Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design.

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.

A lower X-gate in TASK channels traps inhibitors within the vestibule.

TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K2P) channel family-are found in neurons1, cardiomyocytes2-4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8-11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12-15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate-which we designate as an 'X-gate'-created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.

Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters.

Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.

Large-scale structural analysis of the classical human protein tyrosine phosphatome.

Protein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a "head-to-toe" dimerization model for RPTPgamma/zeta that is distinct from the "inhibitory wedge" model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.

Evaluation of bioelectrical impedance analysis in measuring body fat in 6-to-12-year-old boys compared with air displacement plethysmography.

Air displacement plethysmography (ADP) has been considered as the 'standard' method to determine body fat in children due to superior validity and reliability compared with bioelectrical impedance analysis (BIA). However, ADP and BIA are often used interchangeably despite few studies comparing measures of percentage body fat by ADP (%FMADP) with BIA (%FMBIA) in children with and without obesity. The objective of this study was to measure concurrent validity and reliability of %FMADP and %FMBIA in 6-to-12-year-old boys with and without obesity. Seventy-one boys (twenty-five with obesity) underwent body composition assessment. Ten boys participated in intra-day reliability analysis. %FMADP was estimated by Bodpod using sex- and age-specific equations of body density. %FMBIA was estimated by a multi-frequency, hand-to-foot device using child-specific equations based on impedance. Validity was assessed by t tests, correlation coefficients and limits of agreement (LoA); and reliability by technical error of measurement (TEM) and intraclass correlation coefficients (ICC). Compared with %FMADP, %FMBIA was significantly underestimated in the cohort (-3·4 ± 5·6 %; effect size = 0·42) and in both boys with obesity (-5·2 ± 5·5 %; ES = 0·90) and without obesity (-2·4 ± 5·5 %; ES = 0·52). A strong, significant positive correlation was found between %FMADP and %FMBIA (r = 0·80). Across the cohort, LoA were 22·3 %, and no proportional bias was detected. For reliability, TEM were 0·65 % and 0·55 %, and ICC were 0·93 and 0·95 for %FMBIA and %FMADP, respectively. Whilst both %FMADP and %FMBIA are highly reliable methods, considerable differences indicated that the devices cannot be used interchangeably in boys age 6-to-12 years.

Quality of Life in Post-Surgical Hypoparathyroidism (PoSH) in Thyroid and Parathyroid Surgery.

BACKGROUND: Post-surgical hypoparathyroidism (PoSH) is often long term, with significant associated morbidity and ongoing treatment. A recent systematic review found impaired quality of life (QoL) in patients with PoSH, despite stable treatment. Most studies did not include an appropriate control arm and further studies were recommended, taking into account underlying disease and comorbidities. This study aims to compare QoL in patients with PoSH with appropriate control groups. METHODS: This was a cross-sectional observational study using the general quality of life SF-36 tool and a hypocalcaemia symptom score (HcSS) to assess QoL in patients with PoSH and controls (who had similar surgery but without PoSH). Participants were identified from two patient groups (the Butterfly Thyroid Cancer Trust and the Association for Multiple Endocrine Neoplasia Disorders) and a single tertiary centre in the UK. RESULTS: Four hundred and thirty-nine responses (female n = 379, PoSH n = 89) were included with a median (range) age of 52 (19-92) years. Reported dates of surgery ranged from 1973 to 2019. HcSS scores showed significantly more associated symptoms in patients with PoSH than those without (p < 0.001). Although there was no overall difference in QoL between groups, patients with PoSH consistently had lower scores (p = 0.008) in the energy/fatigue subdomain of the SF-36. CONCLUSION: Patients with PoSH reported significantly more fatigue and loss of energy compared to appropriately matched controls, but overall QoL was not significantly different. Standardised QoL measures may not be sensitive enough to highlight the impact on QoL in these patients. A disease-specific tool may be required.

Behaviour change practices in exercise referral schemes: developing realist programme theory of implementation.

