The apoptotic and proliferation rate of tumour budding cells in colorectal cancer outlines a heterogeneous population of cells with various impacts on clinical outcome

AIMS In colorectal cancer (CRC), tumour buds represent an aggressive cell type at the invasive front with apparently low proliferation. The aim of this study was to determine proliferation and apoptotic rates of buds in comparison to tumour centre, front and mucosa. METHODS AND RESULTS Whole tissue sections from 188 CRC patients underwent immunohistochemistry for Ki67. Ten high-power fields (HPFs) were evaluated in mucosa, tumour centre, tumour front and tumour buds (total = 40 HPFs/case). Caspase-3 and M30 immunohistochemistry were performed on a multipunch tissue microarray from the same cohort. Ki67, caspase-3 and M30 immunoreactivity were correlated with outcome. The average percentage of cells showing Ki67 positivity was 5.2% in mucosa, and was not significantly different between the centre and front of the tumour (38.2% and 34.9%; P < 0.0001); 0.3% of buds showed Ki67 positivity (P < 0.0001). Caspase-3 expression was similar in mucosa, tumour centre and tumour front, but lower in tumour buds (<0.1%; P < 0.0001). M30 staining in buds was decreased (0.01%; P < 0.0001) in comparison to other areas. Ki67 positivity in buds was detrimental to survival in univariate (P = 0.0352) and multivariate (P = 0.0355) analysis. Caspase-3-positive tumours showed better outcome than negative tumours (P = 0.0262); but tumours with caspase-3-positive buds showed a worse outcome than those with caspase-3-negative buds (P = 0.0235). CONCLUSIONS Ki67, caspase-3 and M30 staining is absent in most tumour buds, suggesting decreased proliferation and apoptosis. However, the fact that Ki67 and caspase-3 immunoreactivity was associated with unfavourable prognosis points to a heterogeneous population of tumour buds.

Diagnostic and prognostic implications of the PAX8-PPARγ translocation in thyroid carcinomas-a TMA-based study of 226 cases

AIMS Follicular thyroid carcinoma (FTC) has been a diagnostic challenge for decades. The PAX8-PPARγ rearrangement has been detected in FTC and classic papillary thyroid carcinomas (PTCs). The aims of this study were to assess the presence of PAX8-PPARγ by using tissue microarrays in a large cohort of different thyroid neoplasms, and to assess its diagnostic and prognostic implications. METHODS AND RESULTS Fluorescence in-situ hybridization (FISH) analysis for PAX8-PPARγ was performed on 226 thyroid tumours, comprising FTCs (n = 59), PTCs (n = 126), poorly differentiated thyroid carcinomas (PDs; n = 34), follicular thyroid adenomas (FTAs; n = 5), and follicular tumours of unknown malignant potential (FTUMPs; n = 2). PAX8-PPARγ was detected in 12% of FTCs, 1% of PTCs, 7% of PDs, and in both cases of FTUMP. There was no correlation between the extent of capsular or vascular invasion and PAX8-PPARγ, or between lymph node or haematogenous metastasis and PAX8-PPARγ. Overall survival (OS), tumour-specific survival (TSS) and relapse-free-survival (RFS) were not influenced by PAX8-PPARγ. CONCLUSIONS In this study, we demonstrate for the first time the presence of PAX8-PPARγ in PDs and FTUMPs, whereas in FTCs and PTCs the prevalence of PAX8-PPARγ is lower than previously reported. PAX8-PPARγ did not correlate with invasiveness or affect prognosis in any tumour type.

In vitro metabolism of testosterone in the horse liver and involvement of equine CYPs 3A89, 3A94 and 3A95

Testosterone (TES) 6-β-hydroxylation is a significant metabolic step in the biotransformation of TES in human liver microsomes and reflects cytochrome P450 (CYP) 3A4/5 specific metabolic activity. Several CYP3A enzymes have been annotated in the horse genome, but functional characterization is missing. This descriptive study investigates TES metabolism in the horse liver in vitro and the qualitative contribution of three CYP3A isoforms of the horse. Metabolism of TES was investigated by using equine hepatocyte primary cultures and liver microsomes. Chemical inhibitors were used to determine the CYPs involved in TES biotransformation in equine microsomes. Single CYPs 3A89, 3A94, and 3A95, recombinantly expressed in V79 hamster lung fibroblasts, were incubated with TES and the fluorescent metabolite 7-benzyloxy-4-trifluoromethylcoumarin (BFC). The effect of ketoconazole and troleandomycin was evaluated on single CYPs. Testosterone metabolites were analyzed by HPLC and confirmed by GC/MS. In hepatocyte primary cultures, the most abundant metabolite was androstenedione (AS), whereas in liver microsomes, 6-β-hydroxytestosterone showed the largest peak. Formation of 6-β-hydroxytestosterone and 11-β-hydroxytestosterone in liver microsomes was inhibited by ketoconazole, troleandomycin, and quercetin. Equine recombinant CYP3A95 catalyzed 11-β-hydroxylation of testosterone (TES). Metabolism of BFC was significantly inhibited by ketoconazole in CYP3A95, whereas troleandomycin affected the activities of CYP3A94 and CYP3A95. Both inhibitors had no significant effect on CYP3A89. Metabolic reactions and effects of inhibitors differed between the equine CYP3A isoforms investigated. This has to be considered in future in vitro studies.

