How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.     

A Sexual Ornament in Chickens Is Affected by Pleiotropic Alleles at HAO1 and BMP2, Selected during Domestication

Domestication is one of the strongest forms of short-term, directional selection. Although selection is typically only exerted on one or a few target traits, domestication can lead to numerous changes in many seemingly unrelated phenotypes. It is unknown whether such correlated responses are due to pleiotropy or linkage between separate genetic architectures. Using three separate intercrosses between wild and domestic chickens, a locus affecting comb mass (a sexual ornament in the chicken) and several fitness traits (primarily medullary bone allocation and fecundity) was identified. This locus contains two tightly-linked genes, BMP2 and HAO1, which together produce the range of pleiotropic effects seen. This study demonstrates the importance of pleiotropy (or extremely close linkage) in domestication. The nature of this pleiotropy also provides insights into how this sexual ornament could be maintained in wild populations.     

The interplay between toxin-releasing β-glucosidase and plant iridoid glycosides impairs larval development in a generalist caterpillar, Grammia incorrupta (Arctiidae)

Herbivores with polyphagous feeding habits must cope with a diet that varies in quality. One of the most important sources of this variation in host plant suitability is plant secondary chemistry. We examined how feeding on plants containing one such group of compounds, the iridoid glycosides, might affect the growth and enzymatic activity in a polyphagous caterpillar that feeds on over 80 plant species in 50 different families. Larvae of the polyphagous arctiid, Grammia incorrupta, were reared exclusively on one of two plant species, one of which contains iridoid glycosides (Plantago lanceolata, Plantaginaceae) while the other does not (Taraxacum officinale, Asteraceae). Larval weight was measured on the two host plants, and midgut homogenates of last instar larvae were then assayed for activity and kinetic properties of β-glucosidases, using both a standard substrate, 4-nitrophenyl-β-D-glucose (NPβGlc), and the iridoid glycoside aucubin, one of the two main iridoid glycosides in P. lanceolata. Larvae feeding on P. lanceolata weighed significantly less and developed more slowly compared to larvae on T. officinale. While the larval midgut β-glucosidase activity determined with NPβGlc was significantly decreased when fed on P. lanceolata, aucubin was substantially hydrolyzed and the larval β-glucosidase activity towards both substrates correlated negatively with larval weight. Our results demonstrate that host plants containing high concentrations of iridoid glycosides have a negative impact on larval development of this generalist insect herbivore. This is most likely due to the hydrolysis of plant glycosides in the larval midgut which results in the release of toxic aglycones. Linking the reduced larval weight to the toxin-releasing action of an iridoid glycoside cleaving β-glucosidase, our results thus support the detoxification limitation hypothesis, suggesting fitness costs for the larvae feeding solely on P. lanceolata. Thus, in addition to the adaptive regulation of midgut β-glucosidase activity, host plant switching as a behavioral adaptation might be a prerequisite for generalist herbivores that allows them to circumvent the negative effects of plant secondary compounds.     

Intestinal concentrations of free and encapsulated dietary medium-chain fatty acids and effects on gastric microbial ecology and bacterial metabolic products in the digestive tract of piglets

The influence of low dietary levels of free and encapsulated medium-chain fatty acids on their concentrations in the digesta, the gastric microbial ecology and bacterial metabolic products in the gastrointestinal tract (GIT) in weaned piglets was studied. Starting after weaning, 36 piglets were fed a diet without (Control) or with medium-chain fatty acids uncoated (MCFA) or coated with vegetable fat and lecithin (MCFAc). After 4 weeks, the animals were killed, and digesta from the stomach and different sections of the GIT were collected. The concentrations of caprylic (p < 0.001) and capric (p = 0.001) acids were higher in the stomachs of piglets fed diets MCFA and MCFAc compared to the Control group. The concentrations dropped rapidly along the GIT, regardless of encapsulation, and tended to be higher in groups MCFA and MCFAc compared to the Control. Compared to the Control group, ingestion of diet MCFAc led to an increase in the number of eubacteria (p = 0.001), enterobacteriaceae (p < 0.001), clostridial clusters I (p = 0.001) and IV (p = 0.019), Lactobacillus johnsonii (p < 0.001) and Lactobacillus amylovorus (p = 0.001) in gastric contents. A similar trend was seen with diet MCFA. Relative concentrations of short-chain fatty acids were characterised by lower propionic acid levels (p = 0.045), numerically (p < 0.1) higher acetic, lower n-butyric and i-valeric acid concentrations in the small intestine. Lactic acid concentrations were not significantly changed in the GIT, but ammonia concentrations increased (p < 0.001) in the distal small intestine in the MCFA and MCFAc groups. In conclusion, medium-chain fatty acids affected microbial ecology parameters in the gastric contents and bacterial metabolites in the small intestine. At low dietary levels, medium-chain fatty acids may be regarded as modulators of the gastric microbiota in weaned piglets.     

