The unexplored role of sedentary time and physical activity in glucose and lipid metabolism-related placental mRNAs in pregnant women who are obese: the DALI lifestyle randomised controlled trial.

OBJECTIVE: We aimed to explore: (i) the association of sedentary time (ST) and physical activity (PA) during pregnancy with the placental expression of genes related to glucose and lipid metabolism in pregnant women who are obese; (ii) maternal metabolic factors mediating changes in these placental transcripts; and (iii) cord blood markers related to the mRNAs mediating neonatal adiposity. DESIGN: Multicentre randomised controlled trial. SETTING: Hospitals in nine European countries. POPULATION: A cohort of 112 pregnant women with placental tissue. METHODS: Both ST and moderate-to-vigorous PA (MVPA) levels were measured objectively using accelerometry at three time periods during pregnancy. MAIN OUTCOME MEASURES: Placental mRNAs (FATP2, FATP3, FABP4, GLUT1 and PPAR-γ) were measured with NanoString technology. Maternal and fetal metabolic markers and neonatal adiposity were assessed. RESULTS: Longer periods of ST, especially in early to middle pregnancy, was associated with lower placental FATP2 and FATP3 expression (P < 0.05), whereas MVPA at baseline was inversely associated with GLUT1 mRNA (P = 0.02). Although placental FATP2 and FATP3 expression were regulated by the insulin-glucose axis (P < 0.05), no maternal metabolic marker mediated the association of ST/MVPA with placental mRNAs (P > 0.05). Additionally, placental FATP2 expression was inversely associated with cord blood triglycerides and free fatty acids (FFAs; P < 0.01). No cord blood marker mediated neonatal adiposity except for cord blood leptin, which mediated the effects of PPAR-γ on neonatal sum of skinfolds (P < 0.05). CONCLUSIONS: In early to middle pregnancy, ST is associated with the expression of placental genes linked to lipid transport. PA is hardly related to transporter mRNAs. Strategies aimed at reducing sedentary behaviour during pregnancy could modulate placental gene expression, which may help to prevent unfavourable fetal and maternal pregnancy outcomes. TWEETABLE ABSTRACT: Reducing sedentary behaviour in pregnancy might modulate placental expression of genes related to lipid metabolism in women who are obese.

Hemostasis Techniques for Non-variceal Upper GI Hemorrhage: Beyond Injection and Cautery.

B. Nulsen D. M. Jensen.

Quality of life and sexuality in women with lichen sclerosus: a cross-sectional study.

BACKGROUND: Lichen sclerosus (LS) affects the female anogenital area, causing anatomical changes. Reported symptoms include itching, soreness and dyspareunia. AIM: To evaluate the quality of life and sexual functioning in women with LS. METHODS: In total, 158 women aged over 18 years, diagnosed with LS, and referred to North Denmark Regional Hospital from January 2018 to November 2019, were included. Three questionnaires, the Female Sexual Function Index (FSFI), Dermatology Life Quality Index (DLQI) and the World Health Organization Five-Item Well-Being Index (WHO-5), were completed. RESULTS: The women (mean age 47 years, range 18-76) presented a low score on all FSFI scales, with a mean score of 13.83 (95% CI 12.46-15.20), indicating reduced sexual functioning. The subgroup evaluation scored for desire 2.32, arousal 2.23, lubrication 2.39, orgasm 2.28, satisfaction 3.02 and pain 1.59. The DLQI results revealed a mean score of 7.88 (95% CI 7.02-8.74), indicating a moderate effect on the women's everyday life. The mean subscores were treatment 0.32, sexual difficulties 1.56, relations 1.02, work/study 0.34, sport 0.45, social activities 0.54, clothing 0.89, shopping 0.22, embarrassment 0.99 and itching, soreness and pain 1.55. The mean score for the WHO-5 was 56.66 (95% CI 53.48-59.84) indicating that 40% of the women had signs of depression. CONCLUSION: LS has a considerable influence on the sexual functioning and quality of life of women. Healthcare professionals must not only consider the biological aspects but also the psychological and social aspects.

