Development of a fast, objective, quantitative methodology to monitor angiogenesis in the chicken chorioallantoic membrane during development

Recent research investigates the role of different gas concentrations during incubation, on chicken growth, quality and health post hatch. One of the parameters of chicken development which changes under different gas concentrations is angiogenesis in the chorioallantoic membrane (CAM). To be able to perform large incubation experiments under different conditions, angiogenesis in the whole CAM must be quantified objectively and easily. In this paper, a fast, objective, quantitative methodology to assess changes in the overall vascular development in the CAM of chicken embryos is presented. Samples were taken with minimal disturbance by emptying the egg, so that the CAM stayed attached to the shell, which was then cut in pieces. We employed a commercial digital camera and a macro lens set at 5x magnification to take pictures with sufficient contrast and resolution (2.64 mm/pixel). These were processed with computer algorithms to calculate the vascular fraction (VF) and the fractal dimension (FD) automatically on binary images. The ratio of the repeatability and reproducibility variation compared to the parts variation was 0.32 for VF and 0.21 for FD. In a validation experiment (n=284), one group was incubated under hypoxic conditions and the other under normoxic conditions. It was shown that early hypoxia stimulated angiogenesis, while chronic hypoxia impeded growth with significant differences between both groups, which is in accordance with literature data. Thus, we report here a method to asses overall angiogenesis in the CAM under different incubation conditions.     

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser²⁰⁵⁶, Thr²⁶⁴⁷ and Thr²⁶⁰⁹ in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser³²⁰⁵ and Thr³⁹⁵⁰ in mitosis. Phosphorylation of Thr³⁹⁵⁰ is DNA-PK-dependent, whereas phosphorylation of Ser³²⁰⁵ requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser³²⁰⁵ in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser³²⁰⁵ in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr⁶⁸ in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ.     

Role of the Lower and Upper Intestine in the Production and Absorption of Gut Microbiota-Derived PUFA Metabolites

In vitro studies have suggested that isolated gut bacteria are able to metabolize PUFA into CLA (conjugated linoleic acids) and CLnA (conjugated linolenic acids). However, the bioavailability of fatty acid metabolites produced in vivo by the gut microbes remains to be studied. Therefore, we measured intestinal concentration and plasma accumulation of bacterial metabolites produced from dietary PUFA in mice, first injected with a lipoprotein lipase inhibitor, then force-fed with either sunflower oil (200 µl) rich in n-6 PUFA or linseed oil (200 µl) rich in n-3 PUFA. The greatest production of bacterial metabolites was observed in the caecum and colon, and at a much lesser extent in the jejunum and ileum. In the caecal content, CLA proportions were higher in sunflower oil force-fed mice whereas CLnA proportions were higher in linseed oil force-fed mice. The accumulation of the main metabolites (CLA cis-9,trans-11-18:2 and CLnA cis-9,trans-11,cis-15-18:3) in the caecal tissue was not associated with their increase in the plasma, therefore suggesting that, if endogenously produced CLA and CLnA have any biological role in host metabolism regulation, their effect would be confined at the intestinal level, where the microbiota is abundant.     

Growth of Azospirillum irakense KBC1 on the Aryl β-Glucoside Salicin Requires either salA or salB

The rhizosphere nitrogen-fixing bacterium Azospirillum irakense KBC1 is able to grow on pectin and beta-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring beta-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl beta-glucosides. A Delta(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB' subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this beta-glucosidase family in the utilization of beta-glucosides for microbial growth.     

Using Manipulated Photographs to Identify Features of Streetscapes That May Encourage Older Adults to Walk for Transport

Experimental evidence of environmental features important for physical activity is challenging to procure in real world settings. The current study aimed to investigate the causal effects of environmental modifications on a photographed street''s appeal for older adults'' walking for transport. Secondly, we examined whether these effects differed according to gender, functional limitations, and current level of walking for transport. Thirdly, we examined whether different environmental modifications interacted with each other. Qualitative responses were also reported to gain deeper insight into the observed quantitative relationships. Two sets of 16 panoramic photographs of a streetscape were created, in which six environmental factors were manipulated (sidewalk evenness, traffic level, general upkeep, vegetation, separation from traffic, and benches). Sixty older adults sorted these photographs on appeal for walking for transport on a 7-point scale and reported qualitative information on the reasons for their rankings. Sidewalk evenness appeared to have the strongest influence on a street''s appeal for transport-related walking. The effect of sidewalk evenness was even stronger when the street''s overall upkeep was good and when traffic was absent. Absence of traffic, presence of vegetation, and separation from traffic also increased a street''s appeal for walking for transport. There were no moderating effects by gender or functional limitations. The presence of benches increased the streetscape''s appeal among participants who already walked for transport at least an hour/week. The protocols and methods used in the current study carry the potential to further our understanding of environment-PA relationships. Our findings indicated sidewalk evenness as the most important environmental factor influencing a street''s appeal for walking for transport among older adults. However, future research in larger samples and in real-life settings is needed to confirm current findings.     

Performance of Microscopy for the Diagnosis of Malaria and Human African Trypanosomiasis by Diagnostic Laboratories in the Democratic Republic of the Congo: Results of a Nation-Wide External Quality Assessment

The present External Quality Assessment (EQA) assessed microscopy of blood parasites among diagnostic laboratories in the Democratic Republic of the Congo. The EQA addressed 445 participants in 10/11 provinces (October 2013–April 2014). Participants were sent a panel of five slides and asked to return a routinely stained slide which was assessed for quality of preparation and staining. Response rate was 89.9% (400/445). For slide 1 (no parasites), 30.6% participants reported malaria, mostly Plasmodium falciparum. Only 11.0% participants reported slide 2 (Plasmodium malariae) correctly, 71.0% reported “malaria” or “Plasmodium falciparum” (considered acceptable). Slide 3 contained Plasmodium falciparum (109/μl) and Trypanosoma brucei brucei trypomastigotes: they were each reported by 32.5% and 16.5% participants respectively, 6.0% reported both. Slide 4 (Trypanosoma) was recognised by 44.9% participants. Slide 5 (Plasmodium ovale) was correctly reported by 6.2% participants, another 68.8% replied “malaria” or “Plasmodium falciparum” (considered acceptable). Only 13.6% of routine slides returned were correctly prepared and stained. The proportion of correct/acceptable scores for at least 4/5 slides was higher among EQA-experienced participants compared to first time participants (40.9% versus 22.4%, p = 0.001) and higher among those being trained < 2 years ago compared to those who were not (42.9% versus 26.3%, p = 0.01). Among diagnostic laboratories in Democratic Republic of the Congo, performance of blood parasite microscopy including non-falciparum species and Trypanosoma was poor. Recent training and previous EQA participation were associated with a better performance.     

An MRI-Compatible High Frequency AC Resistive Heating System for Homeothermic Maintenance in Small Animals

PurposeTo develop an MRI-compatible resistive heater, using high frequency alternating current (AC), for temperature maintenance of anaesthetised animals.ResultsImages and spectra acquired in the presence and absence of 50–100 kHz AC through the wire heater were indistinguishable, whereas DC power created field shifts and lineshape distortions. Temperature lost during induction of anaesthesia was recovered within approximately 20 minutes and a stable temperature was reached as the mouse’s temperature approached the set target.ConclusionThe AC-powered wire heater maintains adequate heat input to the animal to maintain body temperature, and does not compromise image quality.