Gathering “tea” – from necessity to connectedness with nature. Local knowledge about wild plant gathering in the Biosphere Reserve Grosses Walsertal (Austria)

Background

Wild plant gathering is an essential element in livelihood strategies all over the world. However due to changing circumstances in Europe, the reason for gathering has altered from one of necessity in the past to a pleasurable activity today. Wild plant gathering has therefore also received renewed attention as a form of intangible cultural heritage expressing local preferences, habits and man??s relationship with nature. In the Biosphere Reserve Grosses Walsertal (Austria), local people??s knowledge of the gathering of wild plants and their perception of their own gathering activities are being documented. The focus of this paper is on the uses of herbal teas and the informal guidelines for gathering plants that have been issued by the Bergtee (mountain tea) association.

Methods

Thirty-six free-list interviews were conducted with subsequent semi-structured interviews and three focus group meetings held with members of the Bergtee association. Participatory observation (gathering and processing plants, mixing and marketing tea) also allowed for greater understanding of what had been reported.

Results

In total, 140 different gathered plant species were listed by respondents. Herbal tea is the most frequently mentioned use. The Bergtee association, founded by a young man and two middle-aged women in the valley, is a good example of the link between biological and cultural diversity, with the aim of sharing the biosphere reserve??s natural treasures as well as local plant-related knowledge in the form of herbal tea products. The association??s informal guidelines for gathering reflect people??s attitude to nature: monetary income does not play a major role in gathering plants; instead people??s appreciation of the value of the nature around them is to the fore.

Conclusions

Gathering wild plants can be seen as an expression of people??s regional identity. The conscious appreciation of nature and related local knowledge is crucial for the sustainable conservation and use of the Biosphere Reserve??s resources.     

Volume reduction of the jugular foramina in Cavalier King Charles Spaniels with syringomyelia

ABSTRACT: BACKGROUND: Understanding the pathogenesis of the chiari-like malformation in the Cavalier King Charles Spaniel (CKCS) is incomplete, and current hypotheses do not fully explain the development of syringomyelia (SM) in the spinal cords of affected dogs. This study investigates an unconventional pathogenetic theory for the development of cerebrospinal fluid (CSF) pressure waves in the subarachnoid space in CKCS with SM, by analogy with human diseases. In children with achondroplasia the shortening of the skull base can lead to a narrowing of the jugular foramina (JF) between the cranial base synchondroses. This in turn has been reported to cause a congestion of the major venous outflow tracts of the skull and consequently to an increase in the intracranial pressure (ICP). Amongst brachycephalic dog breeds the CKCS has been identified as having an extremely short and wide braincase. A stenosis of the JF and a consequential vascular compromise in this opening could contribute to venous hypertension, raising ICP and causing CSF jets in the spinal subarachnoid space of the CKCS. In this study, JF volumes in CKCSs with and without SM were compared to assess a possible role of this pathologic mechanism in the development of SM in this breed. RESULTS: Computed tomography (CT) scans of 40 CKCSs > 4 years of age were used to create three-dimensional (3D) models of the skull and the JF. Weight matched groups (7--10 kg) of 20 CKCSs with SM and 20 CKCSs without SM were compared. CKCSs without SM presented significantly larger JF -volumes (median left JF: 0.0633 cm3; median right JF: 0.0703 cm3; p < 0.0001) when compared with CKCSs with SM (median left JF: 0.0382 cm3; median right JF: 0.0434 cm3; p < 0.0001). There was no significant difference between the left and right JF within each group. Bland-Altman analysis revealed excellent reproducibility of all volume measurements. CONCLUSION: A stenosis of the JF and consecutive venous congestion may explain the aetiology of CSF pressure waves in the subarachnoid space, independent of cerebellar herniation, as an additional pathogenetic factor for the development of SM in this breed.     

