The effects of diet and caloric restriction on adipose tissue fatty acid signatures of tufted puffin (<Emphasis Type="Italic">Fratercula cirrhata</Emphasis>) nestlings

Fatty acid (FA) signature analysis is a powerful tool to investigate foraging ecology and food web dynamics in marine ecosystems. However, use of FA signatures to qualitatively or quantitatively infer diets is potentially complicated by effects of nutritional state on lipid metabolism. Estimation of diets using the quantitative fatty acid signature analysis (QFASA) model requires the use of calibration coefficients to account for predator metabolism of individual FAs. We conducted a captive feeding experiment to determine the effects of a 50% reduction in food intake on growth rate and adipose tissue FA signatures of tufted puffin (Fratercula cirrhata) nestlings, a species that routinely experiences food restriction during growth. FA signatures of chicks fed low- and high-calorie diets both exhibited a change in composition in response to the dietary shift with the direction of change in the composition of individual FAs matching the direction of change in the dietary FAs. Despite a growth rate in the restricted nestlings that was 38% of those in the well-fed group, rates of FA turnover were not different between high and low-calorie treatments, and turnover was close to, but not entirely complete, after 27 days on both high-calorie and restricted diets. FA signatures of tufted puffin nestlings were significantly affected by caloric restriction, but these effects were much less pronounced than those of dietary turnover, and calibration coefficients of puffins fed low and high-calorie diets were highly correlated. Our results demonstrate that changes in physiological state can affect FA metabolism, but future research is required to better understand whether the size of these effects is sufficient to substantially alter diet estimation using the QFASA model.     

Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity

Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanning transport proteins, many of which are associated with genetic diseases or multidrug resistance of cancers. In this study, we present a comprehensive approach for the cloning, expression, and purification of human ABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstrate that 11 transporters, including ABCC3, ABCB6, ABCD1, ABCG1, ABCG4, ABCG5, ABCG8, ABCE1, ABCF1, ABCF2, and ABCF3, were expressed at high levels comparable to that of ABCB1 (P-glycoprotein). As an example of the purification strategy via tandem affinity chromatography, we purified ABCC3 (MRP3) whose role in the transport of anticancer drugs, bile acids, and glucuronides has been controversial. The yield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activated with PC lipids, exhibited significant ATPase activity with a Vmax of 82 +/- 32 nmol min-1 mg-1. The ATPase activity was stimulated by bile acids and glucuronide conjugates, reaching 170 +/- 28 nmol min-1 mg-1, but was not stimulated by a variety of anticancer drugs. The glucuronide conjugates ethinylestradiol-3-glucuronide and 17beta-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities (apparent Km values of 2 and 3 microM, respectively) in contrast to bile acids (apparent Km values of >130 microM), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availability of a purification system for the production of large quantities of active transporters presents a major step not only toward understanding the role of ABCC3 but also toward future structure-function analysis of other human ABC transporters.     

Surgical castration of pigs affects the behavioural response to a low-dose lipopolysaccharide (LPS) challenge after weaning

The objective of the present study was to evaluate the effect of an acute stressor in the early postnatal life of pigs, surgical castration, on post-weaning behaviour, and on the behavioural, endocrine and immune responses elicited by a low-dose lipopolysaccharide (LPS) challenge after weaning. At 5-days-of-age, 64 male piglets were randomly assigned to undergo surgical castration or were left untreated (treatment). Pigs were weaned at 28 days-of-age. Behaviour post-weaning and mixing was assessed during a 1-h period, during which agonistic interactions were recorded. One day post-weaning, pigs were injected intraperitoneally with a single dose of 0 or 5 μg/kg of BW of LPS from Escherichia coli (challenge). Sickness behaviour was studied by scan sampling every 5 min for 45 min at 0, 1, 2, 3, 4, 6 and 8 h after the challenge. Blood samples were taken at 0, 2, 12 or 24 h after injection and were analysed for plasma concentrations of tumor necrosis factor-alpha (TNF-), interleukin-1beta (IL-1β), C-reactive protein (CRP), serum amyloid A (SAA) and cortisol. Results showed that non-castrated pigs were more aggressive than castrated pigs immediately after weaning (P < 0.05). Administration of LPS provoked behaviours characteristic of sickness including a reduction in general activity, as well as decreased eating and exploratory behaviours (P < 0.05). These altered behaviours occurred predominantly 3-h post injection (P < 0.05). Significant treatment by challenge interactions showed that castration reduced the occurrence of sickness behaviours induced by LPS, such as depressed general activity (P < 0.01), anorexia (P < 0.01) and reduced exploratory behaviours (P < 0.05). LPS administration increased TNF- levels (P < 0.05), with peak concentrations 2 h after injection (P < 0.01). CRP levels of LPS-treated pigs were higher than saline-treated animals at 12 h (P < 0.05). LPS administration tended to increase plasma SAA levels (P < 0.1), but did not increase cortisol levels (P > 0.1). However, castration did not affect the response of pro-inflammatory cytokines, acute phase proteins and cortisol to the challenge. These results show that surgical castration reduces aggressiveness at weaning and affects specific sickness behaviours but not the endocrine and immune responses elicited by low-dose endotoxin challenge in weaned pigs.     

HIF-VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.     

