SPIRE: the SPIDER reconstruction engine

SPIRE is a Python program written to modernize the user interaction with SPIDER, the image processing system for electron microscopical reconstruction projects. SPIRE provides a graphical user interface (GUI) to SPIDER for executing batch files of SPIDER commands. It also lets users quickly view the status of a project by showing the last batch files that were run, as well as the data files that were generated. SPIRE handles the flexibility of the SPIDER programming environment through configuration files: XML-tagged documents that describe the batch files, directory trees, and presentation of the GUI for a given type of reconstruction project. It also provides the capability to connect to a laboratory database, for downloading parameters required by batch files at the start of a project, and uploading reconstruction results at the end of a project.     

Scoring protein interaction decoys using exposed residues (SPIDER): a novel multibody interaction scoring function based on frequent geometric patterns of interfacial residues

Accurate prediction of the structure of protein-protein complexes in computational docking experiments remains a formidable challenge. It has been recognized that identifying native or native-like poses among multiple decoys is the major bottleneck of the current scoring functions used in docking. We have developed a novel multibody pose-scoring function that has no theoretical limit on the number of residues contributing to the individual interaction terms. We use a coarse-grain representation of a protein-protein complex where each residue is represented by its side chain centroid. We apply a computational geometry approach called Almost-Delaunay tessellation that transforms protein-protein complexes into a residue contact network, or an undirectional graph where vertex-residues are nodes connected by edges. This treatment forms a family of interfacial graphs representing a dataset of protein-protein complexes. We then employ frequent subgraph mining approach to identify common interfacial residue patterns that appear in at least a subset of native protein-protein interfaces. The geometrical parameters and frequency of occurrence of each "native" pattern in the training set are used to develop the new SPIDER scoring function. SPIDER was validated using standard "ZDOCK" benchmark dataset that was not used in the development of SPIDER. We demonstrate that SPIDER scoring function ranks native and native-like poses above geometrical decoys and that it exceeds in performance a popular ZRANK scoring function. SPIDER was ranked among the top scoring functions in a recent round of CAPRI (Critical Assessment of PRedicted Interactions) blind test of protein-protein docking methods.     

Scalable remote homology detection and fold recognition in massive protein networks

The global connectivities in very large protein similarity networks contain traces of evolution among the proteins for detecting protein remote evolutionary relations or structural similarities. To investigate how well a protein network captures the evolutionary information, a key limitation is the intensive computation of pairwise sequence similarities needed to construct very large protein networks. In this article, we introduce label propagation on low-rank kernel approximation (LP-LOKA) for searching massively large protein networks. LP-LOKA propagates initial protein similarities in a low-rank graph by Nyström approximation without computing all pairwise similarities. With scalable parallel implementations based on distributed-memory using message-passing interface and Apache-Hadoop/Spark on cloud, LP-LOKA can search protein networks with one million proteins or more. In the experiments on Swiss-Prot/ADDA/CASP data, LP-LOKA significantly improved protein ranking over the widely used HMM-HMM or profile-sequence alignment methods utilizing large protein networks. It was observed that the larger the protein similarity network, the better the performance, especially on relatively small protein superfamilies and folds. The results suggest that computing massively large protein network is necessary to meet the growing need of annotating proteins from newly sequenced species and LP-LOKA is both scalable and accurate for searching massively large protein networks.     

Biodegradation, sorption, and transport of 2,4-dichlorophenoxyacetic acid in saturated and unsaturated soils.

The fate of an organic contaminant in soil depends on many factors, including sorption, biodegradation, and transport. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was used as a model compound to illustrate the impact of these interacting factors on the fate of an organic contaminant. Batch and column experiments performed with a sandy loam soil mixture under saturated and unsaturated conditions were used to determine the effects of sorption and biodegradation on the fate and transport of 2,4-D. Sorption of 2,4-D was found to have a slight but significant effect on transport of 2,4-D under saturated conditions (retardation factor, 1.8) and unsaturated conditions (retardation factor, 3.4). Biodegradation of 2,4-D was extensive under both batch and column conditions and was found to have a significant impact on 2,4-D transport in column experiments. In batch experiments, complete mineralization of 2,4-D (100 mg kg-1) occurred over a 4-day period following a 3-day lag phase under both saturated and unsaturated conditions. The biodegradation rate parameters calculated for batch experiments were found to be significantly different from those estimated for column experiments.     

