Conformational studies of human milk oligosaccharides using (1)H-(13)C one-bond NMR residual dipolar couplings

1H-(13)C one-bond dipolar coupling values were measured for natural abundance samples of the human milk oligosaccharides "lacto-N-fucopentaose" (LNF-1 LNF-2, and LNF-3), "lacto-N-difucohexaose" (LND-1), "lacto-N-tetraose" (LNT), and "lacto-N-neo-tetraose" (LNnT), four of which have Lewis blood group epitopes. Each oligosaccharide was dissolved in a 7.5% solution of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DMPC/DHPC) bicelle liquid crystals oriented in the NMR magnetic field. The dipolar coupling data and NOE were fitted to conformational models with calculations of an optimum orientation tensor which best represents the dipolar coupling values for a fragment hypothesized to adopt a single conformation. In the case of LNF-1, LNF-2, LNF-3, and LND-1, the models confirm previous conformational models for the Lewis epitopes based on NOE and molecular dynamics simulations. Extensions of the model provided new structural data for the remaining residues. In all cases, upper limits for the errors in the glycosidic angles of the models were estimated. Since residual dipolar coupling provides information on long-range order, it is a valuable complement to other types of NMR data such as NOE and scalar coupling for exploring conformations of complex oligosaccharides.     

The biogeochemistry of phosphorus after the first century of soil development on Rakata Island, Krakatau, Indonesia

This study examined the accumulation of organic carbon (C) and fractions ofsoil phosphorus (P) in soils developing in volcanic ash deposited in the1883 eruption of Krakatau. Organic C has accumulated at rates of 45 to 127g/m²/yr during 110 years of soil development, resulting inprofiles with as much as 14 kgC/m². Most soil P is found inthe HCl-extractable forms, representing apatite. A loss of HCl-extractableP from the surface horizons is associated with a marked accumulation ofNaOH-extractable organic P bound to Al. A bioassay with hill rice suggeststhat P is limiting to plant growth in these soils, perhaps as a result ofthe rapid accumulation of P in organic forms.     

Long Term Non-Invasive Ventilation in Children: Impact on Survival and Transition to Adult Care

BackgroundThe number of children receiving domiciliary ventilatory support has grown over the last few decades driven largely by the introduction and widening applications of non-invasive ventilation. Ventilatory support may be used with the intention of increasing survival, or to facilitate discharge home and/or to palliate symptoms. However, the outcome of this intervention and the number of children transitioning to adult care as a consequence of longer survival is not yet clear.MethodsIn this retrospective cohort study, we analysed the outcome in children (<17 years) started on home NIV at Royal Brompton Hospital over an 18 year period 1993-2011. The aim was to establish for different diagnostic groups: survival rate, likelihood of early death depending on diagnosis or discontinuation of ventilation, and the proportion transitioning to adult care.Results496 children were commenced on home non invasive ventilation; follow-up data were available in 449 (91%). Fifty six per cent (n=254) had neuromuscular disease. Ventilation was started at a median age (IQR) 10 (3-15) years. Thirteen percent (n=59) were less than 1 year old. Forty percent (n=181) have transitioned to adult care. Twenty four percent (n=109) of patients have died, and nine percent (n=42) were able to discontinue ventilatory support.ConclusionLong term ventilation is associated with an increase in survival in a range of conditions leading to ventilatory failure in children, resulting in increasing numbers surviving to adulthood. This has significant implications for planning transition and adult care facilities.     

Viewing Complex,Dynamic Scenes “Through the Eyes” of Another Person: The Gaze-Replay Paradigm

We present a novel “Gaze-Replay” paradigm that allows the experimenter to directly test how particular patterns of visual input—generated from people’s actual gaze patterns—influence the interpretation of the visual scene. Although this paradigm can potentially be applied across domains, here we applied it specifically to social comprehension. Participants viewed complex, dynamic scenes through a small window displaying only the foveal gaze pattern of a gaze “donor.” This was intended to simulate the donor’s visual selection, such that a participant could effectively view scenes “through the eyes” of another person. Throughout the presentation of scenes presented in this manner, participants completed a social comprehension task, assessing their abilities to recognize complex emotions. The primary aim of the study was to assess the viability of this novel approach by examining whether these Gaze-Replay windowed stimuli contain sufficient and meaningful social information for the viewer to complete this social perceptual and cognitive task. The results of the study suggested this to be the case; participants performed better in the Gaze-Replay condition compared to a temporally disrupted control condition, and compared to when they were provided with no visual input. This approach has great future potential for the exploration of experimental questions aiming to unpack the relationship between visual selection, perception, and cognition.     

