In Situ Identification of Plant-Invasive Bacteria with MALDI-TOF Mass Spectrometry

Rhizobia form a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes. The study of rhizobial populations in nature involves the collection of large numbers of nodules found on roots or stems of legumes, and the subsequent typing of nodule bacteria. To avoid the time-consuming steps of isolating and cultivating nodule bacteria prior to genotyping, a protocol of strain identification based on the comparison of MALDI-TOF MS spectra was established. In this procedure, plant nodules were considered as natural bioreactors that amplify clonal populations of nitrogen-fixing bacteroids. Following a simple isolation procedure, bacteroids were fingerprinted by analysing biomarker cellular proteins of 3 to 13 kDa using Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry. In total, bacteroids of more than 1,200 nodules collected from roots of three legumes of the Phaseoleae tribe (cowpea, soybean or siratro) were examined. Plants were inoculated with pure cultures of a slow-growing Bradyrhizobium japonicum strain G49, or either of two closely related and fast-growing Sinorhizobium fredii strains NGR234 and USDA257, or with mixed inoculants. In the fully automatic mode, correct identification of bacteroids was obtained for >97% of the nodules, and reached 100% with a minimal manual input in processing of spectra. These results showed that MALDI-TOF MS is a powerful tool for the identification of intracellular bacteria taken directly from plant tissues

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Fractures in myelomeningocele

BACKGROUND: In patients with myelomeningocele (MMC), a high number of fractures occur in the paralyzed extremities, affecting mobility and independence. The aims of this retrospective cross-sectional study are to determine the frequency of fractures in our patient cohort and to identify trends and risk factors relevant for such fractures. MATERIALS AND METHODS: Between March 1988 and June 2005, 862 patients with MMC were treated at our hospital. The medical records, surgery reports, and X-rays from these patients were evaluated. RESULTS: During the study period, 11% of the patients (n = 92) suffered one or more fractures. Risk analysis showed that patients with MMC and thoracic-level paralysis had a sixfold higher risk of fracture compared with those with sacral-level paralysis. Femoral-neck z-scores measured by dual-energy X-ray absorptiometry (DEXA) differed significantly according to the level of neurological impairment, with lower z-scores in children with a higher level of lesion. Furthermore, the rate of epiphyseal separation increased noticeably after cast immobilization. Mainly patients who could walk relatively well were affected. CONCLUSIONS: Patients with thoracic-level paralysis represent a group with high fracture risk. According to these results, fracture and epiphyseal injury in patients with MMC should be treated by plaster immobilization. The duration of immobilization should be kept to a minimum (<4 weeks) because of increased risk of secondary fractures. Alternatively, patients with refractures can be treated by surgery, when nonoperative treatment has failed

Altered Metabolism and Persistent Starvation Behaviors Caused by Reduced AMPK Function in Drosophila

Organisms must utilize multiple mechanisms to maintain energetic homeostasis in the face of limited nutrient availability. One mechanism involves activation of the heterotrimeric AMP-activated protein kinase (AMPK), a cell-autonomous sensor to energetic changes regulated by ATP to AMP ratios. We examined the phenotypic consequences of reduced AMPK function, both through RNAi knockdown of the gamma subunit (AMPKγ) and through expression of a dominant negative alpha (AMPKα) variant in Drosophila melanogaster. Reduced AMPK signaling leads to hypersensitivity to starvation conditions as measured by lifespan and locomotor activity. Locomotor levels in flies with reduced AMPK function were lower during unstressed conditions, but starvation-induced hyperactivity, an adaptive response to encourage foraging, was significantly higher than in wild type. Unexpectedly, total dietary intake was greater in animals with reduced AMPK function yet total triglyceride levels were lower. AMPK mutant animals displayed starvation-like lipid accumulation patterns in metabolically key liver-like cells, oenocytes, even under fed conditions, consistent with a persistent starved state. Measurements of O2 consumption reveal that metabolic rates are greater in animals with reduced AMPK function. Lastly, rapamycin treatment tempers the starvation sensitivity and lethality associated with reduced AMPK function. Collectively, these results are consistent with models that AMPK shifts energy usage away from expenditures into a conservation mode during nutrient-limited conditions at a cellular level. The highly conserved AMPK subunits throughout the Metazoa, suggest such findings may provide significant insight for pharmaceutical strategies to manipulate AMPK function in humans

