iRFP (near-infrared fluorescent protein) imaging of subcutaneous and deep tissue tumours in mice highlights differences between imaging platforms.

BACKGROUND: In vivo imaging using fluorescence is used in cancer biology for the detection, measurement and monitoring of tumours. This can be achieved with the expression of fluorescent proteins such as iRFP, which emits light at a wavelength less attenuated in biological tissues compared to light emitted by other fluorescent proteins such as GFP or RFP. Imaging platforms capable of detecting fluorescent tumours in small animals have been developed but studies comparing the performance of these platforms are scarce. RESULTS: Through access to three platforms from Xenogen, Bruker and Li-Cor, we compared their ability to detect iRFP-expressing subcutaneous tumours as well as tumours localised deeper within the body of female NSG mice. Each platform was paired with proprietary software for image analyse, but the output depends on subjective decisions from the user. To more objectively compare platforms, we developed an 'in house' software-based approach which results in lower measured variability between mice. CONCLUSIONS: Our comparisons showed that all three platforms allowed for reliable detection and monitoring of subcutaneous iRFP tumour growth. The biggest differences between platforms became apparent when imaging deeper tumours with the Li-Cor platform detecting most tumours and showing the highest dynamic range.

Asymmetry indicates more severe and active disease in Graves' orbitopathy: results from a prospective cross-sectional multicentre study.

PURPOSE: Patients with Graves' orbitopathy can present with asymmetric disease. The aim of this study was to identify clinical characteristics that distinguish asymmetric from unilateral and symmetric Graves' orbitopathy. METHODS: This was a multi-centre study of new referrals to 13 European Group on Graves' Orbitopathy (EUGOGO) tertiary centres. New patients presenting over a 4 month period with a diagnosis of Graves' orbitopathy were included. Patient demographics were collected and a clinical examination was performed based on a previously published protocol. Patients were categorized as having asymmetric, symmetric, and unilateral Graves' orbitopathy. The distribution of clinical characteristics among the three groups was documented. RESULTS: The asymmetric group (n = 83), was older than the symmetric (n = 157) group [mean age 50.9 years (SD 13.9) vs 45.8 (SD 13.5), p = 0.019], had a lower female to male ratio than the symmetric and unilateral (n = 29) groups (1.6 vs 5.0 vs 8.7, p < 0.001), had more active disease than the symmetric and unilateral groups [mean linical Activity Score 3.0 (SD 1.6) vs 1.7 (SD 1.7), p < 0.001 vs 1.3 (SD 1.4), p < 0.001] and significantly more severe disease than the symmetric and unilateral groups, as measured by the Total Eye Score [mean 8.8 (SD 6.6) vs 5.3 (SD 4.4), p < 0.001, vs 2.7 (SD 2.1), p < 0.001]. CONCLUSION: Older age, lower female to male ratio, more severe, and more active disease cluster around asymmetric Graves' orbitopathy. Asymmetry appears to be a marker of more severe and more active disease than other presentations. This simple clinical parameter present at first presentation to tertiary centres may be valuable to clinicians who manage such patients.

Differences in gene regulation in a tephritid model of prezygotic reproductive isolation.

The two tephritid fruit fly pests, Bactrocera tryoni and Bactrocera neohumeralis, are unusually well suited to the study of the genetics of reproductive isolating mechanisms. Sequence difference between the species is no greater than between a pair of conspecific Drosophila melanogaster populations. The two species exist in close sympatry, yet do not hybridize in the field, apparently kept separate by a strong premating isolation mechanism involving the time of day at which mating occurs. This spurred us to search for key genes for which time of day expression is regulated differently between the species. Using replicated, quantitative transcriptomes from head tissues of males of the two species, sampled in the day and night, we identified 141 transcripts whose abundance showed a significant interaction between species and time of day, indicating a difference in gene regulation. The brain transcripts showing this interaction were enriched for genes with a neurone function and 90% of these were more abundant at night than day in B. tryoni. Features of the expression patterns suggest that there may be a difference in the regulation of sleep-wake cycles between the species. In particular several genes, which in D. melanogaster are expressed in circadian pacemaker cells, are promising candidates to further explore the genetic differentiation involved in this prezygotic reproductive isolation mechanism.

The perceptions of teenagers, young adults and professionals in the participation of bone cancer clinical trials.