Decoding the Traumatic Memory among Women with PTSD: Implications for Neurocircuitry Models of PTSD and Real-Time fMRI Neurofeedback

Posttraumatic Stress Disorder (PTSD) is characterized by intrusive recall of the traumatic memory. While numerous studies have investigated the neural processing mechanisms engaged during trauma memory recall in PTSD, these analyses have only focused on group-level contrasts that reveal little about the predictive validity of the identified brain regions. By contrast, a multivariate pattern analysis (MVPA) approach towards identifying the neural mechanisms engaged during trauma memory recall would entail testing whether a multivariate set of brain regions is reliably predictive of (i.e., discriminates) whether an individual is engaging in trauma or non-trauma memory recall. Here, we use a MVPA approach to test 1) whether trauma memory vs neutral memory recall can be predicted reliably using a multivariate set of brain regions among women with PTSD related to assaultive violence exposure (N=16), 2) the methodological parameters (e.g., spatial smoothing, number of memory recall repetitions, etc.) that optimize classification accuracy and reproducibility of the feature weight spatial maps, and 3) the correspondence between brain regions that discriminate trauma memory recall and the brain regions predicted by neurocircuitry models of PTSD. Cross-validation classification accuracy was significantly above chance for all methodological permutations tested; mean accuracy across participants was 76% for the methodological parameters selected as optimal for both efficiency and accuracy. Classification accuracy was significantly better for a voxel-wise approach relative to voxels within restricted regions-of-interest (ROIs); classification accuracy did not differ when using PTSD-related ROIs compared to randomly generated ROIs. ROI-based analyses suggested the reliable involvement of the left hippocampus in discriminating memory recall across participants and that the contribution of the left amygdala to the decision function was dependent upon PTSD symptom severity. These results have methodological implications for real-time fMRI neurofeedback of the trauma memory in PTSD and conceptual implications for neurocircuitry models of PTSD that attempt to explain core neural processing mechanisms mediating PTSD.     

iTILLING: A Personalized Approach to the Identification of Induced Mutations in Arabidopsis