Dietary Modulation of Drosophila Sleep-Wake Behaviour

Background A complex relationship exists between diet and sleep but despite its impact on human health, this relationship remains uncharacterized and poorly understood. Drosophila melanogaster is an important model for the study of metabolism and behaviour, however the effect of diet upon Drosophila sleep remains largely unaddressed. Methodology/Principal Findings Using automated behavioural monitoring, a capillary feeding assay and pharmacological treatments, we examined the effect of dietary yeast and sucrose upon Drosophila sleep-wake behaviour for three consecutive days. We found that dietary yeast deconsolidated the sleep-wake behaviour of flies by promoting arousal from sleep in males and shortening periods of locomotor activity in females. We also demonstrate that arousal from nocturnal sleep exhibits a significant ultradian rhythmicity with a periodicity of 85 minutes. Increasing the dietary sucrose concentration from 5% to 35% had no effect on total sucrose ingestion per day nor any affect on arousal, however it did lengthen the time that males and females remained active. Higher dietary sucrose led to reduced total sleep by male but not female flies. Locomotor activity was reduced by feeding flies Metformin, a drug that inhibits oxidative phosphorylation, however Metformin did not affect any aspects of sleep. Conclusions We conclude that arousal from sleep is under ultradian control and regulated in a sex-dependent manner by dietary yeast and that dietary sucrose regulates the length of time that flies sustain periods of wakefulness. These findings highlight Drosophila as an important model with which to understand how diet impacts upon sleep and wakefulness in mammals and humans

In the Laboratory and during Free-Flight: Old Honey Bees Reveal Learning and Extinction Deficits that Mirror Mammalian Functional Decline

Loss of brain function is one of the most negative and feared aspects of aging. Studies of invertebrates have taught us much about the physiology of aging and how this progression may be slowed. Yet, how aging affects complex brain functions, e.g., the ability to acquire new memory when previous experience is no longer valid, is an almost exclusive question of studies in humans and mammalian models. In these systems, age related cognitive disorders are assessed through composite paradigms that test different performance tasks in the same individual. Such studies could demonstrate that afflicted individuals show the loss of several and often-diverse memory faculties, and that performance usually varies more between aged individuals, as compared to conspecifics from younger groups. No comparable composite surveying approaches are established yet for invertebrate models in aging research. Here we test whether an insect can share patterns of decline similar to those that are commonly observed during mammalian brain aging. Using honey bees, we combine restrained learning with free-flight assays. We demonstrate that reduced olfactory learning performance correlates with a reduced ability to extinguish the spatial memory of an abandoned nest location (spatial memory extinction). Adding to this, we show that learning performance is more variable in old honey bees. Taken together, our findings point to generic features of brain aging and provide the prerequisites to model individual aspects of learning dysfunction with insect models

A Conserved Role for Syndecan Family Members in the Regulation of Whole-Body Energy Metabolism