Different reticuloruminal pH metrics of high-yielding dairy cattle during the transition period in relation to metabolic health, activity, and feed intake.

The measurement of pH in the reticulorumen in combination with a time-pH threshold has been widely applied in research to diagnose subacute ruminal acidosis. However, other pH metrics also have biological value. In this study, 44 animals were monitored during the transition period using reticuloruminal pH boluses. Traditional and more complex pH characteristics were calculated to characterize the reticuloruminal pH profile: time pH <6, slope of a logistic cumulative pH curve (ß0), and deviations [squared error (SqEr)] from pH predictions based on a harmonic static model. In this study, we aimed to examine the associations between those pH metrics and metabolic health parameters, feed intake, and activity. Finally, to describe the reticuloruminal pH dynamically, we also constructed a dynamic linear model. The results of this model were studied in relation to feed intake. All pH parameters were mutually correlated (particularly ß0 and SqEr; mean Pearson correlation of -0.52). pH patterns, rather than time pH <6, were associated with metabolic health and feed intake: high variation in daily pH (ß0 parameter) was reflected in higher blood concentrations of nonesterified fatty acids. Moreover, pH deviations of the harmonic model were negatively associated with feed intake and rumination behavior. This research confirms the biological importance of pH metrics focusing on pH variation and pH deviations and provides deeper insight into its associations with metabolic health status, feed intake, and activity during early lactation.

Metabolic phenotypes of early gestational diabetes mellitus and their association with adverse pregnancy outcomes.

AIMS: To describe the metabolic phenotypes of early gestational diabetes mellitus and their association with adverse pregnancy outcomes. METHODS: We performed a post hoc analysis using data from the Vitamin D And Lifestyle Intervention for gestational diabetes prevention (DALI) trial conducted across nine European countries (2012-2014). In women with a BMI ≥29 kg/m2 , insulin resistance and secretion were estimated from the oral glucose tolerance test values performed before 20 weeks, using homeostatic model assessment of insulin resistance and Stumvoll first-phase indices, respectively. Women with early gestational diabetes, defined by the International Association of Diabetes and Pregnancy Study Groups criteria, were classified into three groups: GDM-R (above-median insulin resistance alone), GDM-S (below-median insulin secretion alone), and GDM-B (combination of both) and the few remaining women were excluded. RESULTS: Compared with women in the normal glucose tolerance group (n = 651), women in the GDM-R group (n = 143) had higher fasting and post-load glucose values and insulin levels, with a greater risk of having large-for-gestational age babies [adjusted odds ratio 3.30 (95% CI 1.50-7.50)] and caesarean section [adjusted odds ratio 2.30 (95% CI 1.20-4.40)]. Women in the GDM-S (n = 37) and GDM-B (n = 56) groups had comparable pregnancy outcomes with those in the normal glucose tolerance group. CONCLUSIONS: In overweight and obese women with early gestational diabetes, higher degree of insulin resistance alone was more likely to be associated with adverse pregnancy outcomes than lower insulin secretion alone or a combination of both.

Timing and climate forcing of volcanic eruptions for the past 2,500 years.

Volcanic eruptions contribute to climate variability, but quantifying these contributions has been limited by inconsistencies in the timing of atmospheric volcanic aerosol loading determined from ice cores and subsequent cooling from climate proxies such as tree rings. Here we resolve these inconsistencies and show that large eruptions in the tropics and high latitudes were primary drivers of interannual-to-decadal temperature variability in the Northern Hemisphere during the past 2,500 years. Our results are based on new records of atmospheric aerosol loading developed from high-resolution, multi-parameter measurements from an array of Greenland and Antarctic ice cores as well as distinctive age markers to constrain chronologies. Overall, cooling was proportional to the magnitude of volcanic forcing and persisted for up to ten years after some of the largest eruptive episodes. Our revised timescale more firmly implicates volcanic eruptions as catalysts in the major sixth-century pandemics, famines, and socioeconomic disruptions in Eurasia and Mesoamerica while allowing multi-millennium quantification of climate response to volcanic forcing.