A Load Balancing Tool for Distributed Parallel Loops

Large scale applications typically contain parallel loops with many iterates. The iterates of a parallel loop may have variable execution times which translate into performance degradation of an application due to load imbalance. This paper describes a tool for load balancing parallel loops on distributed-memory systems. The tool assumes that the data for a parallel loop to be executed is already partitioned among the participating processors. The tool utilizes the MPI library for interprocessor coordination, and determines processor workloads by loop scheduling techniques. The tool was designed independent of any application; hence, it must be supplied with a routine that encapsulates the computations for a chunk of loop iterates, as well as the routines to transfer data and results between processors. Performance evaluation on a Linux cluster indicates that the tool reduces the cost of executing a simulated irregular loop without load balancing by up to 81%. The tool is useful for parallelizing sequential applications with parallel loops, or as an alternate load balancing routine for existing parallel applications.     

Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential 'mini-barcodes' for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation.     

Release from Cross-Orientation Suppression Facilitates 3D Shape Perception

Cross-orientation suppression (COS) in striate cortex has been implicated in the efficient encoding of visual stimuli. We show that release from COS facilitates the decoding of 3-D shape. In planar surfaces overlaid with textures, slanting the surface can increase the visibility of the component parallel to the slant. Since this component provides the orientation flows that signify 3-D shape, the enhancement of visibility facilitates 3-D slant perception. Contrast thresholds reveal that this enhancement results from a decrease in COS when 3-D slant creates a frequency mismatch between texture components. We show that coupling compressive nonlinearities in LGN neurons with expansive nonlinearities in cortical neurons can model the frequency-specific component of suppression.     

Implementation and performance evaluation of reconstruction algorithms on graphics processors

The high-throughput needs in electron tomography and in single particle analysis have driven the parallel implementation of several reconstruction algorithms and software packages on computing clusters. Here, we report on the implementation of popular reconstruction algorithms as weighted backprojection, simultaneous iterative reconstruction technique (SIRT) and simultaneous algebraic reconstruction technique (SART) on common graphics processors (GPUs). The speed gain achieved on the GPUs is in the order of sixty (60x) to eighty (80x) times, compared to the performance of a single central processing unit (CPU), which is comparable to the acceleration achieved on a medium-range computing cluster. This acceleration of the reconstruction is caused by the highly specialized architecture of the GPU. Further, we show that the quality of the reconstruction on the GPU is comparable to the CPU. We present detailed flow-chart diagrams of the implementation. The reconstruction software does not require special hardware apart from the commercially available graphics cards and could be easily integrated in software packages like SPIDER, XMIPP, TOM-Package and others.     

Removing batch effects in analysis of expression microarray data: an evaluation of six batch adjustment methods

The expression microarray is a frequently used approach to study gene expression on a genome-wide scale. However, the data produced by the thousands of microarray studies published annually are confounded by "batch effects," the systematic error introduced when samples are processed in multiple batches. Although batch effects can be reduced by careful experimental design, they cannot be eliminated unless the whole study is done in a single batch. A number of programs are now available to adjust microarray data for batch effects prior to analysis. We systematically evaluated six of these programs using multiple measures of precision, accuracy and overall performance. ComBat, an Empirical Bayes method, outperformed the other five programs by most metrics. We also showed that it is essential to standardize expression data at the probe level when testing for correlation of expression profiles, due to a sizeable probe effect in microarray data that can inflate the correlation among replicates and unrelated samples.     

Protein microarrays on hybrid polymeric thin films prepared by self-assembly of polyelectrolytes for multiple-protein immunoassays

We report here the development and characterization of protein microarrays fabricated on nanoengineered 3-D polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes via self-assembly technique. Antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of porous structure of the 3-D polymer film and thus establishing a platform for parallel analysis. Both antigen and antibody microarrays were fabricated on the PET-coated slides, and direct and indirect immunoassays on protein microarrays for multiple-analyte detection were demonstrated. Microarrays produced on these PET-coated slides have consistent spot morphology and provide performance features needed for proteomic analysis. The protein microarrays on the PET films provide LOD as low as 6 pg/mL and dynamic ranges up to three orders of magnitude, which are wider than the protein microarrays fabricated on aldehyde and poly-L-lysine functionalized slides. The PET films constructed by self-assembly technique in aqueous solution is green chemistry based, cost-effective method to generate 3-D thin film coatings on glass surface, and the coated slide is well suited for immobilizing many types of biological molecules so that a wide variety of microarray formats can be developed on this type of slide.     

Open-Source Syringe Pump Library

This article explores a new open-source method for developing and manufacturing high-quality scientific equipment suitable for use in virtually any laboratory. A syringe pump was designed using freely available open-source computer aided design (CAD) software and manufactured using an open-source RepRap 3-D printer and readily available parts. The design, bill of materials and assembly instructions are globally available to anyone wishing to use them. Details are provided covering the use of the CAD software and the RepRap 3-D printer. The use of an open-source Rasberry Pi computer as a wireless control device is also illustrated. Performance of the syringe pump was assessed and the methods used for assessment are detailed. The cost of the entire system, including the controller and web-based control interface, is on the order of 5% or less than one would expect to pay for a commercial syringe pump having similar performance. The design should suit the needs of a given research activity requiring a syringe pump including carefully controlled dosing of reagents, pharmaceuticals, and delivery of viscous 3-D printer media among other applications.     