The reasons why teenagers and young adults (TYA) with cancer do, or do not, participate in clinical trials is not wholly understood. We explored the perceptions and experiences of young people with bone cancer, and health professionals involved in their care, with regard to participation in two clinical trials. We conducted semi-structured interviews using narrative inquiry with 21 young people aged 15-24 years and 18 health professionals. New understandings emerged about perceptions of, and factors that influence participation in, clinical trials. These include perceptions about the importance and design of the clinical trial, communicating with young people in an age-specific manner, using language young people are comfortable with, support from family, peers and specialists in teenage and young adult cancer care. We conclude that addressing these factors may increase acceptability of clinical trials and the trial design for TYA with cancer and ultimately improve their participation. Qualitative research has an important role in making explicit the perceptions and practices that ensure trials are patient-centred, appropriate and communicated effectively to TYA. Translating knowledge gained into routine practice, will go some way in ensuring that the disparities affecting this population are more fully understood.

Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species.

BACKGROUND: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. RESULTS: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. CONCLUSIONS: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

Expanding the genotypic spectrum of Perrault syndrome.

Perrault syndrome is a rare autosomal recessive disorder characterized by sensorineural hearing loss (SNHL) in both sexes and primary ovarian insufficiency in 46, XX karyotype females. Biallelic variants in five genes are reported to be causative: HSD17B4, HARS2, LARS2, CLPP and C10orf2. Here we present eight families affected by Perrault syndrome. In five families we identified novel or previously reported variants in HSD17B4, LARS2, CLPP and C10orf2. The proband from each family was whole exome sequenced and variants confirmed by Sanger sequencing. A female was compound heterozygous for a known, p.(Gly16Ser) and novel, p.(Val82Phe) variant in D-bifunctional protein (HSD17B4). A family was homozygous for mitochondrial leucyl aminocyl tRNA synthetase (mtLeuRS) (LARS2) p.(Thr522Asn), previously associated with Perrault syndrome. A further family was compound heterozygous for mtLeuRS, p.(Thr522Asn) and a novel variant, p.(Met117Ile). Affected individuals with LARS2 variants had low frequency SNHL, a feature previously described in Perrault syndrome. A female with significant neurological disability was compound heterozygous for p.(Arg323Gln) and p.(Asn399Ser) variants in Twinkle (C10orf2). A male was homozygous for a novel variant in CLPP, p.(Cys144Arg). In three families there were no putative pathogenic variants in these genes confirming additional disease-causing genes remain unidentified. We have expanded the spectrum of disease-causing variants associated with Perrault syndrome.

Serum 25-hydroxyvitamin D concentration and its determinants in the very old: the Newcastle 85+ Study.

SUMMARY: Data on vitamin D status in very old adults are lacking. The aim of this study was to assess 25-hydroxyvitamin D [25(OH)D] concentrations and its predictors in 775 adults aged 85 years old living in North-East England. Low 25(OH)D was alarmingly high during winter/spring months, but its biological significance is unknown. INTRODUCTION: Despite recent concerns about the high prevalence of vitamin D deficiency in much of the British adult and paediatric population, there is a dearth of data on vitamin D status and its predictors in very old adults. The objective of the present study was to describe vitamin D status and its associated factors in a broadly representative sample of very old men and women aged 85 years living in the North East of England (55° N). METHODS: Serum concentrations of 25-hydroxyvitamin D [25(OH)D] were analysed in 775 participants in the baseline phase of the Newcastle 85+ cohort study. Season of blood sampling, dietary, health, lifestyle and anthropometric data were collected and included as potential predictors of vitamin D status in ordinal regression models. RESULTS: Median serum 25(OH)D concentrations were 27, 45, 43 and 33 nmol/L during spring, summer, autumn and winter, respectively. The prevalence of vitamin D deficiency according to North American Institute of Medicine guidelines [serum 25(OH)D <30 nmol/L] varied significantly with season with the highest prevalence observed in spring (51%) and the lowest prevalence observed in autumn (23%; P < 0.001). Reported median (inter-quartile range) dietary intakes of vitamin D were very low at 2.9 (1.2-3.3) µg/day. In multivariate ordinal regression models, non-users of either prescribed or non-prescribed vitamin D preparations and winter and spring blood sampling were associated with lower 25(OH)D concentrations. Dietary vitamin D intake, disability score and disease count were not independently associated with vitamin D status in the cohort. CONCLUSION: There is an alarming high prevalence of vitamin D deficiency (<30 nmol/L) in 85-year-olds living in North East England at all times of the year but particularly during winter and spring. Use of vitamin D containing preparations (both supplements and medications) appeared to be the strongest predictor of 25(OH)D concentrations in these very old adults.

How young people describe the impact of living with and beyond a cancer diagnosis: feasibility of using social media as a research method.