Neotethys closure history of Anatolia: insights from 40Ar–39Ar geochronology and P–T estimation in high pressure metasedimentary rocks

The multiple high-pressure (HP), low-temperature (LT) metamorphic units of Western and Central Anatolia offer a great opportunity to investigate the subduction- and continental accretion-related evolution of the eastern limb of the long-lived Aegean subduction system. Recent reports of the HP–LT index mineral Fe-Mg-carpholite in three metasedimentary units of the Gondwana-derived Anatolide–Tauride continental block (namely the Afyon Zone, the Ören Unit and the southern Menderes Massif) suggest a more complicated scenario than the single-continental accretion model generally put forward in previous studies. This study presents the first isotopic dates (white mica 40Ar–39Ar geochronology), and where possible are combined with P–T estimates (chlorite thermometry, phengite barometry, multi-equilibrium thermobarometry), on carpholite-bearing rocks from these three HP–LT metasedimentary units. It is shown that, in the Afyon Zone, carpholite-bearing assemblages were retrogressed through greenschist-facies conditions at c. 67–62 Ma. Early retrograde stages in the Ören Unit are dated to 63–59 Ma. In the Kurudere–Nebiler Unit (HP Mesozoic cover of the southern Menderes Massif), HP retrograde stages are dated to c. 45 Ma, and post-collisional cooling to c. 26 Ma. These new results support that the Ören Unit represents the westernmost continuation of the Afyon Zone, whereas the Kurudere–Nebiler Unit correlates with the Cycladic Blueschist Unit of the Aegean Domain. In Western Anatolia, three successive HP–LT metamorphic belts thus formed: the northernmost Tavşanlı Zone (c. 88–82 Ma), the Ören–Afyon Zone (between 70 and 65 Ma), and the Kurudere–Nebiler Unit (c. 52–45 Ma). The southward younging trend of the HP–LT metamorphism from the upper and internal to the deeper and more external structural units, as in the Aegean Domain, points to the persistence of subduction in Western Anatolia between 93–90 and c. 35 Ma. After the accretion of the Menderes–Tauride terrane, in Eocene times, subduction stopped, leading to continental collision and associated Barrovian-type metamorphism. Because, by contrast, the Aegean subduction did remain active due to slab roll-back and trench migration, the eastern limb (below Southwestern Anatolia) of the Hellenic slab was dramatically curved and consequently teared. It therefore is suggested that the possibility for subduction to continue after the accretion of buoyant (e.g. continental) terranes probably depends much on palaeogeography.

Endothelial function and sleep: associations of flow-mediated dilation with perceived sleep quality and rapid eye movement (REM) sleep.

Endothelial function typically precedes clinical manifestations of cardiovascular disease and provides a potential mechanism for the associations observed between cardiovascular disease and sleep quality. This study examined how subjective and objective indicators of sleep quality relate to endothelial function, as measured by brachial artery flow-mediated dilation (FMD). In a clinical research centre, 100 non-shift working adults (mean age: 36 years) completed FMD testing and the Pittsburgh Sleep Quality Index, along with a polysomnography assessment to obtain the following measures: slow wave sleep, percentage rapid eye movement (REM) sleep, REM sleep latency, total arousal index, total sleep time, wake after sleep onset, sleep efficiency and apnea-hypopnea index. Bivariate correlations and follow-up multiple regressions examined how FMD related to subjective (i.e., Pittsburgh Sleep Quality Index scores) and objective (i.e., polysomnography-derived) indicators of sleep quality. After FMD showed bivariate correlations with Pittsburgh Sleep Quality Index scores, percentage REM sleep and REM latency, further examination with separate regression models indicated that these associations remained significant after adjustments for sex, age, race, hypertension, body mass index, apnea-hypopnea index, smoking and income (Ps < 0.05). Specifically, as FMD decreased, scores on the Pittsburgh Sleep Quality Index increased (indicating decreased subjective sleep quality) and percentage REM sleep decreased, while REM sleep latency increased (Ps < 0.05). Poorer subjective sleep quality and adverse changes in REM sleep were associated with diminished vasodilation, which could link sleep disturbances to cardiovascular disease.