BACKGROUND: Exercise Referral Schemes have been delivered worldwide in developed countries to augment physical activity levels in sedentary patients with a range of health issues, despite their utility being questioned. Understanding the implementation mechanisms of behaviour change practices is important to avoid inappropriate decommissioning and support future service planning. The aim of this study was to develop initial theories to understand what influences the behaviour change practices of Exercise Referral practitioners within the United Kingdom. METHODS: An eight-month focused ethnography was undertaken, to carry out the first phase of a realist evaluation, which included participant observation, interviews, document analysis, and reflexive journaling. A comprehensive implementation framework (Consolidated Framework for Implementation Research) was adopted providing an extensive menu of determinants. Mechanisms were categorised based on the Theoretical Domains Framework (within the Capability, Opportunity, Motivation, Behaviour model) providing an explanatory tool linking the levels of the framework. RESULTS: Three programme theories are proposed. Firstly, motivation and capability are influenced when behaviour change oriented planning and training are in place. Secondly, motivation is influenced if leadership is supportive of behaviour change practice. Lastly, integration between health professionals and practitioners will influence motivation and capability. The conditions necessary to influence motivation and capability include a person-centred climate, cognizant practitioners, and established communities of practice. CONCLUSIONS: The findings are the first to articulate the necessary elements for the implementation of behaviour change practices in Exercise Referral services. These results outline emerging theories about the conditions, resources, and explanations of behaviour change implementation that can inform service development.

"Ask Parents What They Want to Do" - Mothers Seeking Help with Parenting and Their Perspectives on Barriers and Potential Enablers for Help with Smoking Cessation: A Qualitative Study.

PURPOSE: To describe parents' perceptions of opportunities and barriers to smoking cessation in early parenting and the support needed to achieve this. DESIGN AND METHODS: A qualitative approach, using semi-structured interviews with mothers, and inductive thematic analysis. RESULTS: Barriers to cessation intervention included readiness to quit, lack of knowledge, and the shame and stigma of seeking help. Mothers' perceived opportunities for support from early parenting services included approaches to effecting change, leveraging opportunities to explore smoking cessation, and tailoring interventions to family strengths, with a focus on promoting attachment. CONCLUSIONS: Mothers may be struggling with parenting, but are also open to opportunities to explore smoking behaviours and intervention. Nurses and other health care professionals working in this setting may be well situated to support parents with their decisions to consider smoking cessation and avoid smoking relapse. PRACTICE IMPLICATIONS: The findings can assist nurses and other early parenting service clinicians to consider their approach to parents who continue to smoke or are at risk of smoking relapse.

Mechanical Heterogeneity in the Bone Microenvironment as Characterized by Atomic Force Microscopy.

Bones are structurally heterogeneous organs with diverse functions that undergo mechanical stimuli across multiple length scales. Mechanical characterization of the bone microenvironment is important for understanding how bones function in health and disease. Here, we describe the mechanical architecture of cortical bone, the growth plate, metaphysis, and marrow in fresh murine bones, probed using atomic force microscopy in physiological buffer. Both elastic and viscoelastic properties are found to be highly heterogeneous with moduli ranging over three to five orders of magnitude, both within and across regions. All regions include extremely compliant areas, with moduli of a few pascal and viscosities as low as tens of Pa·s. Aging impacts the viscoelasticity of the bone marrow strongly but has a limited effect on the other regions studied. Our approach provides the opportunity to explore the mechanical properties of complex tissues at the length scale relevant to cellular processes and how these impact aging and disease.

Once-daily single-inhaler versus twice-daily multiple-inhaler triple therapy in patients with COPD: lung function and health status results from two replicate randomized controlled trials.