Syndecans are a family of type-I transmembrane proteins that are involved in cell-matrix adhesion, migration, neuronal development, and inflammation. Previous quantitative genetic studies pinpointed Drosophila Syndecan (dSdc) as a positional candidate gene affecting variation in fat storage between two Drosophila melanogaster strains. Here, we first used quantitative complementation tests with dSdc mutants to confirm that natural variation in this gene affects variability in Drosophila fat storage. Next, we examined the effects of a viable dSdc mutant on Drosophila whole-body energy metabolism and associated traits. We observed that young flies homozygous for the dSdc mutation had reduced fat storage and slept longer than homozygous wild-type flies. They also displayed significantly reduced metabolic rate, lower expression of spargel (the Drosophila homologue of PGC-1), and reduced mitochondrial respiration. Compared to control flies, dSdc mutants had lower expression of brain insulin-like peptides, were less fecund, more sensitive to starvation, and had reduced life span. Finally, we tested for association between single nucleotide polymorphisms (SNPs) in the human SDC4 gene and variation in body composition, metabolism, glucose homeostasis, and sleep traits in a cohort of healthy early pubertal children. We found that SNP rs4599 was significantly associated with resting energy expenditure (P = 0.001 after Bonferroni correction) and nominally associated with fasting glucose levels (P = 0.01) and sleep duration (P = 0.044). On average, children homozygous for the minor allele had lower levels of glucose, higher resting energy expenditure, and slept shorter than children homozygous for the common allele. We also observed that SNP rs1981429 was nominally associated with lean tissue mass (P = 0.035) and intra-abdominal fat (P = 0.049), and SNP rs2267871 with insulin sensitivity (P = 0.037). Collectively, our results in Drosophila and humans argue that syndecan family members play a key role in the regulation of body metabolism

Hmgcr in the Corpus Allatum Controls Sexual Dimorphism of Locomotor Activity and Body Size via the Insulin Pathway in Drosophila

The insulin signaling pathway has been implicated in several physiological and developmental processes. In mammals, it controls expression of 3-Hydroxy-3-Methylglutaryl CoA Reductase (HMGCR), a key enzyme in cholesterol biosynthesis. In insects, which can not synthesize cholesterol de novo, the HMGCR is implicated in the biosynthesis of juvenile hormone (JH). However, the link between the insulin pathway and JH has not been established. In Drosophila, mutations in the insulin receptor (InR) decrease the rate of JH synthesis. It is also known that both the insulin pathway and JH play a role in the control of sexual dimorphism in locomotor activity. In studies here, to demonstrate that the insulin pathway and HMGCR are functionally linked in Drosophila, we first show that hmgcr mutation also disrupts the sexual dimorphism. Similarly to the InR, HMGCR is expressed in the corpus allatum (ca), which is the gland where JH biosynthesis occurs. Two p[hmgcr-GAL4] lines were therefore generated where RNAi was targeted specifically against the HMGCR or the InR in the ca. We found that RNAi-HMGCR blocked HMGCR expression, while the RNAi-InR blocked both InR and HMGCR expression. Each RNAi caused disruption of sexual dimorphism and produced dwarf flies at specific rearing temperatures. These results provide evidence: (i) that HMGCR expression is controlled by the InR and (ii) that InR and HMGCR specifically in the ca, are involved in the control of body size and sexual dimorphism of locomotor activity

Characterization of a Drosophila Alzheimer's Disease Model: Pharmacological Rescue of Cognitive Defects

Transgenic models of Alzheimer's disease (AD) have made significant contributions to our understanding of AD pathogenesis, and are useful tools in the development of potential therapeutics. The fruit fly, Drosophila melanogaster, provides a genetically tractable, powerful system to study the biochemical, genetic, environmental, and behavioral aspects of complex human diseases, including AD. In an effort to model AD, we over-expressed human APP and BACE genes in the Drosophila central nervous system. Biochemical, neuroanatomical, and behavioral analyses indicate that these flies exhibit aspects of clinical AD neuropathology and symptomology. These include the generation of Aβ40 and Aβ42, the presence of amyloid aggregates, dramatic neuroanatomical changes, defects in motor reflex behavior, and defects in memory. In addition, these flies exhibit external morphological abnormalities. Treatment with a γ-secretase inhibitor suppressed these phenotypes. Further, all of these phenotypes are present within the first few days of adult fly life. Taken together these data demonstrate that this transgenic AD model can serve as a powerful tool for the identification of AD therapeutic interventions