How does the taxonomic status of allopatric populations influence species richness within African cichlid fish assemblages?

Aim  Current estimates of species richness within rapidly evolving species flocks are often highly dependent on the species status of allopatric populations that differ in phenotypic traits. These traits may be unreliable indicators of biological species status and systematists may have inconsistently assigned species among lineages or locations on the basis of these traits, thus hampering comparative studies of regional species richness and speciation rates. Our aim was to develop a method of generating standardized estimates of regional species richness suitable for comparative analysis, and to use these estimates to examine the extent and consistency of species assignment of allopatric populations within rapidly evolving cichlid fish flocks present in three east African lakes. Location  Lakes Malawi, Victoria and Tanganyika. Methods  Using published taxon co-occurrence data, a novel approach was employed to calculate standardized ‘minimum’ estimates of regional species richness for hard substrate associated complexes of cichlids within each of the lakes. Minimum estimates were based on an explicit assumption that if taxa present on equivalent habitats have disjunct distributions, then they are allopatric forms of the same species. These estimates were compared with current observed ‘high-end’ regional species richness estimates for those complexes to determine the consistency of species assignment of allopatric populations between lineages within a lake. A ‘sympatry’ index was developed to enable comparisons of levels of species assignment of allopatric populations between-lakes to be made. Results  Within each lake, the minimum and high-end estimates for species richness were significantly correlated across complexes, indicating that the complexes that contain more recognized species contain the most genuine biological species. However, comparisons of complexes among lakes revealed considerable differences. For equivalent geographical areas, substantially higher proportions of recognized species were totally allopatric within the studied Lake Malawi and Lake Victoria complexes, than those of Lake Tanganyika. Main Conclusions  Among African lakes, levels of assignment to species status of allopatric populations were found to be distinctly different. It is unclear whether the discrepancies are a consequence of differences between the lake faunas in degrees of phenotypic divergence among allopatric populations, or are simply the result of inconsistent taxonomic practices. In either case, these results have considerable wider relevance for they emphasize that quantitative measures of regional and beta diversity are critically dependent on the species status of allopatric populations, an issue usually neglected in comparative studies of species richness. The technique introduced here can be used to standardize measures of regional diversity of lineages for comparative analyses, potentially enabling more accurate identification of processes influencing rates of speciation.

Sleep respiratory disturbances and arousals at moderate altitude have overlapping electroencephalogram spectral signatures

An ascent to altitude has been shown to result in more central apneas and a shift towards lighter sleep in healthy individuals. This study employs spectral analysis to investigate the impact of respiratory disturbances (central/obstructive apnea and hypopnea or periodic breathing) at moderate altitude on the sleep electroencephalogram (EEG) and to compare EEG changes resulting from respiratory disturbances and arousals. Data were collected from 51 healthy male subjects who spent 1 night at moderate altitude (2590 m). Power density spectra of Stage 2 sleep were calculated in a subset (20) of these participants with sufficient artefact-free data for (a) epochs with respiratory events without an accompanying arousal, (b) epochs containing an arousal and (c) epochs of undisturbed Stage 2 sleep containing neither arousal nor respiratory events. Both arousals and respiratory disturbances resulted in reduced power in the delta, theta and spindle frequency range and increased beta power compared to undisturbed sleep. The similarity of the EEG changes resulting from altitude-induced respiratory disturbances and arousals indicates that central apneas are associated with micro-arousals, not apparent by visual inspection of the EEG. Our findings may have implications for sleep in patients and mountain tourists with central apneas and suggest that respiratory disturbances not accompanied by an arousal may, none the less, impact sleep quality and impair recuperative processes associated with sleep more than previously believed.

Optional course "Homeopathically potentised substances: placebos or effective?" for medical students at the University of Bern, Switzerland