BACKGROUND: The comparative efficacy of inhaled corticosteroid/long-acting muscarinic antagonist/long-acting ß2-agonist (ICS/LAMA/LABA) triple therapy administered via single or multiple inhalers in patients with chronic obstructive pulmonary disease (COPD) has not been evaluated comprehensively. We conducted two replicate trials comparing single- with multiple-inhaler ICS/LAMA/LABA combination in COPD. METHODS: 207608 and 207609 were Phase IV, 12-week, randomized, double-blind, triple-dummy non-inferiority trials comparing once-daily fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) 100/62.5/25 µg via Ellipta inhaler, with twice-daily budesonide/formoterol (BUD/FOR) 400/12 µg via metered-dose inhaler plus once-daily tiotropium (TIO) 18 µg via HandiHaler. Patients had symptomatic COPD and forced expiratory volume in 1 s (FEV1) < 50% predicted, or FEV1 < 80% predicted and ≥ 2 moderate or 1 severe exacerbations in the prior year. The primary endpoint in both trials was weighted mean change from baseline (wmCFB) in 0-24-h FEV1 at Week 12. Secondary endpoints included CFB in trough FEV1 at Day 84 and 85. Other endpoints included serial FEV1 and health status outcomes at Week 12. Safety was evaluated descriptively. RESULTS: The modified per-protocol population included 720 and 711 patients in studies 207608 and 207609 (intent-to-treat population: 728 and 732). FF/UMEC/VI was non-inferior to BUD/FOR+TIO for wmCFB in 0-24-h FEV1 at Week 12 (Study 207608 treatment difference [95% confidence interval]: 15 mL [- 13, 43]; Study 207609: 11 mL [- 20, 41]). FF/UMEC/VI improved trough FEV1 CFB versus BUD/FOR+TIO at Day 84 and 85 (Day 85 treatment difference: Study 207608: 38 mL [10, 66]; Study 207609: 51 mL [21, 82]) and FEV1 at 12 and 24 h post-morning dose at Week 12 in both studies. No treatment differences were seen in health status outcomes. Safety profiles were similar between treatments; pneumonia occurred in 7 (< 1%) patients with FF/UMEC/VI and 9 (1%) patients with BUD/FOR+TIO, across both studies. CONCLUSIONS: FF/UMEC/VI was non-inferior to BUD/FOR+TIO for wmCFB in 0-24-h FEV1 at Week 12 in patients with COPD. Greater improvements in trough and serial FEV1 measurements at Week 12 with FF/UMEC/VI versus BUD/FOR+TIO, together with similar health status improvements and safety outcomes including the incidence of pneumonia, suggest that once-daily single-inhaler FF/UMEC/VI triple therapy is a viable option for patients looking to simplify their treatment regimen. TRIAL REGISTRATION: GSK (207608/207609; NCT03478683/NCT03478696).

Bone marrow osteoprogenitors are depleted whereas osteoblasts are expanded independent of the osteogenic vasculature in response to zoledronic acid.

Zoledronic acid (ZOL) is an antiresorptive drug used to prevent bone loss in a variety of conditions, acting mainly through suppression of osteoclast activity. There is growing evidence that ZOL can also affect cells of the mesenchymal lineage in bone. We present novel data revealing significant changes in the abundance of perivascular mesenchymal stromal cells (MSCs)/osteoprogenitors and osteoblasts following the injection of ZOL, in vivo. In young mice with high bone turnover and an abundance of perivascular osteoprogenitors, ZOL significantly (P < 0.0001) increased new bone formation. This was accompanied by a decline in osterix-positive osteoprogenitors and a corresponding increase in osteoblasts. However, these effects were not observed in mature mice with low bone turnover. Interestingly, the ZOL-induced changes in cells of the mesenchymal lineage occurred independently of effects on the osteogenic vasculature. Thus, we demonstrate that a single, clinically relevant dose of ZOL can induce new bone formation in microenvironments enriched for perivascular MSC/osteoprogenitors and high osteogenic potential. This arises from the differentiation of perivascular osterix-positive MSC/osteoprogenitors into osteoblasts at sites that are innately osteogenic. Collectively, our data demonstrate that ZOL affects multiple cell types in bone and has differential effects depending on the level of bone turnover.-Hughes, R., Chen, X., Hunter, K. D., Hobbs, J. K., Holen, I., Brown, N. J. Bone marrow osteoprogenitors are depleted whereas osteoblasts are expanded independent of the osteogenic vasculature in response to zoledronic acid.

Use of Electrical Impedance Spectroscopy for Intraoperative Tissue Differentiation During Thyroid and Parathyroid Surgery.

BACKGROUND: Electrical impedance (EI) measures tissue resistance to alternating current across several frequencies and may help identify tissue type. A recent rabbit model demonstrated that electrical impedance spectroscopy (EIS) may facilitate identification of parathyroid glands and potentially improve outcomes following surgery. This study looks at the EI patterns of soft tissues in the human neck to determine whether parathyroid tissue can be accurately identified. METHODS: This was a phase 1, single-arm interventional study involving 56 patients undergoing thyroid and/or parathyroid surgery. Up to 12 EI readings were taken from in vivo and ex vivo thyroid and parathyroid glands, adipose tissue and muscle of each patient. Each reading consists of a series of measurements over 14 frequencies from each tissue. EI patterns were analysed. Two patients were excluded due to data loss due to device malfunction. RESULTS: The median age of participants was 53.5 (range 20-85) years. Thirty-five participants had surgery for thyroid pathology, 17 for parathyroid pathology and four for both. Six hundred and six EIS spectra were reviewed for suitability. One hundred and eighty-four spectra were rejected leaving 422 spectra for analysis. The impedance patterns of the soft tissues differed by histological type. The EI ratio of low (152 Hz) to high (312 kHz) frequencies demonstrated a significant difference between the soft tissues (p = 0.006). Using appropriate thresholds, parathyroid tissue can be distinguished from thyroid tissue with a sensitivity of 76% and specificity of 60%. CONCLUSIONS: This study demonstrates the feasibility of using EIS to aid parathyroid identification and preservation. Further changes to the device and modelling of the EI patterns across the range of frequencies may improve accuracy and facilitate intraoperative use. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02901873).