Outcomes in Severe Upper GI Hemorrhage from Dieulafoy's Lesion with Monitoring of Arterial Blood Flow.

BACKGROUND: Dieulafoy's lesion (DL) is a rare but increasingly recognized cause of severe upper GI hemorrhage (SUGIH). There is little consensus regarding the endoscopic approach to management of bleeding from DL. AIMS: Our purposes were to compare 30-day outcomes of patients with SUGIH from DL with Doppler endoscopic probe (DEP) monitoring of blood flow and guided treatment versus standard visually guided hemostasis (VG). METHODS: Eighty-two consecutive DL patients with SUGIH were identified in a large CURE Hemostasis database from previous prospective cohort studies and two recent RCTs at two university-based medical centers. 30-day outcomes including rebleeding, surgery, angiography, death, and severe medical complications were compared between the two treatment groups. RESULTS: 40.2% of DL bleeds occurred in inpatients. 43.9% of patients had cardiovascular disease, and 48.7% were taking medications associated with bleeding. For the entire cohort, 41.3% (26/63) of patients treated with VG had a composite 30-day outcome as compared to 10.5% (2/19) of patients treated with DEP (p = 0.017). Rebleeding occurred within 30 days in 33.3% and 10.5% of those treated with VG and DEP, respectively (p = 0.051). After propensity score matching, the adjusted 30-day composite outcome occurred in 39.0% in the VG group compared to 2.6% in the DEP group (p < 0.001). Adjusted 30-day rebleeding occurred in 25.3% in the VG group versus 2.6% in the DEP group (p < 0.001). DISCUSSION: DL patients with SUGIH were frequently inpatients and had severe cardiovascular comorbidities and recurrent bleeding. Lesion arterial blood flow monitoring and obliteration are an effective way to treat bleeding from DL which reduces negative 30-day clinical outcomes.

Invited review: Toward a common language in data-driven mastitis detection research.

Sensor technologies for mastitis detection have resulted in the collection and availability of a large amount of data. As a result, scientific publications reporting mastitis detection research have become less driven by approaches based on biological assumptions and more by data-driven modeling. Most of these approaches try to predict mastitis events from (combinations of) raw sensor data to which a wide variety of methods are applied originating from machine learning and classical statistical approaches. However, an even wider variety in terminologies is used by researchers for methods that are similar in nature. This makes it difficult for readers from other disciplines to understand the specific methods that are used and how these differ from each other. The aim of this paper was to provide a framework (filtering, transformation, and classification) for describing the different methods applied in sensor data-based clinical mastitis detection research and use this framework to review and categorize the approaches and underlying methods described in the scientific literature on mastitis detection. We identified 40 scientific publications between 1992 and 2020 that applied methods to detect clinical mastitis from sensor data. Based on these publications, we developed and used the framework and categorized these scientific publications into the 2 data processing techniques of filtering and transformation. These data processing techniques make raw data more amendable to be used for the third step in our framework, that of classification, which is used to distinguish between healthy and nonhealthy (mastitis) cows. Most publications (n = 34) used filtering or transformation, or a combination of these 2, for data processing before classification, whereas the remaining publications (n = 6) classified the observations directly from raw data. Concerning classification, applying a simple threshold was the most used method (n = 19 publications). Our work identified that within approaches several different methods and terminologies for similar methods were used. Not all publications provided a clear description of the method used, and therefore it seemed that different methods were used between publications, whereas in fact just a different terminology was used, or the other way around. This paper is intended to serve as a reference for people from various research disciplines who need to collaborate and communicate efficiently about the topic of sensor-based mastitis detection and the methods used in this context. The framework used in this paper can support future research to correctly classify approaches and methods, which can improve the understanding of scientific publication. We encourage future research on sensor-based animal disease detection, including that of mastitis detection, to use a more coherent terminology for methods, and clearly state which technique (e.g., filtering) and approach (e.g., moving average) are used. This paper, therefore, can serve as a starting point and further stimulates the interdisciplinary cooperation in sensor-based mastitis research.

