Ultrastructural characterization of host-parasite interactions of Plasmodium coatneyi in rhesus macaques.

Plasmodium coatneyi has been proposed as an animal model for human Plasmodium falciparum malaria as it appears to replicate many aspects of pathogenesis and clinical symptomology. As part of the ongoing evaluation of the rhesus macaque model of severe malaria, a detailed ultrastructural analysis of the interaction between the parasite and both the host erythrocytes and the microvasculature was undertaken. Tissue (brain, heart and kidney) from splenectomized rhesus macaques and blood from spleen-intact animals infected with P. coatneyi were examined by electron microscopy. In all three tissues, similar interactions (sequestration) between infected red blood cells (iRBC) and blood vessels were observed with evidence of rosette and auto-agglutinate formation. The iRBCs possessed caveolae similar to P. vivax and knob-like structures similar to P. falciparum. However, the knobs often appeared incompletely formed in the splenectomized animals in contrast to the intact knobs exhibited by spleen intact animals. Plasmodium coatneyi infection in the monkey replicates many of the ultrastructural features particularly associated with P. falciparum in humans and as such supports its use as a suitable animal model. However, the possible effect on host­parasite interactions and the pathogenesis of disease due to the use of splenectomized animals needs to be taken into consideration.

Breast Angiosarcoma Surveillance Study: UK national audit of management and outcomes of angiosarcoma of the breast and chest wall.

BACKGROUND: Breast angiosarcomas are rare tumours of vascular origin. Secondary angiosarcoma occurs following radiotherapy for breast cancer. Angiosarcomas have high recurrence and poor survival rates. This is concerning owing to the increasing use of adjuvant radiotherapy for the treatment of invasive breast cancer and ductal cancer in situ (DCIS), which could explain the rising incidence of angiosarcoma. Outcome data are limited and provide a poor evidence base for treatment. This paper presents a national, trainee-led, retrospective, multicentre study of a large angiosarcoma cohort. METHODS: Data for patients with a diagnosis of breast/chest wall angiosarcoma between 2000 and 2015 were collected retrospectively from 15 centres. RESULTS: The cohort included 183 patients with 34 primary and 149 secondary angiosarcomas. Median latency from breast cancer to secondary angiosarcoma was 6 years. Only 78.9 per cent of patients were discussed at a sarcoma multidisciplinary team meeting. Rates of recurrence were high with 14 of 28 (50 per cent ) recurrences in patients with primary and 80 of 124 (64.5 per cent ) in those with secondary angiosarcoma at 5 years. Many patients had multiple recurrences: total of 94 recurrences in 162 patients (58.0 per cent). Median survival was 5 (range 0-16) years for patients with primary and 5 (0-15) years for those with secondary angiosarcoma. Development of secondary angiosarcoma had a negative impact on predicted breast cancer survival, with a median 10-year PREDICT prognostic rate of 69.6 per cent, compared with 54.0 per cent in the observed cohort. CONCLUSION: A detrimental impact of secondary angiosarcoma on breast cancer survival has been demonstrated. Although not statistically significant, almost all excess deaths were attributable to angiosarcoma. The increased use of adjuvant radiotherapy to treat low-risk breast cancer and DCIS is a cause for concern and warrants further study.

The provision of a time-critical elective surgical service during the COVID-19 Crisis: a UK experience.

INTRODUCTION: With the emergence of the COVID-19 pandemic, all elective surgery was temporarily suspended in the UK, allowing for diversion of resource to manage the anticipated surge of critically unwell patients. Continuing to deliver time-critical surgical care is important to avoid excess morbidity and mortality from pathologies unrelated to COVID-19. We describe the implementation and short-term surgical outcomes from a system to deliver time-critical elective surgical care to patients during the COVID-19 pandemic. MATERIALS AND METHODS: A protocol for the prioritisation and safe delivery of time-critical surgery at a COVID-19 'clean' site was implemented at the Nuffield Health Exeter Hospital, an independent sector hospital in the southwest of England. Outcomes to 30 days postoperatively were recorded, including unplanned admissions after daycase surgery, readmissions and complications, as well as the incidence of perioperative COVID-19 infection in patients and staff. RESULTS: A total of 128 surgical procedures were performed during a 31-day period by a range of specialties including breast, plastics, urology, gynaecology, vascular and cardiology. There was one unplanned admission and and two readmissions. Six complications were identified, and all were Clavien-Dindo grade 1 or 2. All 128 patients had preoperative COVID-19 swabs, one of which was positive and the patient had their surgery delayed. Ten patients were tested for COVID-19 postoperatively, with none testing positive. CONCLUSION: This study has demonstrated the implementation of a safe system for delivery of time-critical elective surgical care at a COVID-19 clean site. Other healthcare providers may benefit from implementation of similar methodology as hospitals plan to restart elective surgery.