Identification of dropout predictors to a community-based physical activity programme that uses motivational interviewing.

BACKGROUND: Participant dropout reduces intervention effectiveness. Predicting dropout has been investigated for Exercise Referral Schemes, but not physical activity (PA) interventions with Motivational Interviewing (MI). METHODS: Data from attendees (n = 619) to a community-based PA programme utilizing MI techniques were analysed using a chi-squared test to determine dropout and attendance group differences. Binary logistic regression investigated the likelihood of dropout before 12 weeks. RESULTS: A total of 44.7% of participants dropped out, with statistical (P < 0.05) differences between groups for age, PA and disability. Regression for each variable showed participants aged 61-70 years (OR = 0.28, CI = 0.09-0.79; P = 0.018), >70 years (OR = 0.30, CI = 0.09-0.90; P = 0.036), and high PA (OR = 0.40, CI = 0.20-0.75; P = 0.006) reduced dropout likelihood. Endocrine system disorders (OR = 4.24, CI = 1.19-19.43; P = 0.036) and musculoskeletal disorders (OR = 3.14, CI = 1.84-5.45; P < 0.001) increased dropout likelihood. Significant variables were combined in a single regression model. Dropout significantly reduced for 61-70 years old (OR = 0.31, CI = 0.10-0.90; P = 0.035), and high PA (OR = 0.39, CI = 0.19-0.76; P = 0.008). Musculoskeletal disorders increased dropout (OR = 2.67, CI = 1.53-4.75; P < 0.001). CONCLUSIONS: Age, PA and disability type significantly influence dropout at 12 weeks. These are the first results specific to MI based programmes indicating the inclusion of MI and highlighting the need for further research.

Strength and Conditioning Habits of Competitive Distance Runners.

Blagrove, RC, Brown, N, Howatson, G, and Hayes, PR. Strength and conditioning habits of competitive distance runners. J Strength Cond Res 34(5): 1392-1399, 2020-Targeted strength and conditioning (S&C) programs can potentially improve performance and reduce injury risk factors in competitive runners. However, S&C practices of distance runners are unknown. This study aimed to explore S&C practices of competitive middle- and long-distance runners and examined whether reported frequency of injuries was influenced by training behaviors. One thousand eight hundred eighty-three distance runners (≥15 years old) completed an online survey. All runners who raced competitively were included in data analysis (n = 667). Distance runners mainly engaged with S&C activities to lower risk of injury (63.1%) and improve performance (53.8%). The most common activities used were stretching (86.2%) and core stability exercises (70.2%). Resistance training (RT) and plyometric training (PT) were used by 62.5 and 35.1% of runners, respectively. Junior (under-20) runners include PT, running drills, and circuit training more so than masters runners. Significantly more international standard runners engaged in RT, PT, and fundamental movement skills training compared with competitive club runners. Middle-distance (800-3,000 m) specialists were more likely to include RT, PT, running drills, circuit training, and barefoot exercises in their program than longer-distance runners. Injury frequency was associated with typical weekly running volume and run frequency. Strength and conditioning did not seem to confer a protection against the number of injuries the runners experienced. Practitioners working with distance runners should critically evaluate the current S&C practices of their athletes, to ensure that activities prescribed have a sound evidence-based rationale.

A facile and regioselective multicomponent synthesis of chiral aryl-1,2-mercaptoamines in water followed by monoamine oxidase (MAO-N) enzymatic resolution.

A facile microwave assisted three-component protocol allows the synthesis of chiral aryl-1,2-mercaptoamines in water in a few minutes with high yields, bypassing the use of toxic aziridine intermediates. The chiral 1,2-mercaptoamines were then deracemized through enzymatic resolution of the racemates using monoamine oxidase (MAO-N) biocatalysts.

Human SNM1A and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair.

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.

Structural analysis of human KDM5B guides histone demethylase inhibitor development.

Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe(2+)-dependent oxygenases that act as histone H3K4 demethylases, thereby regulating cell proliferation and stem cell self-renewal and differentiation. Here we report crystal structures of the catalytic core of the human KDM5B enzyme in complex with three inhibitor chemotypes. These scaffolds exploit several aspects of the KDM5 active site, and their selectivity profiles reflect their hybrid features with respect to the KDM4 and KDM6 families. Whereas GSK-J1, a previously identified KDM6 inhibitor, showed about sevenfold less inhibitory activity toward KDM5B than toward KDM6 proteins, KDM5-C49 displayed 25-100-fold selectivity between KDM5B and KDM6B. The cell-permeable derivative KDM5-C70 had an antiproliferative effect in myeloma cells, leading to genome-wide elevation of H3K4me3 levels. The selective inhibitor GSK467 exploited unique binding modes, but it lacked cellular potency in the myeloma system. Taken together, these structural leads deliver multiple starting points for further rational and selective inhibitor design.

Use of methylene blue and near-infrared fluorescence in thyroid and parathyroid surgery.

PURPOSE: Intraoperative localisation and preservation of parathyroid glands improves outcomes following thyroid and parathyroid surgery. This can be facilitated by fluorescent imaging and methylene blue; a fluorophore is thought to be taken up avidly by parathyroid glands. This preliminary study aims to identify the optimum dose of methylene blue (MB), fluorescent patterns of thyroid and parathyroid glands and develop a protocol for the use of intravenous MB emitted fluorescence to enable parathyroid identification. METHODS: This is a phase 1b, interventional study (NCT02089542) involving 41 patients undergoing thyroid and/or parathyroid surgery. After exposure of the thyroid and/or parathyroid gland(s), intravenous boluses of between 0.05 and 0.5 mg/kg of MB were injected. Fluobeam® (a hand held fluorescence real-time imager) was used to record fluorescence from the operating field prior and up to 10 min following administration. RESULTS: The optimum dose of MB to visualise thyroid and parathyroid glands was 0.4 mg/kg body weight. The median time to onset of fluorescence was 23 and 22 s and the median time to peak fluorescence was 41.5 and 40 s, respectively. The peak fluorescence for thyroid and parathyroid glands compared to muscle were 2.6 and 4.3, respectively. Parathyroid auto-fluorescence prior to methylene blue injection was commonly observed. CONCLUSIONS: A clinical protocol for detection of fluorescence from MB during thyroid and parathyroid surgery is presented. Parathyroids (especially enlarged glands) fluoresce more intensely than thyroid glands. Auto-fluorescence may aid parathyroid detection, but MB fluorescence is needed to demonstrate viability.

Breast education for schoolgirls; why, what, when, and how?

Schools are fundamental settings for health education and adolescent females are an important group for promoting positive breast habits. We surveyed 2089 schoolgirls (11-18 years) to provide evidence for, and guidance on, breast education for schoolgirls. 26% reported negative feelings about their breasts and 87% reported ≥ one breast concern. 72% wanted to know more about breast cancer (69% rating this extremely important). >50% wanted to know more about breast sag and breast pain. Preferred delivery format was age eleven (50%), girls only taught sessions (41%) with female teachers (43%). A need for breast education and delivery preferences was identified.

Parathyroid Hormone (PTH) Increases Skeletal Tumour Growth and Alters Tumour Distribution in an In Vivo Model of Breast Cancer.

Breast cancer cells colonize the skeleton by homing to specific niches, but the involvement of osteoblasts in tumour cell seeding, colonization, and progression is unknown. We used an in vivo model to determine how increasing the number of cells of the osteoblast lineage with parathyroid hormone (PTH) modified subsequent skeletal colonization by breast cancer cells. BALB/c nude mice were injected for five consecutive days with PBS (control) or PTH and then injected with DiD-labelled breast cancer cells via the intra-cardiac route. Effects of PTH on the bone microenvironment and tumour cell colonization and growth was analyzed using bioluminescence imaging, two-photon microscopy, and histological analysis. PTH treatment caused a significant, transient increase in osteoblast numbers compared to control, whereas bone volume/structure in the tibia was unaffected. There were no differences in the number of tumour cells seeding to the tibias, or in the number of tumours in the hind legs, between the control and PTH group. However, animals pre-treated with PTH had a significantly higher number of tumour colonies distributed throughout skeletal sites outside the hind limbs. This is the first demonstration that PTH-induced stimulation of osteoblastic cells may result in alternative skeletal sites becoming available for breast cancer cell colonization.