Optimization of biotinyl-tyramide-based in situ hybridization for sensitive background-free applications on formalin-fixed, paraffin-embedded tissue specimens

BACKGROUND: Over the past five years in situ hybridization techniques employing tyramide amplification reagents have been developed and promise the potential detection of low/single-copy nucleic acid sequences. However the increased sensitivity that tyramide amplification brings about may also lead to problems of background staining that confound data interpretation. METHODS: In this study those factors enabling background-free biotinyl-tyramide based in situ hybridization assay of formalin-fixed paraffin-embedded tissues have been examined. SiHa, HeLa and CaSki cell lines known to contain HPV integrated into the cell genome, and archival cervical pre-invasive lesions and carcinomas have been successfully assessed using biotinylated HPV and centromeric probes. RESULTS: The single most important factor both for sensitivity and clean background was a tissue unmasking regimen that included treatment with 10 mM sodium citrate pH 6.0 at 95°C followed by digestion with pepsin/0.2 M HCl. Concentrations both of probe and primary streptavidin-peroxidase conjugate and pH of hybridization mix and stringency washes were also critical for sensitivity. Certain probes were more associated with background staining than others. This problem was not related to probe purity or size. In these instances composition of hybridization mix solution was especially critical to avoid background. 3-amino-9-ethylcarbazole was preferred over 3,3'-diaminobenzidene as a chromogen because background was cleaner and the 1–2 copies of HPV16 integrated in SiHa cells were readily demonstrable. HPV detection on metaphase spreads prepared from SiHa cells was only successful when a fluorescent detection method was combined with tyramide reagent. 'Punctate' and 'diffuse' signal patterns were identified amongst tissues consistent with the former representing integration and 'diffuse' representing episomal HPV. Only punctate signals were detected amongst the cell lines and were common amongst high-grade pre-invasive lesions and carcinomas. However it remains to be determined why single/low-copy episomal HPV in basal/parabasal cells of low-grade lesions is not also detectable using tyramide-based techniques and whether every punctate signal represents integration. CONCLUSIONS: A tyramide-based in situ hybridization methodology has been established that enables sensitive, background-free assay of clinical specimens. As punctate signals characterize HPV in high-grade cervical lesions the method may have potential for clinical applications

A comparison between direct and pan-derived measurements of the isotopic composition of atmospheric waters

The stable isotopes of water can be used to examine and quantify the contribution to atmospheric moisture from evaporation, transpiration and surface-waters. However, obtaining extensive and ongoing time series data of the isotopic composition of atmospheric moisture has been difficult. Presented here is an alternate method using an isotope mass balance approach to estimate the isotopic composition of atmospheric moisture using water samples collected from class A evaporation pans. While this evaporation pan method does not provide the high-resolution time series data that can be obtained from an isotope analyser taking in-situ measurements of atmospheric moisture, the method is relatively simple and inexpensive to set-up and maintain. In this preliminary investigation, a comparison between the isotopic composition of atmospheric moisture estimated from the evaporation pan method and in-situ measurements of the isotopic composition of water vapour using a Fourier Transform Infrared (FTIR) spectrometer deployed at the Lucas Heights weather station in New South Wales is undertaken. Through comparison of the two series of hydrogen isotope data, an assessment of the evaporation pan method can be made. Although there was some agreement between the isotopic composition of vapour measured by the FTIR spectrometer and the estimation for the atmospheric moisture (R2 = 0.49), the comparison is sensitive to climatic parameters that vary significantly within a 24-hour period such as the relative humidity of air and the air and pan temperatures. Inverting the model to use the FTIR spectrometer measurements at an hourly resolution improved the performance of the model (R2 =0.57). However, this also revealed that the model produced more depleted values of the evaporation pan water isotopes than those observed. In contrast, there was a variable relationship between the modelled and observed isotope values of atmospheric moisture. These conflicting results will need to be resolved before the evaporation pan method is broadly applied in isotope hydrology. © 2011 The Modelling and Simulation Society of Australia and New Zealand Inc

Incidence of major complications from embolo-sclerotherapy of head and neck vascular malformations in a single specialist centre