An interdisciplinary knowledge translation intervention in long-term care: Study protocol for the vitamin D and osteoporosis study (ViDOS) pilot cluster randomized controlled trial

ABSTRACT: BACKGROUND: Knowledge translation (KT) research in long-term care (LTC) is still in its early stages. This protocol describes the evaluation of a multifaceted, interdisciplinary KT intervention aimed at integrating evidence-based osteoporosis and fracture prevention strategies into LTC care processes. Methods and design The Vitamin D and Osteoporosis Study (ViDOS) is underway in 40 LTC homes (n = 21 intervention, n = 19 control) across Ontario, Canada. The primary objectives of this study are to assess the feasibility of delivering the KT intervention, and clinically, to increase the percent of LTC residents prescribed [greater than or equal to]800 IU of vitamin D daily. Eligibility criteria are LTC homes that are serviced by our partner pharmacy provider and have more than one prescribing physician. The target audience within each LTC home is the Professional Advisory Committee (PAC), an interdisciplinary team who meets quarterly. The key elements of the intervention are three interactive educational sessions led by an expert opinion leader, action planning using a quality improvement cycle, audit and feedback reports, nominated internal champions, and reminders/point-of-care tools. Control homes do not receive any intervention, however both intervention and control homes received educational materials as part of the Ontario Osteoporosis Strategy. Primary outcomes are feasibility measures (recruitment, retention, attendance at educational sessions, action plan items identified and initiated, internal champions identified, performance reports provided and reviewed), and vitamin D ([greater than or equal to]800 IU/daily) prescribing at 6 and 12 months. Secondary outcomes include the proportion of residents prescribed calcium supplements and osteoporosis medications, and falls and fractures. Qualitative methods will examine the experience of the LTC team with the KT intervention. Homes are centrally randomized to intervention and control groups in blocks of variable size using a computer generated allocation sequence. Randomization is stratified by home size and profit/nonprofit status. Prescribing data retrieval and analysis are performed by blinded personnel. DISCUSSION: Our study will contribute to an improved understanding of the feasibility and acceptability of a multifaceted intervention aimed at translating knowledge to LTC practitioners. Lessons learned from this study will be valuable in guiding future research and understanding the complexities of translating knowledge in LTC. Trial registration ClinicalTrials.gov NCT01398527.     

Assessing the complex sponge microbiota: core,variable and species-specific bacterial communities in marine sponges

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world''s oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.     

Portable bacterial identification system based on elastic light scatter patterns

ABSTRACT: BACKGROUND: Conventional diagnosis and identification of bacteria requires shipment of samples to a laboratory for genetic and biochemical analysis. This process can take days and imposes significant delay to action in situations where timely intervention can save lives and reduce associated costs. To enable faster response to an outbreak, a low-cost, small-footprint, portable microbial-identification instrument using forward scatterometry has been developed. RESULTS: This device, weighing 9 lb and measuring 12 [MULTIPLICATION SIGN] 6 [MULTIPLICATION SIGN] 10.5 in., utilizes elastic light scatter (ELS) patterns to accurately capture bacterial colony characteristics and delivers the classification results via (non-italic form) wireless access. The overall system consists of two CCD cameras, one rotational and one translational stage, and a 635-nm laser diode. Various software algorithms such as Hough transform, 2-D geometric moments, and the traveling salesman problem (TSP) have been implemented to provide colony count and circularity, centering process, and minimized travel time among colonies. CONCLUSIONS: Experiments were conducted with four bacteria genera using pure and mixed plate and as proof of principle a field test was conducted in four different locations where the average classification rate ranged between 95 and 100%.     

A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax)

Western Palearctic water frogs in the genus Pelophylax are a set of morphologically similar anuran species that form hybridogenetic complexes. Fully reliable identification of species and especially of hybrid ploidy depends on karyological and molecular methods. In central Europe, native water frog populations consist of the Pelophylax esculentus complex, that is, P. lessonae (LL), P. ridibundus (RR) and the hybrid form P. esculentus that can have different karyotypes (RL, LLR and RRL). We developed existing molecular methods further and propose a simple PCR method based on size‐differences in the length of the serum albumin intron‐1 and the RanaCR1, a non‐LTR retrotransposon of the chicken repeat (CR) family. This PCR yields taxon‐specific banding patterns that can easily be screened by standard agarose gel electrophoresis and correctly identify species in all of the 160 samples that had been identified to karyotype with other methods. To distinguish ploidy levels in LR, LLR and RRL specimens, we used the ratio of the peak heights of the larger (ridibundus specific) to the smaller (lessonae specific) bands of fluorescently labelled PCR products resolved on a capillary DNA sequencer and obtained a correct assignment of the karyotype in 93% of cases. Our new method will cut down time and expenses drastically for a reliable identification of water frogs of the P. esculentus complex and potentially for identification of other hybridogenetic complexes and/or taxa, and it even serves as a good indicator of the ploidy status of hybrid individuals.