TILLING (for Targeting Induced Local Lesions IN Genomes) is a well-established method for identifying plants carrying point mutations in genes of interest. A traditional TILLING project requires a significant investment of time and resources to establish the mutant population and screening infrastructure. Here, we describe a modified TILLING procedure that substantially reduces the investment needed to perform mutation screening. Our motivation for developing iTILLING was to make it practical for individual laboratories to rapidly perform mutation screens using specialized genetic backgrounds. With iTILLING, M2 seeds are collected in bulk from the mutagenized population of plants, greatly reducing the labor needed to manage the mutant lines. Growth of the M2 seedlings for mutation screening, tissue collection, and DNA extraction are all performed in 96-well format. Mutations are then identified using high-resolution melt-curve analysis of gene-specific polymerase chain reaction products. Individual plants carrying mutations of interest are transferred from the 96-well growth plates to soil. One scientist can complete an iTILLING screen in less than 4 months. As a proof-of-principle test, we applied iTILLING to Arabidopsis (Arabidopsis thaliana) plants that were homozygous for the mekk1-1 (for MAPK/ERK kinase kinase 1) mutation and also carried a MEKK1 rescue construct. The goal of our screen was to identify mutations in the closely linked MEKK2 and MEKK3 loci. We obtained five mutations in MEKK2 and seven mutations in MEKK3, all located within 20 kb of the mekk1-1 T-DNA insertion. Using repeated iterations of the iTILLING process, mutations in three or more tandemly duplicated genes could be generated.The process of reverse genetics has been widely used by plant biologists to study gene function. In Arabidopsis (Arabidopsis thaliana), three approaches that have been used to generate populations of plants for reverse genetic analysis are insertional mutagenesis (Wisman et al., 1998; Alonso et al., 2003), fast neutron mutagenesis to induce deletions (Li et al., 2001), and chemical mutagenesis to induce point mutations (McCallum et al., 2000). In order to find individual plants carrying point mutations of interest, a process called TILLING (for Targeting Induced Local Lesions IN Genomes) was developed whereby genes are screened for mutations using a PCR-based assay (McCallum et al., 2000). Although originally developed for use with Arabidopsis, the TILLING process has been subsequently applied to a wide range of plants, including barley (Hordeum vulgare; Caldwell et al., 2004), Brassica napus (Wang et al., 2008), Brassica oleracea (Himelblau et al., 2009), Brassica rapa (Stephenson et al., 2010), Lotus japonicus (Perry et al., 2009), maize (Zea mays; Till et al., 2004), Medicago truncatula (Le Signor et al., 2009), oat (Avena sativa; Chawade et al., 2010), pea (Pisum sativum; Triques et al., 2007), potato (Solanum tuberosum; Elias et al., 2009), rice (Oryza sativa; Till et al., 2007), sorghum (Sorghum bicolor; Xin et al., 2008), soybean (Glycine max; Cooper et al., 2008), tomato (Solanum lycopersicum ; Gady et al., 2009), and wheat (Triticum aestivum; Dong et al., 2009). TILLING has also been used in Drosophila (Winkler et al., 2005), zebrafish (Wienholds et al., 2003), and Caenorhabditis elegans (Gilchrist et al., 2006).The chemical mutagen most commonly used to create the mutant populations used for TILLING is ethyl methanesulfonate (EMS). When working with plants, seeds are soaked in EMS to induce mutations throughout the genome. Mutagenized seeds are then planted on soil, and the resulting plants are grown to maturity to produce M2 seeds, which are collected from the plants individually or in small pools. Next, M2 seed samples from each individual plant are germinated and grown to produce tissue from which DNA can be extracted. The resulting large collection of ordered DNA samples and the corresponding M2 seeds constitute the infrastructure of a TILLING population. PCR-based screening can then be used to find individual plants in the population carrying mutations in genes of interest (McCallum et al., 2000). Once established, this type of TILLING infrastructure can serve the needs of an entire research community through a fee-for-service screening operation (Colbert et al., 2001; Martín et al., 2009).Several different strategies have been developed for identifying the mutations present in a TILLING population, but all of them involve detecting heteroduplex PCR products. A heteroduplex is formed when a mixture of wild-type and mutant PCR products are melted and reannealed, resulting in DNA duplexes that contain a single-base mismatch. TILLING was originally described using denaturing HPLC to identify mutations based on the differential retention times of heteroduplexes and homoduplexes in the chromatography column (McCallum et al., 2000). TILLING has since been modified so that endonucleases are used to cleave PCR products containing a heteroduplex. Cleavage products are then separated via gel electrophoresis to identify banding patterns indicative of mutations (Colbert et al., 2001).More recently, high-resolution melting analysis of PCR products has been used to identify heteroduplexes when performing TILLING (Dong et al., 2009; Gady et al., 2009). High-resolution melting analysis was originally developed for use in clinical settings to identify known single-nucleotide polymorphisms and small insertions/deletions potentially linked to genetic diseases (Erali et al., 2008). With high-resolution melting, the mismatch in a heteroduplex is visualized as a melting event that occurs more rapidly or at a lower temperature than the corresponding homoduplex. Montgomery et al. (2007) demonstrated that mutation scanning with high-resolution melting is a robust technique with greater than 95% sensitivity in distinguishing heteroduplexes from homoduplexes. It has also been observed that the sensitivity with which mutations in PCR products can be identified using DNA melting analysis depends on the resolution of the instrumentation used for collecting the melt-curve data (Zhou et al., 2005; Herrmann et al., 2006).Although traditional TILLING is a high-throughput method for mutation screening, the establishment of the initial screening population and the corresponding ordered DNA samples requires a substantial up-front investment of time and money. Because of this situation, TILLING resources are available for only two genetic backgrounds in Arabidopsis: wild-type Columbia-0 and Landsberg erecta (Greene et al., 2003; Martín et al., 2009). If a scientist is interested in identifying mutations in a more specialized genetic background, the costs associated with establishing a TILLING population can be prohibitive. Therefore, we were interested in determining if a modified version of the TILLING process could be developed that would substantially reduce the investment of time and resources necessary to perform mutation screening. The individualized TILLING procedure, or iTILLING, which we describe in this paper provides one solution to this challenge.     

Evolution of Novel Signal Traits in the Absence of Female Preferences in Neoconocephalus Katydids (Orthoptera,Tettigoniidae)

Background Significance

Communication signals that function to bring together the sexes are important for maintaining reproductive isolation in many taxa. Changes in male calls are often attributed to sexual selection, in which female preferences initiate signal divergence. Natural selection can also influence signal traits if calls attract predators or parasitoids, or if calling is energetically costly. Neutral evolution is often neglected in the context of acoustic communication.

Methodology/Principal Findings

We describe a signal trait that appears to have evolved in the absence of either sexual or natural selection. In the katydid genus Neoconocephalus, calls with a derived pattern in which pulses are grouped into pairs have evolved five times independently. We have previously shown that in three of these species, females require the double pulse pattern for call recognition, and hence the recognition system of the females is also in a derived state. Here we describe the remaining two species and find that although males produce the derived call pattern, females use the ancestral recognition mechanism in which no pulse pattern is required. Females respond equally well to the single and double pulse calls, indicating that the derived trait is selectively neutral in the context of mate recognition.

Conclusions/Significance

These results suggest that 1) neutral changes in signal traits could be important in the diversification of communication systems, and 2) males rather than females may be responsible for initiating signal divergence.     

Liver-specific knockdown of JNK1 up-regulates proliferator-activated receptor gamma coactivator 1 beta and increases plasma triglyceride despite reduced glucose and insulin levels in diet-induced obese mice

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.