Objective: Current data on the nature and rate of major complications for embolo-sclerotherapy (EST) of vascular malformations are scarce. However, even fewer studies focus on vascular malformations specific to the head and neck, which confer an increased specific risk of airway compromise, neurologic and ophthalmologic injury. More understanding is required surrounding the type and incidence of complications to improve treatment planning and informed consent. Therefore, this study aimed to review major complications secondary to EST of head and neck vascular malformations over a 5-year period in a single specialized multidisciplinary centre for vascular anomalies. / Methods: All interventions were decided by the multidisciplinary team. Demographic, procedural and complication data between 1st January 2013 and 31st December 2017 were prospectively documented in a dedicated database and analysed. EST of high-flow vascular malformations (HFVMs) was performed by selective catheter angiography or direct injection, and by direct injection only for low-flow vascular malformations (LFVMs). Major complications were defined as any tissue or functional damage caused by direct injection, distal embolization or tissue reaction and were decided by the multidisciplinary team. / Results: Forty-eight patients (median age of 35 years; range of 14–70 years; 18 men and 30 women) had 100 EST procedures for head and neck vascular malformation. Of these, 14 patients had EST for HFVM and 34 patients for LFVM, total 43 and 57 procedures, respectively. Overall, five patients with HFVM developed major complications from EST when compared with two patients with LFVM (p = 0.0167). Two patients required pre-emptive tracheostomy due to risk of post-operative airway compromise. Overall, seven (14.6%) patients experienced major complication from EST. In the HFVM group, major complications from EST occurred in five patients; four cases of tissue ulceration and necrosis (two needed debridement, one healed with resultant fibrosis that impeded speech and one resolved spontaneously) and one post-procedural airway compromise requiring tracheostomy. Meanwhile, in the LFVM group, major complications occurred in two patients; one case of severe necrosis involving the alar cartilage, lip and cheek requiring debridement and reconstruction under plastics and one simple cellulitis. No patients sustained stroke or vision impairment. / Conclusions: EST is relatively safe for head and neck vascular malformations in a high-volume experienced centre. Our major complication rate of 14.6% per patient (35.7% for HFVM; 5.9% for LFVM) or 7% per procedure (11.6% for HFVM; 3.5% LFVM) compares favourably with published data from other centres. These data will improve treatment planning and informed consent for EST for both HFVM and LFVM of the head and neck

Patient radiation exposure from embolo-sclerotherapy of peripheral vascular malformations

Objective: Embolo-sclerotherapy (EST) is the mainstay therapy for peripheral vascular malformations which involves the exposure of patients to ionizing radiation. We aimed to analyze the radiation exposure to patients from EST of peripheral vascular malformations over five years in a single specialist center. / Methods: All patients who had EST performed in a single specialist center for peripheral vascular malformations between January 1st 2013 and January 8th 2018 were identified from a prospectively collected database. Data collection included basic demographics, procedure date, anatomical site, type of vascular malformations and procedural details. Radiation exposure, measured in dose-area product (DAP) and fluoroscopy times, of all patients identified to have EST during the period were retrospectively reviewed. Statistical analysis was performed using Mann-Whitney U and Kruskal-Wallis tests for comparison between subgroups. P<0.05 was considered significant. / Results: A total of 237 patients (median age 30 years; range 1 – 73 years) underwent 419 ESTs during the study period. Of these, 61 (25.7%) patients had arteriovenous malformations (AVM) and underwent 140 (33.4%) ESTs. Meanwhile, 176 (74.3%) patients had venous and lymphatic malformations and underwent 279 ESTs (66.6%). Patients with AVMs had a median of 2 procedures (range 1 - 13), compared to a median of 1 (range 1 - 6) for venous and lymphatic malformations within the study period. The median DAP for single and cumulative EST for peripheral vascular malformations were 1.26 Gycm2 (range 0.00 – 698.36 Gycm2) and 1.91 Gycm2 (range 0.00 – 1300.24 Gycm2), respectively. Whereas, the median fluoroscopy time for single and cumulative EST were 19 seconds (range 1 – 3846 seconds) and 30 seconds (range 1 – 5843 seconds), respectively. Significantly higher patient radiation exposure, in DAP and fluoroscopy times, were measured for single and cumulative EST for AVM when compared with venous and lymphatic malformation (both p <0.01; Mann Whitney U). A significant difference in DAP but not fluoroscopy time was found when anatomical areas of vascular malformations were compared. / Conclusions: Patient radiation exposure for EST of peripheral vascular malformations, measured in DAP and fluoroscopy times, appeared to be generally less than those reported for endovascular arterial and deep venous interventions in the literature. However, some patients with peripheral vascular malformations received relatively high radiation doses. Further studies to investigate the risk factors and long-term side-effects of radiation exposure in these patients and strategies to reduce it are required