Short pulse intense pulsed light versus pulsed dye laser for the treatment of facial redness.

Treating diffuse facial redness with an intense pulsed light (IPL) source usually requires multiple sessions and may not achieve complete clearance. The 595 nm pulsed dye laser (PDL) enjoys a good reputation for reducing facial redness with non-purpuric settings. The objective of this study was to compare facial redness reduction using these two devices. After establishing the lowest light dose able to achieve transient purpura for the same pulse width of 1,5 ms with each technology, right and left sides of the face were randomly assigned for each type of treatment. There were two treatment sessions 4 weeks apart and the final evaluation was performed 8 weeks after thesecond treatment. Four blinded experienced dermatologists analyzed pre and post-treatment photographs, which demonstrated an average of 60% improvement on the segment treated with the IPL as opposed to 45% on the other segment. Pain level was described as mild and the procedure was well tolerated for both types of treatment. In this study we showed that short-pulsed intense pulsed light and PDL are similar in decreasing facial redness when non-purpuric low fluence settings are used. The IPL was faster and did not have consumables.

Search for Sterile Neutrinos in MINOS and MINOS+ Using a Two-Detector Fit.

A search for mixing between active neutrinos and light sterile neutrinos has been performed by looking for muon neutrino disappearance in two detectors at baselines of 1.04 and 735 km, using a combined MINOS and MINOS+ exposure of 16.36×10^{20} protons on target. A simultaneous fit to the charged-current muon neutrino and neutral-current neutrino energy spectra in the two detectors yields no evidence for sterile neutrino mixing using a 3+1 model. The most stringent limit to date is set on the mixing parameter sin^{2}θ_{24} for most values of the sterile neutrino mass splitting Δm_{41}^{2}>10^{-4} eV^{2}.

Comparing patient-reported outcomes entered at home versus at hospital, and testing touch screens for initial recruitment to scientific trials in arthritis patients.

OBJECTIVES: Touch screens for entering patient-reported outcomes (PROs) are available at all Danish departments of rheumatology reporting to the nationwide DANBIO registry. This project comprises two substudies in patients with rheumatoid arthritis (RA) or axial spondyloarthritis (AxSpA), aiming to (A) investigate the feasibility of first line patient recruitment for research via touch screens, and (B) compare PROs collected at hospital versus at home, including patient preferences. METHOD: Substudy A: using a touch screen, patients answered whether we could contact them about a clinical research project (yes/no). Characteristics of patients who accepted/declined were explored using chi-squared and Mann-Whitney U-tests. Substudy B (randomized crossover agreement study): a random sample of patients from the accepting group in substudy A was contacted by telephone. According to prespecified power and sample size estimation, 56 patients were included. After randomization, 50% of patients entered PROs and information on comorbidities and lifestyle from home and then at hospital, and 50% first from hospital and then at home. Finally, they stated their preference for data entry (hospital/home/equally good). Differences in PROs entered from home and in the hospital were compared (limits of agreement, 95% confidence intervals, and intraclass correlation coefficients). RESULTS: The touch-screen invitation was accepted by 428/952 patients (45%). Patients who accepted and those who declined had similar PROs and demographics. Substudy B was completed by 42 patients (22 RA, 20 AxSpA). They had no significant differences between PROs and lifestyle/comorbidity data entered from home and hospital, except for AxSpA patients on the Bath Ankylosing Spondylitis Functional Index and Bath Ankylosing Spondylitis Disease Activity Index item 5. The preferred method of data entry was hospital (10%), home (50%), and equally good (40%). CONCLUSION: Touch screens seem feasible for first line research recruitment. PROs collected from home were similar to the touch-screen solution. Patients preferred data entry from home.