Significantly reducing implant loss rates in immediate implant-based breast reconstruction: A protocol and completed audit of quality assurance.

INTRODUCTION: Immediate implant-based breast reconstruction (IBR) rates have increased considerably with the advent of acellular dermal matrices. Implant loss is a significant complication and is costly to patients and the NHS. National Mastectomy and Breast Reconstruction Audit and Implant-Based Breast Reconstruction Audit data have demonstrated national implant loss rate of 9% at 3 months. National Oncoplastic Guidelines for Best Practice cite a < 5% target. We aimed to reduce implant loss by introducing a protocol with pre-, intra- and post-operative interventions. METHODS: Audit of IBR at a single oncoplastic breast unit was commenced and implant loss at 3 months was recorded (May 2012-July 2014). Patients were identified from a prospectively maintained database, and case notes were examined by identifying factors associated with implant loss. A team involving microbiology, theatre staff, infection control and surgeons was established. A novel, evidence-based intervention bundle, including more than 25 protocol changes, was introduced. Prospective re-audit of IBR (April 2015-December 2017) was completed following introduction of the new protocol and implant loss was recorded at 3 months. RESULTS: The first retrospective audit of 77 reconstructions (54 patients) demonstrated 11 implant losses at 3 months (14%). Re-audit, post-intervention, comprised 129 reconstructions (106 patients) with no implant loss at 3 months. Fisher's exact analysis revealed statistically significant reduction in implant loss rate (P < 0.00001) following protocol introduction. CONCLUSIONS: Implant loss rate following IBR can be reduced to an exceptionally low level, well below national targets, by adhering to this evidence-based intervention bundle. Our protocol could improve outcomes nationally.

Electron microscopy in the diagnosis of Ehlers-Danlos syndromes: correlation with clinical and genetic investigations.

BACKGROUND: The Ehlers-Danlos syndromes (EDS) consist of 13 subtypes with overlapping features including joint hypermobility, skin and vascular fragility and generalized connective tissue friability. As DNA analysis has become the gold standard for investigation of EDS, transmission electron microscopy (TEM) in clinical practice is decreasing. However, owing to the use of next-generation sequencing, the frequency of variants of uncertain significance (VUS) identified using DNA analysis is increasing. We hypothesized that TEM can provide evidence for or against pathogenicity of VUS. OBJECTIVES: The aim of this study was to evaluate the role of TEM in the diagnosis of EDS subtypes. METHODS: Data were collected from patients who underwent a skin biopsy between October 2012 and March 2017 at the London EDS National Diagnostic Service. TEM biopsies were categorized as 'normal' or 'abnormal' according to the description and conclusion in the TEM reports. Definitive diagnoses were reached via a combination of clinical features, structural and functional studies and DNA investigations. RESULTS: The analysis included 177 patients, comprising 30 abnormal and 147 normal TEM reports. A definitive diagnosis of monogenic EDS subtypes was made in 24 patients. Overall, 17 of these 24 patients (71%) had an abnormal biopsy report and seven (29%) had a normal biopsy report. No TEM findings were specifically associated with any EDS subtype, although collagen flowers were present in most patients with a genetically confirmed diagnosis of classical EDS. CONCLUSIONS: TEM analysis of collagen structure may have the potential to provide evidence for or against the pathogenicity of a VUS, but more work is needed to establish a clear role for TEM in this process. What's already known about this topic? Collagen fibril abnormalities can be seen in several Ehlers-Danlos syndrome (EDS) subtypes. What does this study add? This study provides clinical data, transmission electron microscopy (TEM) data and molecular data of one of the largest groups of patients suspected to have a monogenetic EDS subtype. No TEM findings were specifically associated with an EDS subtype. There was a higher percentage (71%) of abnormal biopsy findings in patients with a definitive diagnosis of a monogenetic EDS subtype and where a class 4/5 genetic variant was present.