Investigation of a New Electrode Array Technology for a Central Auditory Prosthesis

Ongoing clinical studies on patients recently implanted with the auditory midbrain implant (AMI) into the inferior colliculus (IC) for hearing restoration have shown that these patients do not achieve performance levels comparable to cochlear implant patients. The AMI consists of a single-shank array (20 electrodes) for stimulation along the tonotopic axis of the IC. Recent findings suggest that one major limitation in AMI performance is the inability to sufficiently activate neurons across the three-dimensional (3-D) IC. Unfortunately, there are no currently available 3-D array technologies that can be used for clinical applications. More recently, there has been a new initiative by the European Commission to fund and develop 3-D chronic electrode arrays for science and clinical applications through the NeuroProbes project that can overcome the bulkiness and limited 3-D configurations of currently available array technologies. As part of the NeuroProbes initiative, we investigated whether their new array technology could be potentially used for future AMI patients. Since the NeuroProbes technology had not yet been tested for electrical stimulation in an in vivo animal preparation, we performed experiments in ketamine-anesthetized guinea pigs in which we inserted and stimulated a NeuroProbes array within the IC and recorded the corresponding neural activation within the auditory cortex. We used 2-D arrays for this initial feasibility study since they were already available and were sufficient to access the IC and also demonstrate effective activation of the central auditory system. Based on these encouraging results and the ability to develop customized 3-D arrays with the NeuroProbes technology, we can further investigate different stimulation patterns across the ICC to improve AMI performance.     

SPIDER: software for protein identification from sequence tags with de novo sequencing error

For the identification of novel proteins using MS/MS, de novo sequencing software computes one or several possible amino acid sequences (called sequence tags) for each MS/MS spectrum. Those tags are then used to match, accounting amino acid mutations, the sequences in a protein database. If the de novo sequencing gives correct tags, the homologs of the proteins can be identified by this approach and software such as MS-BLAST is available for the matching. However, de novo sequencing very often gives only partially correct tags. The most common error is that a segment of amino acids is replaced by another segment with approximately the same masses. We developed a new efficient algorithm to match sequence tags with errors to database sequences for the purpose of protein and peptide identification. A software package, SPIDER, was developed and made available on Internet for free public use. This paper describes the algorithms and features of the SPIDER software.     

Supporting parallel applications on clusters of workstations: The Virtual Communication Machine‐based architecture

This paper presents a novel networking architecture designed for communication intensive parallel applications running on clusters of workstations (COWs) connected by high speed networks. The architecture addresses what is considered one of the most important problems of cluster-based parallel computing: the inherent inability of scaling the performance of communication software along with the host CPU performance. The Virtual Communication Machine (VCM), resident on the network coprocessor, presents a scalable software solution by providing configurable communication functionality directly accessible at user-level. The VCM architecture is configurable in that it enables the transfer to the VCM of selected communication-related functionality that is traditionally part of the application and/or the host kernel. Such transfers are beneficial when a significant reduction of the host CPU's load translates into a small increase in the coprocessor's load. The functionality implemented by the coprocessor is available at the application level as VCM instructions. Host CPU(s) and coprocessor interact through shared memory regions, thereby avoiding expensive CPU context switches. The host kernel is not involved in this interaction; it simply “connects” the application to the VCM during the initialization phase and is called infrequently to handle exceptional conditions. Protection is enforced by the VCM based on information supplied by the kernel. The VCM-based communication architecture admits low cost and open implementations, as demonstrated by its current ATM-based implementation based on off-the-shelf hardware components and using standard AAL5 packets. The architecture makes it easy to implement communication software that exhibits negligible overheads on the host CPU(s) and offers latencies and bandwidths close to the hardware limits of the underlying network. These characteristics are due to the VCM's support for zero-copy messaging with gather/scatter capabilities and the VCM's direct access to any data structure in an application's address space. This paper describes two versions of an ATM-based VCM implementation, which differ in the way they use the memory on the network adapter. Their performance under heavy load is compared in the context of a synthetic client/server application. The same application is used to evaluate the scalability of the architecture to multiple VCM-based network interfaces per host. Parallel implementations of the Traveling Salesman Problem and of Georgia Tech Time Warp, an engine for discrete-event simulation, are used to demonstrate VCM functionality and the high performance of its implementation. The distributed- and shared-memory versions of these two applications exhibit comparable performance, despite the significant cost-performance advantage of the distributed-memory platform. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

tRNA Dissociation from EF-Tu after GTP Hydrolysis: Primary Steps and Antibiotic Inhibition