OBJECTIVE: Young people with cancer exhibit unique needs. During a time of normal physical and psychological change, multiple disease and treatment-related symptoms cause short and long-term physical and psychosocial effects. Little is known about how young people cope with the impact of cancer and its treatment on daily routines and their strategies to manage the challenges of cancer and treatments. We aimed to determine how young people describe these challenges through a social media site. METHODS: Using the principles of virtual ethnography and watching videos on a social media site we gathered data from young people describing their cancer experience. Qualitative content analysis was employed to analyse and interpret the narrative from longitudinal 'video diaries' by 18 young people equating to 156 films and 27 h and 49 min of recording. Themes were described then organized and clustered into typologies grouping commonalities across themes. RESULTS: Four typologies emerged reflective of the cancer trajectory: treatment and relenting side effects, rehabilitation and getting on with life, relapse, facing more treatment and coming to terms with dying. CONCLUSIONS: This study confirms the need for young people to strive towards normality and creating a new normal, even where uncertainty prevailed. Strategies young people used to gain mastery over their illness and the types of stories they choose to tell provide the focus of the main narrative. Social Media sites can be examined as a source of data, to supplement or instead of more traditional routes of data collection known to be practically challenging with this population. Copyright © 2016 John Wiley & Sons, Ltd.

CTLA-4 as a genetic determinant in autoimmune Addison's disease.

In common with several other autoimmune diseases, autoimmune Addison's disease (AAD) is thought to be caused by a combination of deleterious susceptibility polymorphisms in several genes, together with undefined environmental factors and stochastic events. To date, the strongest genomic association with AAD has been with alleles at the HLA locus, DR3-DQ2 and DR4. The contribution of other genetic variants has been inconsistent. We have studied the association of 16 single-nucleotide polymorphisms (SNPs) within the CD28-CTLA-4-ICOS genomic locus, in a cohort comprising 691 AAD patients of Norwegian and UK origin with matched controls. We have also performed a meta-analysis including 1002 patients from European countries. The G-allele of SNP rs231775 in CTLA-4 is associated with AAD in Norwegian patients (odds ratio (OR)=1.35 (confidence interval (CI) 1.10-1.66), P=0.004), but not in UK patients. The same allele is associated with AAD in the total European population (OR=1.37 (CI 1.13-1.66), P=0.002). A three-marker haplotype, comprising PROMOTER_1661, rs231726 and rs1896286 was found to be associated with AAD in the Norwegian cohort only (OR 2.43 (CI 1.68-3.51), P=0.00013). This study points to the CTLA-4 gene as a susceptibility locus for the development of AAD, and refines its mapping within the wider genomic locus.

Rethinking antithyroid drugs in pregnancy.

Uncontrolled hyperthyroidism in pregnancy poses a risk to both mother and foetus, and the optimal treatment strategy in this setting remains elusive. Instigation of pharmacological therapy or an alternative intervention during pregnancy requires careful consideration, and the evidence that has underpinned our choice of antithyroid drug has not been robust. Recent research developments have prompted us to question our practice, and reconsider our approach to managing this patient group.

Consensus statement on the diagnosis, treatment and follow-up of patients with primary adrenal insufficiency.

Primary adrenal insufficiency (PAI), or Addison's disease, is a rare, potentially deadly, but treatable disease. Most cases of PAI are caused by autoimmune destruction of the adrenal cortex. Consequently, patients with PAI are at higher risk of developing other autoimmune diseases. The diagnosis of PAI is often delayed by many months, and most patients present with symptoms of acute adrenal insufficiency. Because PAI is rare, even medical specialists in this therapeutic area rarely manage more than a few patients. Currently, the procedures for diagnosis, treatment and follow-up of this rare disease vary greatly within Europe. The common autoimmune form of PAI is characterized by the presence of 21-hydroxylase autoantibodies; other causes should be sought if no autoantibodies are detected. Acute adrenal crisis is a life-threatening condition that requires immediate treatment. Standard replacement therapy consists of multiple daily doses of hydrocortisone or cortisone acetate combined with fludrocortisone. Annual follow-up by an endocrinologist is recommended with the focus on optimization of replacement therapy and detection of new autoimmune diseases. Patient education to enable self-adjustment of dosages of replacement therapy and crisis prevention is particularly important in this disease. The authors of this document have collaborated within an EU project (Euadrenal) to study the pathogenesis, describe the natural course and improve the treatment for Addison's disease. Based on a synthesis of this research, the available literature, and the views and experiences of the consortium's investigators and key experts, we now attempt to provide a European Expert Consensus Statement for diagnosis, treatment and follow-up.

Vasoactive Intestinal Peptide Receptor, CRTH2, Antagonist Treatment Improves Eosinophil and Mast Cell-Mediated Esophageal Remodeling and Motility Dysfunction in Eosinophilic Esophagitis.