New stiletto flies in the genus Anabarhynchus Macquart (Diptera: Therevidae) from Kangaroo Island, South Australia.

Anabarhynchus Macquart 1848 is a species-rich genus of stiletto flies (Diptera: Therevidae) belonging to the subfamily Therevinae, with over 113 species described from Australia. These flies are diverse and abundant in Australia's eucalypt woodland and mallee habitats. Here we describe, diagnose and illustrate a further three new Anabarhynchus species in the genus as follows: A. aurantilateralis sp. nov. and A. halmaturinus sp. nov., and A. venabrunneis sp. nov., from Kangaroo Island. These new species bring the total number of described Australian species in the genus to 116, with 13 of these known to occur on Kangaroo Island.

Two patients with Ehlers-Danlos syndrome type VIII with unexpected hoarseness.

Ehlers-Danlos syndrome (EDS) encompasses a genetically and clinically heterogeneous group of connective tissue disorders, characterized by joint hypermobility, skin hyperextensibility and tissue fragility. It is a rare condition, and inheritance is either autosomal dominant or recessive. Previously grouped into 11 different subtypes, with increasing knowledge of the underlying molecular defects, it was reclassified in 1997 into 6 major groups, with type VIII excluded from this classification. Type VIII EDS is a very rare subtype, characterized by severe, early-onset periodontitis, skin fragility and abnormal scarring. Voice abnormalities have occasionally been described in other forms of the condition, and may be due to defects in the collagen of the vocal ligament. We report two cases of patients with EDS type VIII and hoarseness.

Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.

The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of 'standard' 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.

Stable isotope imaging of biological samples with high resolution secondary ion mass spectrometry and complementary techniques.

Stable isotopes are ideal labels for studying biological processes because they have little or no effect on the biochemical properties of target molecules. The NanoSIMS is a tool that can image the distribution of stable isotope labels with up to 50 nm spatial resolution and with good quantitation. This combination of features has enabled several groups to undertake significant experiments on biological problems in the last decade. Combining the NanoSIMS with other imaging techniques also enables us to obtain not only chemical information but also the structural information needed to understand biological processes. This article describes the methodologies that we have developed to correlate atomic force microscopy and backscattered electron imaging with NanoSIMS experiments to illustrate the imaging of stable isotopes at molecular, cellular, and tissue scales. Our studies make it possible to address 3 biological problems: (1) the interaction of antimicrobial peptides with membranes; (2) glutamine metabolism in cancer cells; and (3) lipoprotein interactions in different tissues.

Melting during late-stage rifting in Afar is hot and deep.

Investigations of a variety of continental rifts and margins worldwide have revealed that a considerable volume of melt can intrude into the crust during continental breakup, modifying its composition and thermal structure. However, it is unclear whether the cause of voluminous melt production at volcanic rifts is primarily increased mantle temperature or plate thinning. Also disputed is the extent to which plate stretching or thinning is uniform or varies with depth with the entire continental lithospheric mantle potentially being removed before plate rupture. Here we show that the extensive magmatism during rifting along the southern Red Sea rift in Afar, a unique region of sub-aerial transition from continental to oceanic rifting, is driven by deep melting of hotter-than-normal asthenosphere. Petrogenetic modelling shows that melts are predominantly generated at depths greater than 80 kilometres, implying the existence of a thick upper thermo-mechanical boundary layer in a rift system approaching the point of plate rupture. Numerical modelling of rift development shows that when breakup occurs at the slow extension rates observed in Afar, the survival of a thick plate is an inevitable consequence of conductive cooling of the lithosphere, even when the underlying asthenosphere is hot. Sustained magmatic activity during rifting in Afar thus requires persistently high mantle temperatures, which would allow melting at high pressure beneath the thick plate. If extensive plate thinning does occur during breakup it must do so abruptly at a late stage, immediately before the formation of the new ocean basin.

Impact of age extension to include 47-49 year old women on the workload of the surgical department of a single Breast Cancer Screening Unit--The first non-randomized experience in UK.