Natural and anthropogenic variations in methane sources during the past two millennia.

Methane is an important greenhouse gas that is emitted from multiple natural and anthropogenic sources. Atmospheric methane concentrations have varied on a number of timescales in the past, but what has caused these variations is not always well understood. The different sources and sinks of methane have specific isotopic signatures, and the isotopic composition of methane can therefore help to identify the environmental drivers of variations in atmospheric methane concentrations. Here we present high-resolution carbon isotope data (δ(13)C content) for methane from two ice cores from Greenland for the past two millennia. We find that the δ(13)C content underwent pronounced centennial-scale variations between 100 BC and AD 1600. With the help of two-box model calculations, we show that the centennial-scale variations in isotope ratios can be attributed to changes in pyrogenic and biogenic sources. We find correlations between these source changes and both natural climate variability--such as the Medieval Climate Anomaly and the Little Ice Age--and changes in human population and land use, such as the decline of the Roman empire and the Han dynasty, and the population expansion during the medieval period.

Describing temporal variation in reticuloruminal pH using continuous monitoring data.

Reticuloruminal pH has been linked to subclinical disease in dairy cattle, leading to considerable interest in identifying pH observations below a given threshold. The relatively recent availability of continuously monitored data from pH boluses gives new opportunities for characterizing the normal patterns of pH over time and distinguishing these from abnormal patterns using more sensitive and specific methods than simple thresholds. We fitted a series of statistical models to continuously monitored data from 93 animals on 13 farms to characterize normal variation within and between animals. We used a subset of the data to relate deviations from the normal pattern to the productivity of 24 dairy cows from a single herd. Our findings show substantial variation in pH characteristics between animals, although animals within the same farm tended to show more consistent patterns. There was strong evidence for a predictable diurnal variation in all animals, and up to 70% of the observed variation in pH could be explained using a simple statistical model. For the 24 animals with available production information, there was also a strong association between productivity (as measured by both milk yield and dry matter intake) and deviations from the expected diurnal pattern of pH 2 d before the productivity observation. In contrast, there was no association between productivity and the occurrence of observations below a threshold pH. We conclude that statistical models can be used to account for a substantial proportion of the observed variability in pH and that future work with continuously monitored pH data should focus on deviations from a predictable pattern rather than the frequency of observations below an arbitrary pH threshold.

Circulating human papillomavirus DNA as a surveillance tool in head and neck squamous cell carcinoma: A systematic review and meta-analysis.

BACKGROUND: The incidence of human papillomavirus-induced (HPV+) head and neck squamous cell carcinoma (HNSCC), that is, especially oropharyngeal cancers (OPSCC), is increasing, and a significant proportion of patients encounter disease progression. A simple and sensitive test to identify patients with progression is an unmet need. OBJECTIVE OF REVIEW: To systematically review the literature and carry out a meta-analysis of studies, investigating circulating HPV-DNA as a biomarker for disease progression in patients with HNSCC. TYPE OF REVIEW: A systematic review and meta-analysis. SEARCH STRATEGY: PubMed, EMBASE and the Cochrane Library were systematically searched for articles published in English from January 1980 to November 2017. Search terms used were related to HPV, cancer sites, blood-based biomarkers and terms for specific use settings. EVALUATION METHOD: Articles reviewed and selected by authors and data on study design, demographic variables, location, HPV status, number of pre-treatment blood tests, number of post-treatment blood tests, blood HPV status and number of recurrences and length of follow-up were extracted. A meta-analysis of HPV-DNA as a diagnostic test for recurrence by means of a hierarchical summary receiver operating curve (HSROC) model was performed. RESULTS: We identified 5 studies (n = 600 subjects) examining circulating HPV-DNA in patients with HNSCC. In these 5 studies (n = 411), patients had both pre- and post-treatment blood samples. The pooled sensitivity, in detecting a recurrence, was estimated to be 54% (95% CI: 32%-74%), while the pooled specificity was 98% (95% CI: 93%-99.4%). The pooled false-positive rate is 2% (95% CI: 0.6%-7%). The area under the curve (AUC) of the summary HSROC was 0.93. Positive predictive value was estimated to 93% and the negative predictive value to 94%. CONCLUSIONS: Plasma HPV-DNA is a promising tool for surveillance in patients with HPV-related HNSCC, that is, OPSCC, and has a high specificity. By recent technical advances and by increasing follow-up blood samples, the sensitivity could likely be improved.

