A longitudinal MRI and TSPO PET-based investigation of brain region-specific neuroprotection by diazepam versus midazolam following organophosphate-induced seizures.

Acute poisoning with organophosphorus cholinesterase inhibitors (OPs), such as OP nerve agents and pesticides, can cause life threatening cholinergic crisis and status epilepticus (SE). Survivors often experience significant morbidity, including brain injury, acquired epilepsy, and cognitive deficits. Current medical countermeasures for acute OP poisoning include a benzodiazepine to mitigate seizures. Diazepam was long the benzodiazepine included in autoinjectors used to treat OP-induced seizures, but it is now being replaced in many guidelines by midazolam, which terminates seizures more quickly, particularly when administered intramuscularly. While a direct correlation between seizure duration and the extent of brain injury has been widely reported, there are limited data comparing the neuroprotective efficacy of diazepam versus midazolam following acute OP intoxication. To address this data gap, we used non-invasive imaging techniques to longitudinally quantify neuropathology in a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) with and without post-exposure intervention with diazepam or midazolam. Magnetic resonance imaging (MRI) was used to monitor neuropathology and brain atrophy, while positron emission tomography (PET) with a radiotracer targeting translocator protein (TSPO) was utilized to assess neuroinflammation. Animals were scanned at 3, 7, 28, 65, 91, and 168 days post-DFP and imaging metrics were quantitated for the hippocampus, amygdala, piriform cortex, thalamus, cerebral cortex and lateral ventricles. In the DFP-intoxicated rat, neuroinflammation persisted for the duration of the study coincident with progressive atrophy and ongoing tissue remodeling. Benzodiazepines attenuated neuropathology in a region-dependent manner, but neither benzodiazepine was effective in attenuating long-term neuroinflammation as detected by TSPO PET. Diffusion MRI and TSPO PET metrics were highly correlated with seizure severity, and early MRI and PET metrics were positively correlated with long-term brain atrophy. Collectively, these results suggest that anti-seizure therapy alone is insufficient to prevent long-lasting neuroinflammation and tissue remodeling.

Co-localization of fluorescent signals using deep learning with Manders overlapping coefficient.

Object-based co-localization of fluorescent signals allows the assessment of interactions between two (or more) biological entities using spatial information. It relies on object identification with high accuracy to separate fluorescent signals from the background. Object detectors using convolutional neural networks (CNN) with annotated training samples could facilitate the process by detecting and counting fluorescent-labeled cells from fluorescence photomicrographs. However, datasets containing segmented annotations of colocalized cells are generally not available, and creating a new dataset with delineated masks is label-intensive. Also, the co-localization coefficient is often not used as a component during training with the CNN model. Yet, it may aid with localizing and detecting objects during training and testing. In this work, we propose to address these issues by using a quantification coefficient for co-localization called Manders overlapping coefficient (MOC)1 as a single-layer branch in a CNN. Fully convolutional one-state (FCOS)2 with a Resnet101 backbone served as the network to evaluate the effectiveness of the novel branch to assist with bounding box prediction. Training data were sourced from lab curated fluorescence images of neurons from the rat hippocampus, piriform cortex, somatosensory cortex, and amygdala. Results suggest that using modified FCOS with MOC outperformed the original FCOS model for accuracy in detecting fluorescence signals by 1.1% in mean average precision (mAP). The model could be downloaded from https://github.com/Alphafrey946/Colocalization-MOC.

Mapping geometry in Fizeau transmission spheres with a small f-number.

The ideal mapping geometry in a Fizeau interferometer is to map equal height increments on a flat object and equal angle increments on a spherical surface to equal heights on the detector. So the initial intent of the optical design of Fizeau transmission spheres (TSs) is to provide R-θ mapping geometry for equal angle increments. The corresponding unequal heights mapping will introduce retrace error as coma when linear carrier fringes exist. On the contrary, equal heights mapping with R-sin θ mapping geometry will avoid linear carrier fringes induced coma error. These two different mapping geometries conflict especially for the TS with a small f-number. In this paper, we will first explore the design and the performance of the f/0.75 TS according to the two different mapping geometries, and then evaluate the mapping geometry for the commercial ZYGO f/0.75 TS, and give some engineering notes for the designers, the metrologists, and the fabricators in the optical laboratory.

A scalable and integrative system for pathway bioinformatics and systems biology.

