Delivery of prolamins to the protein storage vacuole in maize aleurone cells

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.     

Analytical and numerical solutions of the potential and electric field generated by different electrode arrays in a tumor tissue under electrotherapy

Background

During the cardiac cycle, the heart normally produces repeatable physiological sounds. However, under pathologic conditions, such as with heart valve stenosis or a ventricular septal defect, blood flow turbulence leads to the production of additional sounds, called murmurs. Murmurs are random in nature, while the underlying heart sounds are not (being deterministic).

Innovation

We show that a new analytical technique, which we call Digital Subtraction Phonocardiography (DSP), can be used to separate the random murmur component of the phonocardiogram from the underlying deterministic heart sounds.

Methods

We digitally recorded the phonocardiogram from the anterior chest wall in 60 infants and adults using a high-speed USB interface and the program Gold Wave http://www.goldwave.com. The recordings included individuals with cardiac structural disease as well as recordings from normal individuals and from individuals with innocent heart murmurs. Digital Subtraction Analysis of the signal was performed using a custom computer program called Murmurgram. In essence, this program subtracts the recorded sound from two adjacent cardiac cycles to produce a difference signal, herein called a "murmurgram". Other software used included Spectrogram (Version 16), GoldWave (Version 5.55) as well as custom MATLAB code.

Results

Our preliminary data is presented as a series of eight cases. These cases show how advanced signal processing techniques can be used to separate heart sounds from murmurs. Note that these results are preliminary in that normal ranges for obtained test results have not yet been established.

Conclusions

Cardiac murmurs can be separated from underlying deterministic heart sounds using DSP. DSP has the potential to become a reliable and economical new diagnostic approach to screening for structural heart disease. However, DSP must be further evaluated in a large series of patients with well-characterized pathology to determine its clinical potential.     

Coexistence of lateral and co-tuned inhibitory configurations in cortical networks

The responses of neurons in sensory cortex depend on the summation of excitatory and inhibitory synaptic inputs. How the excitatory and inhibitory inputs scale with stimulus depends on the network architecture, which ranges from the lateral inhibitory configuration where excitatory inputs are more narrowly tuned than inhibitory inputs, to the co-tuned configuration where both are tuned equally. The underlying circuitry that gives rise to lateral inhibition and co-tuning is yet unclear. Using large-scale network simulations with experimentally determined connectivity patterns and simulations with rate models, we show that the spatial extent of the input determined the configuration: there was a smooth transition from lateral inhibition with narrow input to co-tuning with broad input. The transition from lateral inhibition to co-tuning was accompanied by shifts in overall gain (reduced), output firing pattern (from tonic to phasic) and rate-level functions (from non-monotonic to monotonically increasing). The results suggest that a single cortical network architecture could account for the extended range of experimentally observed response types between the extremes of lateral inhibitory versus co-tuned configurations.     

The value of semi-natural grasslands for the conservation of carabid beetles in long-term managed forested landscapes

Species rich semi-natural grasslands are disappearing across Europe, affecting invertebrate diversity negatively. In NW Spain, the recent abandonment of traditional farming practices and the gradual decrease in grazing pressures are reducing the number and extent of montane grasslands. In this context, we investigated the composition of carabid beetle (Coleoptera, Carabidae) assemblages that inhabit semi-natural grasslands situated in long-term managed oak and beech forested landscapes. According to their spatial arrangement, the studied grasslands were classified into: (1) interior or gap grasslands (small and completely surrounded by continuous forest) and (2) exterior grasslands (large and connected to a variety of habitat types). Our results indicate that, within each forested landscape, the gap and exterior grasslands harboured particular carabid assemblages (i.e. exclusive or abundantly collected species), which were also distinct from the surrounding forest carabid fauna. Dissimilarities between gap and exterior grasslands in each landscape suggest great carabid diversity at the regional scale. We also detected species-specific responses as several carabids were mainly associated with gap or exterior grasslands. Consequently, in highly modified forested landscapes, semi-natural grassland remnants may constitute great value for the protection of the carabid fauna. Specifically, we recommend conservation strategies that preserve variety in grassland features and maintain proper management activities to prevent the loss of specialised species and a decrease in regional carabid diversity.     

Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli

Background

n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance.

Methodology/Principal Findings

Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%.

Conclusions/Significance

We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the rational engineering of more robust biofuel producers.     

Localization of ORC1 during the cell cycle in human leukemia cells

The interaction of the origin recognition complex (ORC) with replication origins is a critical parameter in eukaryotic replication initiation. In mammals the ORC remains bound except during mitosis, thus the localization of ORC complexes allows localization of origins. A monoclonal antibody that recognizes human ORC1 was used to localize ORC complexes in populations of human MOLT-4 cells separated by cell cycle position using centrifugal elutriation. ORC1 staining in cells in early G1 is diffuse and primarily peripheral. As the cells traverse G1, ORC1 accumulates and becomes more localized towards the center of the nucleus, however around the G1/S boundary the staining pattern changes and ORC1 appears peripheral. By mid to late S phase ORC1 immunofluorescence is again concentrated at the nuclear center. During anaphase, ORC1 staining is localized mainly in the pericentriolar regions. These findings suggest that concerted movements of origin DNA sequences in addition to the previously documented assembly and disassembly of protein complexes are an important aspect of replication initiation loci in eukaryotes.     

Innovative approaches for effective selection of lipase-producing microorganisms as whole cell catalysts for biodiesel production

The high cost of commercial lipases limits their industrial application in the production of biodiesel or fatty acid methyl esters (FAME). This disadvantage has encouraged the search for lipase-producing microorganisms (LPMs) as potential whole cell catalysts for FAME production. The aim of this study, therefore, was to evaluate innovative procedures for easy selection and testing of LPMs as a low-cost whole cell catalyst, based on catalytic performance, methanol tolerance and physico-chemical cell surface properties. The latter (in particular the cell surface hydrophobicity and charge) were analyzed because of their crucial role in microbial adhesion to surfaces and the concomitant increase in cell immobilization and bioavailability of hydrophobic substrates. Biocatalysis experiments performed in the presence of nutrient, rapeseed oil and methanol were an effective tool for studying and identifying, in just two experiments, the capacity of different LPMs as biocatalysts in organic media, as well as the methanol tolerance of the cell and the lipase. This indicates the potential for using live microorganisms for FAME production. Another finding was that the inhibitory effect of methanol is more significant for lipase activity than LPM growth, indicating that the way in which alcohol is supplied to the reaction is a crucial step in FAME production by biocatalysts. According to these results, the application of these innovative assessments should simplify the search for new strains which are able to effectively catalyze the FAME production process.