Single Center Experience of Sirolimus Therapy in Head and Neck Low-flow Vascular Malformations

OBJECTIVE: Recently, studies have shown that sirolimus is clinically efficacious in the treatment of some low-flow vascular malformations (LFVM). This study aimed to assess the efficacy and safety of sirolimus in treating complex head and neck (H&N) LFVM that were challenging and/or refractory to standard treatment. METHODS: Each patient had baseline and 6-months assessments consisting of clinical history and examination, quality of life (QoL) questionnaires, laboratory investigations, MRI and medical photography. Patients were followed up 1-week and then 1-monthly for 6-months. Wilcoxon signed-rank test was used to compare pre-and 6-months treatment in all 8 domains of RAND 36-Item Short Form Health Survey (SF-36), hospital anxiety and depression scale (HADS), and visual analog score for pain (VAS-P). P < 0.05 was considered significant. RESULTS: Seven patients (median age 43 years, range 23-65 years) were recruited. Six patients completed the six-months course of therapy with 1 patient withdrawing due to intolerable side effects. All six patients reported reduction of swelling with and without other symptom improvement related to the vascular malformations while on treatment. However, at 1-month review after discontinuation of sirolimus, 5 patients reported return of initial symptoms. Overall, patients demonstrated an improvement in QoL six-months treatment but there was no statistical significance (P > 0.05) in all 8 domains of SF-36, HADS and VAS-P. Five patients demonstrated a minimum 10% decrease in lesion size six-months treatment (median 21%, range 13-40%). A Wilcoxon signed-rank test showed that sirolimus treatment did elicit a statistically significant change in lesion size in either direction (Z = -1.992, P = 0.046). The most common side effects found were dyslipidaemia (n-4) and mouth ulcers (n = 2). CONCLUSIONS: In our preliminary experience, sirolimus is effective and safe in treating patients with complex H&N LFVM. This provides an alternative treatment where standard treatment is challenging and/or refractory

Spontaneous Parity Violation in SUSY Strong Gauge Theory

We suggest simple models of spontaneous parity violation in supersymmetric strong gauge theory. We focus on left-right symmetric model and investigate vacuum with spontaneous parity violation. Non-perturbative effects are calculable in supersymmetric gauge theory, and we suggest two new models. The first model shows confinement, and the second model has a dual description of the theory. The left-right symmetry breaking and electroweak symmetry breaking are simultaneously occurred with the suitable energy scale hierarchy. The second model also induces spontaneous supersymmetry breaking.Comment: 14 page

Quality of life and mental health of patients with vascular malformations in a single specialist center in the United Kingdom

OBJECTIVE: Patients with vascular malformations suffer from chronic debilitating symptoms that have been shown to contribute negatively to their quality of life (QoL) and mental health. Despite this, the current literature evaluating the QoL and mental health of patients with vascular malformations remains scarce. Our aim was to evaluate the QoL and mental health of patients with vascular malformations. METHODS: We prospectively analyzed the validated health-related QoL (HRQoL) questionnaires: the RAND Health Care 36-Item Short Form Survey (SF-36), Hospital Anxiety and Depression Scale (HADS), and visual analogue score for pain reported by 253 patients with vascular malformations in a specialist center of vascular anomalies in the UK over two years. RESULTS: Patients with vascular malformations reported significantly poorer SF-36 scores in all domains compared with the UK general population. Patients with low-flow vascular malformations and arteriovenous malformations reported little variations in SF-36, HADS, and visual analogue score for pain scores. No significant association was found between age and any of the health-related QoL scores, other than the physical functioning in SF-36. Female patients reported significantly lower physical and social functioning of SF-36 and worse HADS-Depression than their male counterparts. Patients with syndromic vascular malformations reported significantly lower SF-36 scores in role-physical, role-emotional and bodily pain than nonsyndromic vascular malformations. CONCLUSIONS: This study concluded that patients with vascular malformations reported worse QoL than the UK general population. Therefore, the assessment and management of QoL and mental health should be incorporated into the overall treatment strategies of patients with vascular malformations

Which doctors and with what problems contact a specialist service for doctors? A cross sectional investigation