MOTIVATION: Progress in systems biology depends on developing scalable informatics tools to predictively model, visualize, and flexibly store information about complex biological systems. Scalability of these tools, as well as their ability to integrate within larger frameworks of evolving tools, is critical to address the multi-scale and size complexity of biological systems. RESULTS: Using current software technology, such as self-generation of database and object code from UML schemas, facilitates rapid updating of a scalable expert assistance system for modeling biological pathways. Distribution of key components along with connectivity to external data sources and analysis tools is achieved via a web service interface. AVAILABILITY: All sigmoid modeling software components and supplementary information are available through: http://www.igb.uci.edu/servers/sb.html.

Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site.

Alternative pre-mRNA splicing may be the most efficient and widespread mechanism to generate multiple protein isoforms from single genes. Here, we describe the genomic analysis of one of the most frequent types of alternative pre-mRNA splicing, alternative 5'- and 3'-splice-site selection. Using an EST-based alternative splicing database recording >47,000 alternative splicing events, we determined the frequency and location of alternative 5'- and 3'-splice sites within the human genome. The most common alternative splice sites used in the human genome are located within 6 nucleotides (nt) of the dominant splice site. We show that the EST database overrepresents alternative splicing events that maintain the reading frame, thus supporting the concept that RNA quality-control steps ensure that mRNAs that encode for potentially harmful protein products are destroyed and do not serve as templates for translation. The most frequent location for alternative 5'-splice sites is 4 nt upstream or downstream from the dominant splice site. Sequence analysis suggests that this preference is a consequence of the U1 snRNP binding sequence at the 5'-splice site, which frequently contains a GU dinucleotide 4 nt downstream from the dominant splice site. Surprisingly, approximately 50% of duplicated 3'-YAG splice junctions are subject to alternative splicing. This high probability of alternative 3'-splice-site activation in close proximity of the dominant 3'-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity.

A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.

Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for two-step purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 m urea, by Ni(2+) chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein complexes, thereby expanding proteomic analyses to the description of weakly or transiently associated protein complexes.

The architecture of pre-mRNAs affects mechanisms of splice-site pairing.

The exon/intron architecture of genes determines whether components of the spliceosome recognize splice sites across the intron or across the exon. Using in vitro splicing assays, we demonstrate that splice-site recognition across introns ceases when intron size is between 200 and 250 nucleotides. Beyond this threshold, splice sites are recognized across the exon. Splice-site recognition across the intron is significantly more efficient than splice-site recognition across the exon, resulting in enhanced inclusion of exons with weak splice sites. Thus, intron size can profoundly influence the likelihood that an exon is constitutively or alternatively spliced. An EST-based alternative-splicing database was used to determine whether the exon/intron architecture influences the probability of alternative splicing in the Drosophila and human genomes. Drosophila exons flanked by long introns display an up to 90-fold-higher probability of being alternatively spliced compared with exons flanked by two short introns, demonstrating that the exon/intron architecture in Drosophila is a major determinant in governing the frequency of alternative splicing. Exon skipping is also more likely to occur when exons are flanked by long introns in the human genome. Interestingly, experimental and computational analyses show that the length of the upstream intron is more influential in inducing alternative splicing than is the length of the downstream intron. We conclude that the size and location of the flanking introns control the mechanism of splice-site recognition and influence the frequency and the type of alternative splicing that a pre-mRNA transcript undergoes.

ChemDB: a public database of small molecules and related chemoinformatics resources.

MOTIVATION: The development of chemoinformatics has been hampered by the lack of large, publicly available, comprehensive repositories of molecules, in particular of small molecules. Small molecules play a fundamental role in organic chemistry and biology. They can be used as combinatorial building blocks for chemical synthesis, as molecular probes in chemical genomics and systems biology, and for the screening and discovery of new drugs and other useful compounds. RESULTS: We describe ChemDB, a public database of small molecules available on the Web. ChemDB is built using the digital catalogs of over a hundred vendors and other public sources and is annotated with information derived from these sources as well as from computational methods, such as predicted solubility and three-dimensional structure. It supports multiple molecular formats and is periodically updated, automatically whenever possible. The current version of the database contains approximately 4.1 million commercially available compounds and 8.2 million counting isomers. The database includes a user-friendly graphical interface, chemical reactions capabilities, as well as unique search capabilities. AVAILABILITY: Database and datasets are available on http://cdb.ics.uci.edu.

The absence of favorable aromatic interactions between beta-sheet peptides.