Response to rearing in laboratory of the xylophagous grape pest,Xylotrechus arvicola (Coleoptera: Cerambycidae)

Xylotrechus arvicola (Olivier) (Coleoptera: Cerambycidae) is an important pest in vineyards (Vitis vinifera) in the main Iberian wine‐producing regions. Larvae were reared with Semi‐Synthetic Iglesias (SSI) diet over 27 months and two generations in the laboratory. Larval mortality was highest during the first (49.49 %) and second (9.38 %) month of rearing, increasing to 50.52 % during the first month if F2 reared larvae were obtained from an F1 adult female obtained in laboratory. The diet had sufficient nutrients to enable the pest to complete its life cycle within nine months, with F1 larval viability ranging from 23.49 % to 27.97 % and F2 larval viability reduced to 2.07 %. However, the diet did not allow for the completion of additional life cycles and generations (F3, F4,…). Larval mortality increased as the months of rearing (66.13 %, 69.51 % and 89.50 %) and generations (59.10 % and 76.93 % in F1 and F2, respectively) progressed in the laboratory. The larva–adult period of females obtained in the laboratory was longer than for males. In the laboratory, the life cycle was shortened in relation to the life cycle in the field because larvae did not require a cold period to break diapause and start pupation. This indicates that X. arvicola has the potential to complete its life cycle inside grape wood in vineyards of wine‐producing regions with warmer winters.     

Inference on diversity from forest inventories: a review

A number of international agreements and commitments emphasize the importance of appropriate monitoring protocols and assessments as prerequisites for sound conservation and management of the world’s forest ecosystems. Mandated periodic surveys, like forest inventories, provide a unique opportunity to identify and properly satisfy natural resource management information needs. Distinctively, there is an increasing need for detecting diversity by means of unambiguous diversity measures. Because all diversity measures are functions of tree species abundances, estimation of tree diversity indices and profiles is inevitably performed by estimating tree species abundances and then estimating indices and profiles as functions of the abundance estimates. This strategy can be readily implemented in the framework of current forest inventory approaches, where tree species abundances are routinely estimated by means of plots placed onto the surveyed area in accordance with probabilistic schemes. The purpose of this paper is to assess the effectiveness of this strategy by reviewing theoretical results from published case studies. Under uniform random sampling (URS), that is when plots are uniformly and independently located on the study region, consistency and asymptotic normality of diversity index estimators follow from standard limit theorems as the sampling effort increases. In addition, variance estimation and bias reduction are achieved using the jackknife method. Despite its theoretical simplicity, URS may lead to uneven coverage of the study region. In order to avoid unbalanced sampling, the use of tessellation stratified sampling (TSS) is suggested. TSS involves covering the study region by a polygonal grid and randomly selecting a plot in each polygon. Under TSS, the diversity index estimators are consistent, asymptotically normal and more precise than those achieved using URS. Variance estimation is possible and there is no need to reduce bias.     

Influence of magnetopriming on germination,growth, physiology,oil and essential contents of cumin (Cuminum cyminum L.)

An environment friendly technique is desirable for enhancing of crop production. In this study, seeds of cumin (Cuminum cyminum L.) Yazd accession were treated with 0, 150 mT, 250 mT, 500 mT and 1 T magnetic field strengths for min and germination parameters were determined. All of the treatments improved germination parameters and seedling growth as compared with the control. However, 500 and 150 mT were the most effective treatments and thus selected for field studies. Seeds were exposed to 150 mT and 500 mT static magnetic field of strengths for 12 min, planted in the field during 2010–2012 in Isfahan, Iran. Static magnetic field treatment shortened the days to emergence, flowering and maturity, increased chlorophyll and reduced proline contents. Highest biological, seed and essential oil yield, oil content, and oil yield were recorded under 500 mT followed by 150 mT and control, respectively. The results suggested that the pretreatment of cumin seeds with static magnetic field can improve germination, growth and oil and essential contents of the cumin seed under the field.     

PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation

Mitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.     

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.     

The Natural Antimicrobial Carvacrol Inhibits Quorum Sensing in Chromobacterium violaceum and Reduces Bacterial Biofilm Formation at Sub-Lethal Concentrations

The formation of biofilm by bacteria confers resistance to biocides and presents problems in medical and veterinary clinical settings. Here we report the effect of carvacrol, one of the major antimicrobial components of oregano oil, on the formation of biofilms and its activity on existing biofilms. Assays were carried out in polystyrene microplates to observe (a) the effect of 0–0.8 mM carvacrol on the formation of biofilms by selected bacterial pathogens over 24 h and (b) the effect of 0–8 mM carvacrol on the stability of pre-formed biofilms. Carvacrol was able to inhibit the formation of biofilms of Chromobacterium violaceum ATCC 12472, Salmonella enterica subsp. Typhimurium DT104, and Staphylococcus aureus 0074, while it showed no effect on formation of Pseudomonas aeruginosa (field isolate) biofilms. This inhibitory effect of carvacrol was observed at sub-lethal concentrations (<0.5 mM) where no effect was seen on total bacterial numbers, indicating that carvacrol''s bactericidal effect was not causing the observed inhibition of biofilm formation. In contrast, carvacrol had (up to 8 mM) very little or no activity against existing biofilms of the bacteria described, showing that formation of the biofilm also confers protection against this compound. Since quorum sensing is an essential part of biofilm formation, the effect of carvacrol on quorum sensing of C. violaceum was also studied. Sub-MIC concentrations of carvacrol reduced expression of cviI (a gene coding for the N-acyl-L-homoserine lactone synthase), production of violacein (pigmentation) and chitinase activity (both regulated by quorum sensing) at concentrations coinciding with carvacrol''s inhibiting effect on biofilm formation. These results indicate that carvacrol''s activity in inhibition of biofilm formation may be related to the disruption of quorum sensing.