A method to characterize the roughness of 2-D line features: recrystallization boundaries.

A method is presented, which allows quantification of the roughness of nonplanar boundaries of objects for which the neutral plane is not known. The method provides quantitative descriptions of both the local and global characteristics. How the method can be used to estimate the sizes of rough features and local curvatures is also presented. The potential of the method is illustrated by quantification of the roughness of two recrystallization boundaries in a pure Al specimen characterized by scanning electron microscopy.

Monitoring total-body inflammation and damage in joints and entheses: the first follow-up study of whole-body magnetic resonance imaging in rheumatoid arthritis.

OBJECTIVE: To investigate changes in whole-body magnetic resonance imaging (WBMRI) inflammatory and structural lesions in most joints and entheses in patients with rheumatoid arthritis (RA) treated with adalimumab. METHODS: WBMRI was obtained at weeks 0, 6, 16, and 52 in a 52 week follow-up study of 37 RA patients starting treatment with adalimumab. Readability and reliability of WBMRI were investigated for 76 peripheral joints, 23 discovertebral units, the sacroiliac joints, and 33 entheses. Changes in WBMRI joint and entheses counts were investigated. RESULTS: The readability of peripheral and axial joints was 82-100%, being less for elbows and small joints of the feet. For entheses, 72-100% were readable, except for entheses at the anterior chest wall, elbow, knee, and plantar fascia. The intrareader agreement was high for bone marrow oedema (BMO), bone erosion (80-100%), and enthesitis (77-100%), and slightly lower for synovitis and soft tissue inflammation (50-100%). All synovitis, BMO, and soft tissue inflammation counts decreased numerically during treatment. The 26-joint synovitis WBMRI count decreased significantly during the first 16 weeks for patients with a good European League Against Rheumatism (EULAR) response (from median 6 to 4, p < 0.05), but not for patients with a moderate or no EULAR response. There were no overall changes in structural lesions. CONCLUSIONS: WBMRI allows simultaneous monitoring of most axial and peripheral joints and entheses in RA patients and can visualize a decrease in inflammatory counts during treatment. This first WBMRI follow-up study of patients with RA encourages further investigation of the usefulness of WBMRI in RA.

Surfactant concentration and type affects the removal of Escherichia coli from pig skin during a simulated hand wash.

The effect of surfactant type and concentration on a bland soap formulation's ability to remove bacteria from hands remains largely unstudied. Several combinations of surfactants and water were combined to test bacterial removal efficacy using a hand-washing device (two pieces of pig skin and a mechanical motor) to simulate a hand wash. A nalidixic acid-resistant, nonpathogenic strain of Escherichia coli (ATCC 11229) was used. Two anionic surfactants, sodium lauryl sulphate and sodium stearoyl lactylate, and two nonionic surfactants, poloxamer 407 and sorbitan monostearate, each in concentrations of 2, 5 and 10% were studied. A slight positive (r2  = 0·17) but significant (P = 0·03) correlation was observed between hydrophile-lipophile balance value and mean log reduction. No correlation was observed between pH of the treatment solution and the mean log reduction (r2  = 0·05, P = 0·25). A 10% sodium lauryl sulphate mixture showed the highest log reduction (x¯ = 1·1 log CFU reduction, SD = 0·54), and was the only treatment significantly different from washing with water (P = 0·0005). There was a correlation between increasing surfactant concentrations above the critical micelle concentration, and mean microbial reduction (r2  = 0·62, P = 0·001). SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterizes the role of surfactants in removing microbes during a hand wash. Numerous studies address how surfactants support antimicrobial effect in soap, or cause irritation of skin, but no published studies show which surfactants are best for removing microbes. We used pig skin as a model for human skin and a lathering device to simulate a hand wash. A 10% sodium lauryl sulphate mixture was the only treatment significantly different from a water wash. There was a strong correlation between increasing surfactant concentrations above the critical micelle concentration and mean microbial reduction.