Background: In the United Kingdom, specialist treatment and intervention services for doctors are underdeveloped. The MedNet programme, created in 1997 and funded by the London Deanery, aims to fill this gap by providing a self-referral, face-to-face, psychotherapeutic assessment service for doctors in London and South-East England. MedNet was designed to be a low-threshold service, targeting doctors without formal psychiatric problems. The aim of this study was to delineate the characteristics of doctors utilising the service, to describe their psychological morbidity, and to determine if early intervention is achieved. Methods: A cross-sectional study including all consecutive self-referred doctors (n = 121, 50% male) presenting in 2002–2004 was conducted. Measures included standardised and bespoke questionnaires both self-report and clinician completed. The multi-dimensional evaluation included: demographics, CORE (CORE-OM, CORE-Workplace and CORE-A) an instrument designed to evaluate the psychological difficulties of patients referred to outpatient services, Brief Symptom Inventory to quantify caseness and formal psychiatric illness, and Maslach Burnout Inventory. Results: The most prevalent presenting problems included depression, anxiety, interpersonal, self-esteem and work-related issues. However, only 9% of the cohort were identified as severely distressed psychiatrically using this measure. In approximately 50% of the sample, problems first presented in the preceding year. About 25% were on sick leave at the time of consultation, while 50% took little or no leave in the prior 12 months. A total of 42% were considered to be at some risk of suicide, with more than 25% considered to have a moderate to severe risk. There were no significant gender differences in type of morbidity, severity or days off sick. Conclusion: Doctors displayed high levels of distress as reflected in the significant proportion of those who were at some risk of suicide; however, low rates of severe psychiatric illness were detected. These findings suggest that MedNet clients represent both ends of the spectrum of severity, enabling early clinical engagement for a significant proportion of cases that is of importance both in terms of personal health and protecting patient care, and providing a timely intervention for those who are at risk, a group for whom rapid intervention services are in need and an area that requires further investigation in the UK

A Geographically-Restricted but Prevalent Mycobacterium tuberculosis Strain Identified in the West Midlands Region of the UK between 1995 and 2008

Background: We describe the identification of, and risk factors for, the single most prevalent Mycobacterium tuberculosis strain in the West Midlands region of the UK.Methodology/Principal Findings: Prospective 15-locus MIRU-VNTR genotyping of all M. tuberculosis isolates in the West Midlands between 2004 and 2008 was undertaken. Two retrospective epidemiological investigations were also undertaken using univariable and multivariable logistic regression analysis. The first study of all TB patients in the West Midlands between 2004 and 2008 identified a single prevalent strain in each of the study years (total 155/3,056 (5%) isolates). This prevalent MIRU-VNTR profile (32333 2432515314 434443183) remained clustered after typing with an additional 9-loci MIRU-VNTR and spoligotyping. The majority of these patients (122/155, 79%) resided in three major cities located within a 40 km radius. From the apparent geographical restriction, we have named this the "Mercian" strain. A multivariate analysis of all TB patients in the West Midlands identified that infection with a Mercian strain was significantly associated with being UK-born (OR = 9.03, 95% CI = 4.56-17.87, p 65 years old (OR = 0.25, 95% CI = 0.09-0.67, p < 0.01). A second more detailed investigation analyzed a cohort of 82 patients resident in Wolverhampton between 2003 and 2006. A significant association with being born in the UK remained after a multivariate analysis (OR = 9.68, 95% CI = 2.00-46.78, p < 0.01) and excess alcohol intake and cannabis use (OR = 6.26, 95% CI = 1.45-27.02, p = .01) were observed as social risk factors for infection.Conclusions/Significance: The continued consistent presence of the Mercian strain suggests ongoing community transmission. Whilst significant associations have been found, there may be other common risk factors yet to be identified. Future investigations should focus on targeting the relevant risk groups and elucidating the biological factors that mediate continued transmission of this strain

Confirmation of low genetic diversity and multiple breeding females in a social group of Eurasian badgers from microsatellite and field data

The Eurasian badger ( Meles meles ) is a facultatively social carnivore that shows only rudimentary co-operative behaviour and a poorly defined social hierarchy. Behavioural evidence and limited genetic data have suggested that more than one female may breed in a social group. We combine pregnancy detection by ultrasound and microsatellite locus scores from a well-studied badger population from Wytham Woods, Oxfordshire, UK, to demonstrate that multiple females reproduce within a social group. We found that at least three of seven potential mothers reproduced in a group that contained 11 reproductive age females and nine offspring. Twelve primers showed variability across the species range and only five of these were variable in Wytham. The microsatellites showed a reduced repeat number, a significantly higher number of nonperfect repeats, and moderate heterozygosity levels in Wytham. The high frequency of imperfect repeats and demographic phenomena might be responsible for the reduced levels of variability observed in the badger