Background Apart from compulsory lectures on classical homeopathy (CH), traditional Chinese medicine/acupuncture, neural therapy and anthroposophically extended medicine (AEM), our institute at the University of Bern offers several optional practical courses for medical students. The aim of this course during autumn 2011 and spring 2012 was to discuss basic research, observational and clinical studies in the fields of CH and AEM, so that students i) learned how to read and appraise scientific publications, ii) learned how complementary medicine can be investigated with scientific methods, and iii) were able to form their own opinion about the possible specific effects and effectiveness of homeopathically potentised substances. Methods Introductory lectures on AEM, CH and study design were given to 12 second year medical students. The students appraised 12 research articles and presented the results in class, followed by discussions with experts in the fields of basic and clinical research from our institute. A company producing homeopathic remedies was visited and students could practise potentization procedures and trituration. At the end of the course, students compiled posters with arguments in favour of and against specific effects and effectiveness as well as their own conclusions. The course was evaluated using a written questionnaire with closed and open questions. Results Previous knowledge about CH and AEM was scarce among the students. It slightly increased during the course, and the course itself fostered their interest on the topic. This course was chosen by most students, because they were genuinely interested in the topic (and not because other courses they had wanted to visit were fully booked). The students especially valued the discussions, the various perspectives presented to them, and experiencing a potentization process. Conclusion Medical students were interested to learn more about homeopathically potentised substances. The contradictory study results made it difficult for them to form their own opinion. Apart from appraising articles, the students would have liked to meet and talk to patients.

Actigraphic assessment of periodic leg movements in patients with restless legs syndrome.

The diagnosis of restless legs syndrome (RLS) relies upon diagnostic criteria which are based on history only, and dopaminergic treatment is not normally the first choice of treatment for all patients. It would be worthwhile to identify patients non-responsive to dopaminergic treatment beforehand, because they may suffer from a restless legs-like syndrome and may require alternative treatment. We included retrospectively 24 adult patients fulfilling the four essential criteria for restless legs and 12 age-matched healthy controls. They were investigated by ambulatory actigraphy from both legs over three nights, and patients started treatment with dopamine agonists after this diagnostic work-up. We examined 12 responders to dopaminergic treatment and 12 non-responders and studied the association between response to dopaminergic treatment and the periodic limb movement index (PLMI) as assessed with actigraphy. Demographic characteristics, excessive daytime sleepiness and fatigue at baseline were similar in all three groups. Baseline RLS severity was similar between responders and non-responders [International Restless Legs Severity Scale (IRLS): 25 ± 9 and 24 ± 8]. Group comparisons of PLMI before treatment initiation showed significant differences between the three groups. Post-hoc pairwise comparisons revealed that healthy controls had significantly lower PLMI (4.9 ± 4.5) than responders (29.3 ± 22.7) and non-responders (13.3 ± 11.2). Similarly, the PLMI in responders was lower than in non-responders. PLMI day-to-day variability did not differ between responders and non-responders and there was no correlation between treatment effect, as assessed by the decrease of the IRLS and baseline PLMI. Our retrospective study indicates that actigraphy to assess periodic limb movements may contribute to a better diagnosis of dopamine-responsive restless legs syndrome.

Lack of efficacy of low-dose spironolactone as adjunct treatment to conventional congestive heart failure treatment in dogs.

Aldosterone plays an important role in the pathophysiology of heart failure. Aldosterone receptor blockade has been shown to reduce morbidity and mortality in human patients with advanced congestive left ventricular heart failure. This study was designed to assess the efficacy and tolerance of long-term low-dose spironolactone when added to conventional heart failure treatment in dogs with advanced heart failure. Eighteen client-owned dogs with advanced congestive heart failure due to either degenerative valve disease (n=11) or dilated cardiomyopathy (n=7) were included in this prospective, placebo-controlled, double-blinded, randomized clinical study. After initial stabilization including furosemide, angiotensin-converting enzyme inhibitors, pimobendan and digoxin, spironolactone at a median dose of 0.52 mg/kg (range 0.49-0.8 mg/kg) once daily (n=9) or placebo (n=9) was added to the treatment, and the dogs were reassessed 3 and 6 months later. Clinical scoring, echocardiography, electrocardiogram, systolic blood pressure measurement, thoracic radiography, sodium, potassium, urea, creatinine, alanine aminotransferase, aldosterone and aminoterminal atrial natriuretic propeptide were assessed at baseline, 3 and 6 months. Survival times were not significantly different between the two treatment groups. Spironolactone was well tolerated when combined with conventional heart failure treatment.