BACKGROUND AND AIMS: Ultrasonography has shown that eosinophils accumulate in each segment of the esophageal mucosa in human EoE, ultimately promoting esophageal motility dysfunction; however, no mechanistic evidence explains how or why this accumulation occurs. METHODS: Quantitative PCR, ELISA, flow cytometry, immunostaining, and immunofluorescence analyses were performed using antibodies specific to the related antigens and receptors. RESULTS: In deep esophageal biopsies of EoE patients, eosinophils and mast cells accumulate adjacent to nerve cell-derived VIP in each esophageal segment. qRT-PCR analysis revealed five- to sixfold increases in expression levels of VIP, CRTH2, and VAPC2 receptors and proteins in human blood- and tissue-accumulated eosinophils and mast cells. We also observed a significant correlation between mRNA CRTH2 levels and eosinophil- and nerve cell-derived VIPs in human EoE (p < 0.05). We provide evidence that eosinophil and mast cell deficiency following CRTH2 antagonist treatment improves motility dysfunction in a chronic DOX-inducible CC10-IL-13 murine model of experimental EoE. CONCLUSIONS: CRTH2 antagonist treatment is a novel therapeutic strategy for inflammatory cell-induced esophageal motility dysfunction in IL-13-induced chronic experimental EoE.

Evaluating the efficacy of internal teat sealants at dry-off for the prevention of new intra-mammary infections during the dry-period or clinical mastitis during early lactation in dairy cows: A systematic review update and sequential meta-analysis.

A systematic review and Bayesian sequential pair-wise meta-analyses were conducted to assess the efficacy of internal teat sealants (ITS) administered at dry-off in comparison to no treatment for preventing new intramammary infections (IMI) and clinical mastitis (CM) in dairy cattle. This work updated a previous systematic review and network meta-analysis conducted in 2019 but employed a narrowed scope and eligibility. The updated eligibility included studies that used ITS without concurrent therapy compared to a no treatment control (NTC), a study population of dairy cows or prepartum heifers, controlled trial design, and assessed one of the following outcomes: incidence of new IMI at calving or CM during the first 30 days in milk (DIM). Risk of bias was assessed through the Cochrane Risk of Bias 2.0 tool. Evidence quality was assessed using Grading of Recommendations Assessment, Development, and Evaluation (GRADE). There were 141 potentially relevant records identified from the updated search conducted on April 29, 2021, with a publication date restriction of 2018 or later; one study passed full-text screening and was included. Of the 32 studies included in the previous review, 12 studies were relevant after applying the modified eligibility criteria, totaling 13 studies included in this review (12 addressing IMI at calving outcome, 4 addressing CM at 30 DIM outcome). Sequential meta-analysis was conducted for both outcomes in R 3.6.0. Decisions for stopping were assessed at each analysis for intervention effect or futility in finding an effect based on a priori minimum clinically relevant values (ORδ =0.5, 0.75). ITS at dry-off significantly reduced odds of new IMI at calving compared to NTC at the second meta-analysis (OR2 =0.27, 95% CI=0.22-0.34), and onward (OR12 =0.29, 95% CI=0.27-0.32). For CM at 30 DIM, significance was reached at the second meta-analysis (OR2 =0.59, 95% CI=0.47-0.73), and onward (OR3 =0.47, 95% CI=0.42-0.51). Stopping for effect occurred at the second analysis in both outcomes and ORδs, but low-quality evidence and heterogeneity concerns were noted. A continuity-correction to include zero-event CM studies showed significance at the third meta-analysis (OR3 =0.79, 95% CI=0.73-0.86), stopping for effect at the fourth for ORδ = 0.75 (OR4 =0.77, 95% CI=0.72-0.83), and stopping for futility at the second for ORδ = 0.5 (OR2 =0.94, 95% CI=0.75-1.20), but the main CM analysis was considered more appropriate due to the sensitivity analysis' very low-quality evidence assessment. Based on sequential evidence available, sufficient research currently exists for practical use, and cessation of future research until substantial changes to ITS application occur may be appropriate.

A scoping exercise of favourable characteristics of professionals working in teenage and young adult cancer care: 'thinking outside of the box'.