In each round of ribosomal translation, the translational GTPase elongation factor Tu (EF-Tu) delivers a transfer RNA (tRNA) to the ribosome. After successful decoding, EF-Tu hydrolyzes GTP, which triggers a conformational change that ultimately results in the release of the tRNA from EF-Tu. To identify the primary steps of these conformational changes and how they are prevented by the antibiotic kirromycin, we employed all-atom explicit-solvent molecular dynamics simulations of the full ribosome-EF-Tu complex. Our results suggest that after GTP hydrolysis and P_i release, the loss of interactions between the nucleotide and the switch 1 loop of EF-Tu allows domain D1 of EF-Tu to rotate relative to domains D2 and D3 and leads to an increased flexibility of the switch 1 loop. This rotation induces a closing of the D1-D3 interface and an opening of the D1-D2 interface. We propose that the opening of the D1-D2 interface, which binds the CCA tail of the tRNA, weakens the crucial EF-Tu-tRNA interactions, which lowers tRNA binding affinity, representing the first step of tRNA release. Kirromycin binds within the D1-D3 interface, sterically blocking its closure, but does not prevent hydrolysis. The resulting increased flexibility of switch 1 explains why it is not resolved in kirromycin-bound structures.     

Co-culture of osteoblasts and chondrocytes modulates cellular differentiation in vitro

Biological integration of cartilage grafts with subchondral bone remains a significant clinical challenge. We hypothesize that interaction between osteoblasts and chondrocytes is important in regenerating the osteochondral interface on tissue-engineered osteochondral grafts. We describe here a sequential co-culturing model which permits cell-cell contact and paracrine interaction between osteoblast and chondrocytes in 3-D culture. This model was used to determine the effects of co-culture on the phenotypic maintenance of osteoblasts and chondrocytes. It was found that while chondrocytes synthesized a type II collagen and glycosaminoglycan (GAG) matrix, GAG deposition was significantly lower in co-culture. Alkaline phosphatase activity was maintained in osteoblasts, but cell-mediated mineralization in co-culture was markedly lower compared to osteoblast controls. These results collectively suggest that interactions between osteoblasts and chondrocytes modulate cell phenotypes, and the importance of these interactions on osteochondral interface regeneration will be explored in future studies.     

Effect of integration of a GFP reporter gene on fitness of Ralstonia eutropha during growth with 2,4-dichlorophenoxyacetic acid

Green fluorescent proteins (GFPs) are frequently used as marker and reporter systems to assess the fate and activity of microbial strains with the ability to degrade xenobiotic compounds. To evaluate the potential of this tool for tracking herbicide-degrading microorganisms in the environment a promoterless gfp was linked to the tfd C promoter, which is activated during degradation of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), and integrated into the chromosome of the 2,4-D-degrading strain Ralstonia eutropha JMP 134. The effects of the inserted gfp gene on the kinetics of 2,4-D degradation by R. eutropha in batch and chemostat culture were compared to those of the wild-type strain. In batch culture with 2,4-D as the only carbon and energy source the maximum specific growth rate of the gfp-marked strain did not differ significantly from the wild type. However, compared to the wild type, the 2,4-D steady-state concentration in 2,4-D-limited chemostat cultures of the gfp-marked strain was higher at all dilution rates tested. The reduced competitiveness of the gfp-marked strain at low substrate concentrations was confirmed in a competition experiment for 2,4-D in continuous culture at a dilution rate of 0.075 h-1. Reproducibly, the gfp-marked strain was displaced by the wild-type strain. The study clearly demonstrates that fitness of constructs cannot be assessed by measuring micro max with selected substrates in batch cultures only but that a thorough kinetic analysis is needed, which also considers slow, carbon-limited growth conditions as they occur in the environment.     

3-D warped discrete cosine transform for MRI image compression

Image compression is an application of data compression on digital images. Several lossy/lossless transform coding techniques are used for image compression. Discrete cosine transform (DCT) is one such widely used technique. A variation of DCT, known as warped discrete cosine transform (WDCT), is used for 2-D image compression and it is shown to perform better than the DCT at high bit-rates. We extend this concept and develop the 3-D WDCT, a transform that has not been previously investigated. We outline some of its important properties, which make it especially suitable for image compression. We then propose a complete image coding scheme for volumetric data sets based on the 3-D WDCT scheme. It is shown that the 3-D WDCT-based compression scheme performs better than a similar 3-D DCT scheme for volumetric data sets at high bit-rates.