Long‐term tag retention on two species of small cetaceans

The effects of tagging on small cetaceans are difficult to assess due to logistical difficulties in recapturing the whales. In this study two narwhals, Monodon monoceros, and five harbor porpoises, Phocoena phocoena, were recaptured between 297 and 767 days after instrumentation with satellite transmitters. The transmitters were mounted by pins that were pushed through the fins of the porpoises or the backs of the narwhals. Overall body condition seemed unaffected by the instrumentations. Macroscopical examination revealed that umbilicalization of the tissue surrounding the pins was almost complete. On one of the narwhals the reepithelialization created a closed tunnel where the pins were isolated from the subdermal tissue, however the reepithelialization was incomplete around the middle of the pin and a low‐grade inflammation increased with decreasing thickness of epidermis. The inflammation consisted of mononuclear cells, mainly lymphocytes. With increasing inflammation the number of neutrophils and macrophages increased. In the lymphoid follicular hyperplasia macrophages and a few neutrophils were found, in one case accompanied by Splendore‐Hoeppli material with radiating eosinophilic clubs and Gram‐positive cocci. Immunohistochemical staining of the cocci for Staphylococcus aureus was positive. The observations from the recaptured cetaceans suggest that the instrumentations caused only temporary and low‐grade inflammatory responses.     

An empirical comparison of models for the phenology of bird migration

Bird migration phenology shows strong responses to climate change. Studies of trends and patterns in phenology are typically based on annual summarizing metrics, such as means and quantiles calculated from raw daily count data. However, with irregularly sampled data and large day‐to‐day variation, such metrics can be biased and noisy, and may be analysed using phenological functions fitted to the data. Here we use count data of migration passage from a Finnish bird observatory to compare different models for the phenological distributions of spring migration (27 species) and autumn migration (57 species). We assess parsimony and goodness‐of‐fit in a set of models, with phenological functions of different complexity, optionally with covariates accounting for day‐to‐day variability. The covariates describe migration intensities of related species or relative migration intensities the previous day (autocovariates). We found that parametric models are often preferred over the more flexible generalized additive models with constrained degrees of freedom. Models corresponding to a mixture of two distinct passing populations were frequently preferred over simpler ones, but usually no more complex models are needed. Slightly more complex models were favoured in spring compared to autumn. Related species’ migration activity effectively improves the model by accounting for the large day‐to‐day variation. Autocovariates were usually not that relevant, implying that autocorrelation is generally not a major concern if phenology is modelled properly. We suggest that parametric models are relatively good for studying single‐population migration phenology, or a mix of two groups with distinct phenologies, especially if daily variation in migration intensity can be controlled for. Generalized additive models may be useful when the migrating population composition is unknown. Despite these guidelines, choosing an appropriate model involves case‐by‐case assessment or the biological relevance and rationale for modelling phenology.     

Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3–43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (K_D = 220 pM) and HER3-expressing tumor cells with EC₅₀ values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3–43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3–43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.     

A simple method to induce hypoxia-induced vascular endothelial growth factor-A (VEGF-A) expression in T24 human bladder cancer cells

Angiogenesis is an essential process for the establishment, development, and dissemination of several malignant tumors including bladder cancer. The hypoxic condition promotes the stabilization of hypoxia-inducible factor 1 alpha (HIF-1α), which translocates to the nucleus to mediate angiogenic factors including the vascular endothelial growth factor A (VEGF-A). AnaeroGen system was developed for microbiology area to create a low oxygen tension required to the growth of anaerobic bacteria. Here, we hypothesized the use of AnaeroGen system to induce hypoxia in T24 human bladder carcinoma cells, in order to promote the overexpression of VEGF-A. T24 cells were cultured in six-well plates containing McCoy medium. Exposures of T24 cells to hypoxia for 1, 8, 24, and 48 h were performed using the Oxoid AnaeroGen system, while T24 cells under normoxia were used as control. The expression of VEGF-A and HIF-1α was analyzed by real-time PCR. ELISA for HIF-1α was carried out. The VEGF-A expression increased significantly by Oxoid AnaeroGen-induced hypoxia in a time-depending manner, reaching the peak in 48 h of hypoxia. Although HIF-1α mRNA was not changed, HIF-1α protein was increased in the presence of hypoxia, reaching a peak at 8 h. These results demonstrated that the Oxoid AnaeroGen system is a simple method to expose T24 cells to hypoxia and efficiently to upregulate VEGF expression in T24 cells.     

Idiosyncratic responses to climate‐driven forest fragmentation and marine incursions in reed frogs from Central Africa and the Gulf of Guinea Islands

Organismal traits interact with environmental variation to mediate how species respond to shared landscapes. Thus, differences in traits related to dispersal ability or physiological tolerance may result in phylogeographic discordance among co‐distributed taxa, even when they are responding to common barriers. We quantified climatic suitability and stability, and phylogeographic divergence within three reed frog species complexes across the Guineo‐Congolian forests and Gulf of Guinea archipelago of Central Africa to investigate how they responded to a shared climatic and geological history. Our species‐specific estimates of climatic suitability through time are consistent with temporal and spatial heterogeneity in diversification among the species complexes, indicating that differences in ecological breadth may partly explain these idiosyncratic patterns. Likewise, we demonstrated that fluctuating sea levels periodically exposed a land bridge connecting Bioko Island with the mainland Guineo‐Congolian forest and that habitats across the exposed land bridge likely enabled dispersal in some species, but not in others. We did not find evidence that rivers are biogeographic barriers across any of the species complexes. Despite marked differences in the geographic extent of stable climates and temporal estimates of divergence among the species complexes, we recovered a shared pattern of intermittent climatic suitability with recent population connectivity and demographic expansion across the Congo Basin. This pattern supports the hypothesis that genetic exchange across the Congo Basin during humid periods, followed by vicariance during arid periods, has shaped regional diversity. Finally, we identified many distinct lineages among our focal taxa, some of which may reflect incipient or unrecognized species.     