A scoping exercise to define the preferred competencies of professionals involved in teenage and young adult (TYA) cancer care. Data were generated during two workshops with health professionals. In groups, they ranked skills, knowledge and attitudes, previously identified through a literature search, onto a diamond template. Data were also used from an education day with TYA professionals, who generated lists of key skills, knowledge and attitudes. Individually, professionals then selected the top five areas of competence to care for young people with cancer. The workshops generated three diamonds, which exhibited agreement of 13 principle skills, knowledge and attitudes. The top two being: 'expertise in treating paediatric and adult cancers' and 'understanding cancer'. The data from the education day suggested communication, technical knowledge and teamwork as being core role features for professionals who care for young people with cancer. Integration of both datasets; one derived inductively, the other deductively provides a comprehensive outline of core skills health professionals require to be proficient in young people's cancer care. These results will form the basis of future discussion around workforce strategies and inform a Delphi survey.

Screening of platelets for bacterial contamination at the Welsh Blood Service.

BACKGROUND AND OBJECTIVE: This report details the results of the implementation of a bacterial screening system at the Welsh Blood Service and provides an estimate of the levels of bacterial contamination at the time of sampling. MATERIALS AND METHODS: Apheresis (Caridian BCT) and buffy coat-derived pooled platelet components were sampled on day 1 for bacterial contamination and the sample was monitored throughout the lifespan of the platelet component. Unused platelet components were re-tested to determine the effectiveness of the screening. Results from the BacT/ALERT are uploaded to the in-house Blood Establishment Computer System (BECS) every 12 min. Positive alerts are automatically sent to staff, facilitating a timely intervention. RESULTS: Between February 2003 and March 2010 the screening system tested 54 828 platelets and detected 257 (1 in 213) initial positives of which 35 (1 in 1567, 0·06%) were confirmed [95% confidence interval (CI), 0·04-0·08%]. Additionally, screening of 6438 unused platelet components detected another 6 (1 in 1073, 0·09%) confirmed positives not detected during initial testing (95% CI, 0·02-0·16%). Analysis of the data suggests that on day 1 the number of bacteria in such platelet component packs was between 5 and 62 cfus total. Day 1 culture has a sensitivity of 40%. CONCLUSIONS: The bacterial screening system has removed a significant number, but not all bacterially contaminated platelet components from the supply. The sample volume is an important factor in sensitivity due to the low number of bacteria in a platelet component pack on day 1. An effective notification and recall system is a critical part of the bacterial screening system.

Isolation of genomic DNA from defatted oil seed residue of rapeseed (Brassica napus).

A simple protocol for obtaining pure, restrictable and amplifiable megabase genomic DNA from oil-free seed residue of Brassica napus, an important oil seed plant, has been developed. Oil from the dry seeds was completely recovered in an organic solvent and quantified gravimetrically followed by processing of the residual biomass (defatted seed residue) for genomic DNA isolation. The isolated DNA can be cut by a range of restriction enzymes. The method enables simultaneous isolation and recovery of lipids and genomic DNA from the same test sample, thus allowing two independent analyses from a single sample. Multiple micro-scale oil extraction from the commercial seeds gave approximately 39% oil, which is close to the usual oil recovery from standard oil seed. Most of the amplified fragments were scored in the range of 2.5 to 0.5 kb, best suited for scoring as molecular diagnostics.

Axisymmetric indentation of curved elastic membranes by a convex rigid indenter.

Motivated by applications to seed germination, we consider the transverse deflection that results from the axisymmetric indentation of an elastic membrane by a rigid body. The elastic membrane is fixed around its boundary, with or without an initial pre-stretch, and may be initially curved prior to indentation. General indenter shapes are considered, and the load-indentation curves that result for a range of spheroidal tips are obtained for both flat and curved membranes. Wrinkling may occur when the membrane is initially curved, and a relaxed strain-energy function is used to calculate the deformed profile in this case. Applications to experiments designed to measure the mechanical properties of seed endosperms are discussed.

The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors.

Erythrocytes infected with mature forms of Plasmodium falciparum do not circulate but are withdrawn from the peripheral circulation; they are bound to the endothelial lining and to uninfected erythrocytes in the microvasculature. Blockage of the blood flow, hampered oxygen delivery, and severe malaria may follow if binding is excessive. The NH(2)-terminal head structure (Duffy binding-like domain 1 [DBL1alpha]-cysteine-rich interdomain region [CIDR1alpha]) of a single species of P. falciparum erythrocyte membrane protein 1 (PfEMP1) is here shown to mediate adherence to multiple host receptors including platelet-endothelial cell adhesion molecule 1 (PECAM-1)/CD31, the blood group A antigen, normal nonimmune immunoglobulin M, three virulence-associated receptor proteins, a heparan sulfate-like glucosaminoglycan, and CD36. DBL2delta was found to mediate additional binding to PECAM-1/CD31. The exceptional binding activity of the PfEMP1 head structure and its relatively conserved nature argues that it holds an important role in erythrocyte sequestration and therefore in the virulence of the malaria parasite.