all patients diagnosed with a subsequent primary malignancy. Two matched controls were identified for each case. Odds ratios were calculated to detect and quantify any risk factors in the cases compared to their matched controls. Mean follow-up for the cohort was 5.2 years. A total of 68 patients who developed second cancers at least 6 months after their primary diagnosis were identified, giving a crude incidence of 1.89% overall: 3.21% among the patients treated for HL, 1.32% in those treated for NHL. Most common were bronchial, breast, colorectal and haematological malignancies. High stage at diagnosis almost reached statistical significance in the analysis of just the NHL patients (odds ratio=3.48; P=0.068) after adjustment for other factors. Treatment modality was not statistically significant in any analysis. High stage at diagnosis of NHL may be a risk factor for developing a second primary cancer