Effect of flecainide on atrial fibrillatory rate in a large animal model with induced atrial fibrillation

Background

Atrial fibrillatory cycle length has been considered one of the indices of atrial electrical remodelling during atrial fibrillation (AF), which can be assessed from surface ECG by computer-assisted calculation of atrial fibrillatory rate (AFR). Horses have been suggested as a bona fide model for AF studies since horses too, develop lone AF, however data on AF characteristics in horses are extremely sparse and non-invasive characterization of AF complexity using surface ECG processing has not been reported.

Aim

The aim was to study characteristics of induced AF and its modification by flecainide.

Methods

The study group consisted on 3 horses with spontaneous persistent AF and 13 with pace-induced AF. Seven horses were treated with saline (control) and eight with flecainide (2 mg/kg). ECGs were analysed using spatiotemporal cancellation of QRST complexes and calculation of AFR from the residual atrial signal.

Results

At AF onset, AFR was 295?±?52 fibrillations per minute (fpm) in the horses with induced AF treated with flecainide, 269?±?36 fpm in the control group (ns), and 364?±?26 fpm in the horses with spontaneous persistent AF (P?<?0.05 compared to the control group). Flecainide caused a decrease in AFR in all animals and restored sinus rhythm in the animals with induced AF. In the control animals, AFR increased from 269?±?36 fpm to a plateau of 313?±?14 fpm before decreasing to 288?±?28 fpm during the last 10% of the AF episodes preceding spontaneous conversion (P?<?0.05).

Conclusion

AFR in horses with induced AF resembles AFR in humans with paroxysmal AF. Flecainide caused a rapid decrease in AFR in all horses, further supporting the method to be a non-invasive technique to study the effect of antiarrhythmic compounds.

     

Genotyping‐by‐sequencing‐based genome‐wide association studies on Verticillium wilt resistance in autotetraploid alfalfa (Medicago sativa L.)

Verticillium wilt (VW) is a fungal disease that causes severe yield losses in alfalfa. The most effective method to control the disease is through the development and use of resistant varieties. The identification of marker loci linked to VW resistance can facilitate breeding for disease‐resistant alfalfa. In the present investigation, we applied an integrated framework of genome‐wide association with genotyping‐by‐sequencing (GBS) to identify VW resistance loci in a panel of elite alfalfa breeding lines. Phenotyping was performed by manual inoculation of the pathogen to healthy seedlings, and scoring for disease resistance was carried out according to the standard test of the North America Alfalfa Improvement Conference (NAAIC). Marker–trait association by linkage disequilibrium identified 10 single nucleotide polymorphism (SNP) markers significantly associated with VW resistance. Alignment of the SNP marker sequences to the M. truncatula genome revealed multiple quantitative trait loci (QTLs). Three, two, one and five markers were located on chromosomes 5, 6, 7 and 8, respectively. Resistance loci found on chromosomes 7 and 8 in the present study co‐localized with the QTLs reported previously. A pairwise alignment (blastn ) using the flanking sequences of the resistance loci against the M. truncatula genome identified potential candidate genes with putative disease resistance function. With further investigation, these markers may be implemented into breeding programmes using marker‐assisted selection, ultimately leading to improved VW resistance in alfalfa.     

<Emphasis Type="Italic">In Vitro</Emphasis> Model Simulating Gastro-Intestinal Digestion in the Pediatric Population (Neonates and Young Infants)

The focus on drug delivery for the pediatric population has been steadily increasing in the last decades. In terms of developing in vitro models simulating characteristics of the targeted pediatric population, with the purpose of predicting drug product performance after oral administration, it is important to simulate the gastro-intestinal conditions and processes the drug will encounter upon oral administration. When a drug is administered in the fed state, which is commonly the case for neonates, as they are typically fed every 3 h, the digestion of the milk will affect the composition of the fluid available for drug dissolution/solubilization. Therefore, in order to predict the solubilized amount of drug available for absorption, an in vitro model simulating digestion in the gastro-intestinal tract should be utilized. In order to simulate the digestion process and the drug solubilization taking place in vivo, the following aspects should be considered; physiologically relevant media, media volume, use of physiological enzymes in proper amounts, as well as correct pH and addition of relevant co-factors, e.g., bile salts and co-enzymes. Furthermore, physiological transit times and appropriate mixing should be considered and mimicked as close as possible. This paper presents a literature review on physiological factors relevant for digestion and drug solubilization in neonates. Based on the available literature data, a novel in vitro digestion model simulating digestion and drug solubilization in the neonate and young infant pediatric population (2 months old and younger) was designed.