Seedling resistance, tolerance and escape from herbivores: insights from co-dominant canopy tree species in a resource-poor African rain forest

* Although plants can reduce the impacts of herbivory in multiple ways, these defensive traits are often studied in isolation and an understanding of the resulting strategies is incomplete. * In the study reported here, empirical evidence was simultaneously evaluated for the three main sets of traits available to plants: (i) resistance through constitutive leaf traits, (ii) tolerance to defoliation and (iii) escape in space, for three caesalpiniaceous tree species Microberlinia bisulcata, Tetraberlinia bifoliolata and T. korupensis, which co-dominate groves within the lowland primary rain forest of Korup National Park (Cameroon). * Mesh cages were placed around individual wild seedlings to exclude insect herbivores at 41 paired canopy gap and understorey locations. After following seedling growth and survival for c. 2 years, caged and control treatments were removed, leaves harvested to determine nutrient and phenolic concentrations, leaf mass per area estimated, and seedling performance in gaps followed for a further c. 2 years to quantify tolerance to the leaf harvesting. * The more nutrient-rich leaves of the weakly shade-tolerant M. bisulcata were damaged much more in gaps than the two strongly shade-tolerant Tetraberlinia species, which had higher leaf mass per area and concentrations of total phenols. Conversely, the faster-growing M. bisulcata was better able to tolerate defoliation in terms of height growth (reflushing capacity), but not at maintaining overall leaf numbers, than the other two species. * Across gaps, insect-mediated Janzen–Connell effects were most pronounced for M. bisulcata, less so for T. korupensis, and not detectable for T. bifoliolata. The three species differed distinctly in their secondary metabolic profiles. * Taken together, the results suggested a conceptual framework linking the three sets of traits, one in which the three co-dominant species adopt different strategies towards herbivore pressure depending on their different responses to light availability. This study is one of the first in a natural forest ecosystem to examine resistance to, tolerance of, and escape from herbivory among a group of co-occurring tropical tree species.

Climate and soil attributes determine plant species turnover in global drylands

Aim Geographical, climatic and soil factors are major drivers of plant beta diversity, but their importance for dryland plant communities is poorly known. The aim of this study was to: (1) characterize patterns of beta diversity in global drylands; (2) detect common environmental drivers of beta diversity; and (3) test for thresholds in environmental conditions driving potential shifts in plant species composition. Location Global. Methods Beta diversity was quantified in 224 dryland plant communities from 22 geographical regions on all continents except Antarctica using four complementary measures: the percentage of singletons (species occurring at only one site); Whittaker's beta diversity, β(W); a directional beta diversity metric based on the correlation in species occurrences among spatially contiguous sites, β(R2); and a multivariate abundance-based metric, β(MV). We used linear modelling to quantify the relationships between these metrics of beta diversity and geographical, climatic and soil variables. Results Soil fertility and variability in temperature and rainfall, and to a lesser extent latitude, were the most important environmental predictors of beta diversity. Metrics related to species identity percentage of singletons and β(W) were most sensitive to soil fertility, whereas those metrics related to environmental gradients and abundance (β(R2) and β(MV) were more associated with climate variability. Interactions among soil variables, climatic factors and plant cover were not important determinants of beta diversity. Sites receiving less than 178 mm of annual rainfall differed sharply in species composition from more mesic sites (> 200 mm). Main conclusions Soil fertility and variability in temperature and rainfall are the most important environmental predictors of variation in plant beta diversity in global drylands. Our results suggest that those sites annually receiving c. 178 mm of rainfall will be especially sensitive to future climate changes. These findings may help to define appropriate conservation strategies for mitigating effects of climate change on dryland vegetation.

Speech outcome in complete unilateral cleft lip and palate - a comparison of three methods of the hard palate closure.

The aim of this study was to compare the speech in subjects with cleft lip and palate, in whom three methods of the hard palate closure were used. One hundred and thirty-seven children (96 boys, 41 girls; mean age = 12 years, SD = 1·2) with complete unilateral cleft lip and palate (CUCLP) operated by a single surgeon with a one-stage method were evaluated. The management of the cleft lip and soft palate was comparable in all subjects; for hard palate repair, three different methods were used: bilateral von Langenbeck closure (b-vL group, n = 39), unilateral von Langenbeck closure (u-vL group, n = 56) and vomerplasty (v-p group, n = 42). Speech was assessed: (i) perceptually for the presence of a) hypernasality, b) compensatory articulations (CAs), c) audible nasal air emissions (ANE) and d) speech intelligibility; (ii) for the presence of compensatory facial grimacing, (iii) with clinical intra-oral evaluation and (iv) with videonasendoscopy. A total rate of hypernasality requiring pharyngoplasty was 5·1%; total incidence post-oral compensatory articulations (CAs) was 2·2%. The overall speech intelligibility was good in 84·7% of cases. Oronasal fistulas (ONFs) occurred in 15·7% b-vL subjects, 7·1% u-vL subjects and 50% v-p subjects (P < 0·001). No statistically significant intergroup differences for hypernasality, CAs and intelligibility were found (P > 0·1). In conclusion, the speech after early one-stage repair of CUCLP was satisfactory. The method of hard palate repair affected the incidence of ONFs, which, however, caused relatively mild and inconsistent speech errors.