Split-intein mediated re-assembly of genetically encoded Ca(2+) indicators

While genetically encoded Ca(2+) indicators (GECIs) allow Ca(2+) imaging in model organisms, the gene expression is often under the control of a single promoter that may drive expression beyond, the cell types of interest. To enable more cell-type specific targeting, GECIs can be brought under the, control of the intersecting expression from two promoters. Here, we present the splitting and, reassembly of two representative GECIs (TN-XL and GCaMP2) mediated by the split intein from Nostoc, punctiforme (NpuDnaE). While the split TN-XL biosensor offered ratiometric Ca(2+) imaging, it had a, diminished Ca(2+) response relative to the native TN-XL biosensor. In contrast, the split GCaMP2, biosensor retained similar Ca(2+) response to the native GCaMP2. The split GCaMP2 biosensor was, further targeted to the pharyngeal muscles of Caenorhabditis elegans where Ca(2+) signals from feeding C. elegans, were imaged. Thus, we envision that increased cell-type targetability of GECIs is feasible with two, complementary promoters.     

Simultaneous saccharification and fermentation (SSF) of <Emphasis Type="Italic">Jatropha curcas</Emphasis> shells: utilization of co-products from the biodiesel production process

Jatropha curcas has great potential as an oil crop for use in biodiesel applications, and the outer shell is rich in lignocellulose that may be converted to ethanol, giving rise to the concept of a biorefinery. In this study, two dilute pretreatments of 0.5% H₂SO₄ and 1.0% NaOH were performed on Jatropha shells with subsequent simultaneous saccharification and fermentation (SSF) of the pretreated water-insoluble solids (WIS) to evaluate the effect of inhibitors in the pretreatment slurry. A cellulase loading of 15 FPU/g WIS, complimented with an excess of cellobiase (19.25 U/g), was used for SSF of either the washed WIS or the original slurry to determine the effect of inhibitors. Ethanol and glucose were monitored during SSF of 20 g of pretreated biomass. The unwashed slurry showed to have a positive effect on SSF efficiency for the NaOH-pretreated biomass. Maximum efficiencies of glucan conversion to ethanol in the WIS were 40.43% and 41.03% for the H₂SO₄- and NaOH-pretreated biomasses, respectively.     

Detection of structural variants and indels within exome data

We report an algorithm to detect structural variation and indels from 1 base pair (bp) to 1 Mbp within exome sequence data sets. Splitread uses one end-anchored placements to cluster the mappings of subsequences of unanchored ends to identify the size, content and location of variants with high specificity and sensitivity. The algorithm discovers indels, structural variants, de novo events and copy number-polymorphic processed pseudogenes missed by other methods.     

Second-Order Conditioning and Conditioned Inhibition: Influences of Speed versus Accuracy on Human Causal Learning

In human causal learning, excitatory and inhibitory learning effects can sometimes be found in the same paradigm by altering the learning conditions. This study aims to explore whether learning in the feature negative paradigm can be dissociated by emphasising speed over accuracy. In two causal learning experiments, participants were given a feature negative discrimination in which the outcome caused by one cue was prevented by the addition of another. Participants completed training trials either in a self-paced fashion with instructions emphasising accuracy, or under strict time constraints with instructions emphasising speed. Using summation tests in which the preventative cue was paired with another causal cue, participants in the accuracy groups correctly rated the preventative cue as if it reduced the probability of the outcome. However, participants in the speed groups rated the preventative cue as if it increased the probability of the outcome. In Experiment 1, both speed and accuracy groups later judged the same cue to be preventative in a reasoned inference task. Experiment 2 failed to find evidence of similar dissociations in retrospective revaluation (release from overshadowing vs. mediated extinction) or learning about a redundant cue (blocking vs. augmentation). However in the same experiment, the tendency for the accuracy group to show conditioned inhibition and the speed group to show second-order conditioning was consistent even across sub-sets of the speed and accuracy groups with equivalent accuracy in training, suggesting that second-order conditioning is not merely a consequence of poorer acquisition. This dissociation mirrors the trade-off between second-order conditioning and conditioned inhibition observed in animal conditioning when training is extended.     

FoldEco: a model for proteostasis in E. coli

To gain insight into the interplay of processes and species that maintain a correctly folded, functional proteome, we have developed a computational model called FoldEco. FoldEco models the cellular proteostasis network of the E. coli cytoplasm, including protein synthesis, degradation, aggregation, chaperone systems, and the folding characteristics of protein clients. We focused on E. coli because much of the needed input information--including mechanisms, rate parameters, and equilibrium coefficients--is available, largely from in vitro experiments; however, FoldEco will shed light on proteostasis in other organisms. FoldEco can generate hypotheses to guide the design of new experiments. Hypothesis generation leads to system-wide questions and shows how to convert these questions to experimentally measurable quantities, such as changes in protein concentrations with chaperone or protease levels, which can then be used to improve our current understanding of proteostasis and refine the model. A web version of FoldEco is available at http://foldeco.scripps.edu.     

The sva package for removing batch effects and other unwanted variation in high-throughput experiments

Heterogeneity and latent variables are now widely recognized as major sources of bias and variability in high-throughput experiments. The most well-known source of latent variation in genomic experiments are batch effects-when samples are processed on different days, in different groups or by different people. However, there are also a large number of other variables that may have a major impact on high-throughput measurements. Here we describe the sva package for identifying, estimating and removing unwanted sources of variation in high-throughput experiments. The sva package supports surrogate variable estimation with the sva function, direct adjustment for known batch effects with the ComBat function and adjustment for batch and latent variables in prediction problems with the fsva function.     

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application – SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary “Manual Initialize” and “Hand Draw” tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.     

Fine-root respiration in a loblolly pine (Pinus taeda L.) forest exposed to elevated CO2 and N fertilization

Forest ecosystems release large amounts of carbon to the atmosphere from fine-root respiration (R(r)), but the control of this flux and its temperature sensitivity (Q(10)) are poorly understood. We attempted to: (1) identify the factors limiting this flux using additions of glucose and an electron transport uncoupler (carbonyl cyanide m-chlorophenylhydrazone); and (2) improve yearly estimates of R(r) by directly measuring its Q(10)in situ using temperature-controlled cuvettes buried around intact, attached roots. The proximal limits of R(r) of loblolly pine (Pinus taeda L.) trees exposed to free-air CO(2) enrichment (FACE) and N fertilization were seasonally variable; enzyme capacity limited R(r) in the winter, and a combination of substrate supply and adenylate availability limited R(r) in summer months. The limiting factors of R(r) were not affected by elevated CO(2) or N fertilization. Elevated CO(2 )increased annual stand-level R(r) by 34% whereas the combination of elevated CO(2) and N fertilization reduced R(r) by 40%. Measurements of in situ R(r) with high temporal resolution detected diel patterns that were correlated with canopy photosynthesis with a lag of 1 d or less as measured by eddy covariance, indicating a dynamic link between canopy photosynthesis and root respiration. These results suggest that R(r) is coupled to daily canopy photosynthesis and increases with carbon allocation below ground.