This paper asks whether interactions between phenylalanine (Phe) residues of the non-hydrogen-bonded cross-strand pairs of antiparallel beta-sheets are important and finds that they are not. Peptides 1a-d [o-BuO-C6H4CO-AA1-Orn(i-PrCO-Hao)-Phe-Ile-AA5-NHMe: 1a AA1, AA5 = Phe; 1b AA1, AA5 = Cha (cyclohexylalanine); 1c AA1 = Phe, AA5 = Cha; 1d AA1 = Cha, AA5 = Phe] provide a sensitive system for probing interactions between phenylalanine residues. These peptides form beta-sheet homodimers in organic solvents. When the homodimers of different peptides are mixed, they equilibrate to form heterodimers, as well as homodimers. The position of the equilibrium reflects the propensity of the first (AA1) and fifth (AA5) amino acids to interact within the non-hydrogen-bonded cross-strand pairs of beta-sheets. Mixing peptides 1a-d in all six possible binary combinations provides a measure of the relative propensities of Phe and Cha to pair. Analysis by 1H NMR spectroscopy of the equilibrium constants in CDCl3 solution reveals no significant preference for the formation of Phe-Phe pairs. The equilibria in all six experiments are essentially statistical (K approximately 4), and no (<0.1 kcal/mol) preference is seen for any pairing combination. A survey of Phe-Phe pairs in the Interchain beta-Sheet Database (http://www.igb.uci.edu/servers/icbs/) corroborates that little significant contact occurs between the aromatic rings in the non-hydrogen-bonded cross-strand pairs of antiparallel beta-sheets at the interface between polypeptide chains. Even though contacts between aromatic rings are favorable when they are of suitable geometry, the energetic price of achieving suitable geometries appears to offset the energetic benefits of such contacts in the current model system, as well as in proteins.

Retroviruses and yeast retrotransposons use overlapping sets of host genes.

A collection of 4457 Saccharomyces cerevisiae mutants deleted for nonessential genes was screened for mutants with increased or decreased mobilization of the gypsylike retroelement Ty3. Of these, 64 exhibited increased and 66 decreased Ty3 transposition compared with the parental strain. Genes identified in this screen were grouped according to function by using GOnet software developed as part of this study. Gene clusters were related to chromatin and transcript elongation, translation and cytoplasmic RNA processing, vesicular trafficking, nuclear transport, and DNA maintenance. Sixty-six of the mutants were tested for Ty3 proteins and cDNA. Ty3 cDNA and transposition were increased in mutants affected in nuclear pore biogenesis and in a subset of mutants lacking proteins that interact physically or genetically with a replication clamp loader. Our results suggest that nuclear entry is linked mechanistically to Ty3 cDNA synthesis but that host replication factors antagonize Ty3 replication. Some of the factors we identified have been previously shown to affect Ty1 transposition and others to affect retroviral budding. Host factors, such as these, shared by distantly related Ty retroelements and retroviruses are novel candidates for antiviral targets.

ICBS: a database of interactions between protein chains mediated by beta-sheet formation.

MOTIVATION: Interchain beta-sheet (ICBS) interactions occur widely in protein quaternary structures, interactions between proteins and protein aggregation. These interactions play a central role in many biological processes and in diseases ranging from AIDS and cancer to anthrax and Alzheimer's. RESULTS: We have created a comprehensive database of ICBS interactions that is updated on a weekly basis and allows entries to be sorted and searched by relevance and other criteria through a simple Web interface. We derive a simple ICBS index to quantify the relative contributions of the beta-ladders in the overall interchain interaction and compute first- and second-order statistics regarding amino acid composition and pairing at different relative positions in the beta-strands. Analysis of the database reveals a 15.8% prevalence of significant ICBS interactions, the majority of which involve the formation of antiparallel beta-sheets and many of which involve the formation of dimers and oligomers. The frequencies of amino acids in ICBS interfaces are similar to those in intrachain beta-sheet interfaces. A full range of non-covalent interactions between side chains complement the hydrogen-bonding interactions between the main chains. Polar amino acids pair preferentially with polar amino acids and non-polar amino acids pair preferentially with non-polar amino acids among antiparallel (i, j) pairs. We anticipate that the statistics and insights gained from the database will guide the development of agents that control interchain beta-sheet interactions and that the database will help identify new protein interactions and targets for these agents. AVAILABILITY: The database is available at: http://www.igb.uci.edu/servers/icbs/