INTRODUCTION: The aim of this study was to assess the impact on the surgical unit of the first year (prevalence screening) of non-randomized invitations to 47-49 year old women for breast screening, from a single breast screening unit. METHODS: All women undergoing surgery in the age group 47-49 years, referred via screening were identified and the increased workload analysed. RESULTS: 4250 (76%) women were screened of the 5624 invited. 396 women were recalled, of whom 88 (22%) underwent a core biopsy. 32 patients required surgical intervention. 20 patients (62.5%) were confirmed to have either DCIS (6 patients) or invasive malignancy (14 patients). They required 37 theatres attendances requiring 42 operations. 16 wire guided wide local excisions (14 with sentinel node biopsy), 7 mastectomies (2 with sentinel node biopsy; 1 with axillary clearance), 6 margin re-excisions, 1 tissue expander insertion and removal, 3 Latissimus Dorsi with implant and 2 TRAM reconstructions. Other cases include haematoma drainage, scar revisions and nipple reconstructions. This group generated 100 NHS surgical outpatient consultations (78 breast and 22 plastic surgery). 12 patients (37.5%) underwent surgery for a B3 vacuum result; 10 underwent wire guided and 1 ultrasound guided skin marked excision biopsy. 1 patient was treated privately. This group generated 25 NHS surgical outpatients consultations. CONCLUSIONS: This study highlights the impact of the 47-49 year age extension within the breast screening programme on the workload of the surgical department of a UK Breast Cancer Screening Unit offering non-randomized invitations. The study will inform other surgical units of expected workload when age extension is fully implemented.

Potential of recombinant opa proteins as vaccine candidates against hyperinvasive meningococci.

Neisseria meningitidis causes half a million cases of septicemia and meningitis globally each year. The opacity (Opa) integral outer membrane proteins from N. meningitidis are polymorphic and highly immunogenic. Particular combinations of Opa proteins are associated with the hyperinvasive meningococcal lineages that have caused the majority of serogroup B and C meningococcal disease in industrialized countries over the last 60 years. For the first time, this genetic structuring of a diverse outer membrane protein family has been used to select a novel combination of representative antigens for immunogenicity testing. Fourteen recombinant Opa variants were produced and used in murine immunizations inducing an increase in specific antimeningococcal total IgG levels. All 14 Opa proteins elicited bactericidal antibodies against at least one hyperinvasive meningococcal isolate, and most isolates from each hyperinvasive lineage were killed by at least one Opa antiserum at a titer of 1:16 or greater. Cross-reactive bactericidal antibody responses were observed among clonal complexes. A theoretical coverage of 90% can be achieved by using a particular combination of 6 Opa proteins against an isolate collection of 227 recent United Kingdom disease cases. This study indicates the potential of Opa proteins to provide broad coverage against multiple meningococcal hyperinvasive lineages.

Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo.

Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7(-/-) erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119(+)/CD71(High) stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.

Identification of faecal transmission of Toxoplasma gondii: Small science, large characters.

The first clue to the elucidation of the complete life cycle of Toxoplasma gondii was the identification of an infectious form in cat faeces that could be transmitted orally and could survive in the external environment for extended periods. This personal review describes the scientist (W.M. Hutchison) and the background to the initial discovery and covers the period to the complete elucidation of the life cycle of T. gondii.

Urban trench fever presenting as culture-negative endocarditis.

A young Russian man presented with increasing shortness of breath and signs of worsening aortic regurgitation. A diagnosis of infective endocarditis was made before emergency valve replacement. The infective cause was not discovered by routine culture but was suggested by electron microscopy and confirmed by serology and PCR testing.

Corticotomy-/osteotomy-assisted tooth movement microCTs differ.

Corticotomy-assisted and osteotomy-assisted tooth movement involves surgical incisions through the alveolar bone. To ascertain whether teeth move by distraction osteogenesis or by regional accelerated phenomenon (RAP), we randomly assigned 30 Sprague-Dawley rats to one of 5 experimental groups: corticotomy alone, corticotomy-assisted tooth movement, osteotomy alone, osteotomy-assisted tooth movement, or tooth movement alone. Each animal was imaged by microtomography immediately after surgery, after 21 days, and after 2 months. After 21 days, regional accelerated phenomenon was observed in the alveolar bone of the corticotomy-treated animals and distraction osteogenesis in the osteotomy-assisted tooth movement animals. Pixel count data were analyzed by nested ANOVA for 5 experimental groups, split-mouth controls, 3 levels along the root, and 5 sites per level. The most demineralized sites after 21 days differed for each of the experimental groups. Our study indicates that osteotomies and corticotomies induce different alveolar bone reactions, which can be exploited for tooth movement.

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.