A method for culturing Gram-negative skin microbiota.

BACKGROUND: Commensal Gram-negative (CGN) microbiota have been identified on human skin by DNA sequencing; however, methods to reliably culture viable Gram-negative skin organisms have not been previously described. RESULTS: Through the use of selective antibiotics and minimal media we developed methods to culture CGN from skin swabs. We identified several previously uncharacterized CGN at the species level by optimizing growth conditions and limiting the inhibitory effects of nutrient shock, temperature, and bacterial competition, factors that may have previously limited CGN isolation from skin cultures. CONCLUSIONS: Our protocol will permit future functional studies on the influences of CGN on skin homeostasis and disease.

Search for Sterile Neutrinos Mixing with Muon Neutrinos in MINOS.

We report results of a search for oscillations involving a light sterile neutrino over distances of 1.04 and 735 km in a ν_{µ}-dominated beam with a peak energy of 3 GeV. The data, from an exposure of 10.56×10^{20} protons on target, are analyzed using a phenomenological model with one sterile neutrino. We constrain the mixing parameters θ_{24} and Δm_{41}^{2} and set limits on parameters of the four-dimensional Pontecorvo-Maki-Nakagawa-Sakata matrix, |U_{µ4}|^{2} and |U_{τ4}|^{2}, under the assumption that mixing between ν_{e} and ν_{s} is negligible (|U_{e4}|^{2}=0). No evidence for ν_{µ}→ν_{s} transitions is found and we set a world-leading limit on θ_{24} for values of Δm_{41}^{2}≲1 eV^{2}.

Molecular profiling of tumour budding implicates TGFß-mediated epithelial-mesenchymal transition as a therapeutic target in oral squamous cell carcinoma.

Although tumour budding is an adverse prognostic factor for many cancer types, the molecular mechanisms governing this phenomenon are incompletely understood. Therefore, understanding the molecular basis of tumour budding may provide new therapeutic and diagnostic options. We employ digital image analysis to demonstrate that the number of tumour buds in cytokeratin-stained sections correlates with patients having lymph node metastases at diagnosis. The tumour bud count was also a predictor of overall survival, independent of TNM stage. Tumour buds and paired central tumour areas were subsequently collected from oral squamous cell carcinoma (OSCC) specimens, using laser capture microdissection, and examined with RNA sequencing and miRNA-qPCR arrays. Compared with cells from the central parts of the tumours, budding cells exhibited a particular gene expression signature, comprising factors involved in epithelial-mesenchymal transition (EMT) and activated TGFß signalling. Transcription factors ZEB1 and PRRX1 were up-regulated concomitantly with the decreased expression of mesenchymal-epithelial (MET) transcription factors (eg OVOL1) in addition to Krüppel-like factors and Grainyhead-like factors. Moreover, miR-200 family members were down-regulated in budding tumour cells. We used immunohistochemistry to validate five markers of the EMT/MET process in 199 OSCC tumours, as well as in situ hybridization in 20 OSCC samples. Given the strong relationship between tumour budding and the development of lymph node metastases and an adverse prognosis, therapeutics based on inhibiting the activation of TGFß signalling may prove useful in the treatment of OSCC.