The SAH7 Homologue of the Allergen Ole e 1 Interacts with the Putative Stress Sensor SBP1 (Selenium-Binding Protein 1) in Arabidopsis thaliana.

In this study, we focused on a member of the Ole e 1 domain-containing family, AtSAH7, in Arabidopsis thaliana. Our lab reports for the first time on this protein, AtSAH7, that was found to interact with Selenium-binding protein 1 (AtSBP1). We studied by GUS assisted promoter deletion analysis the expression pattern of AtSAH7 and determined that the sequence 1420 bp upstream of the transcription start can act as a minimal promoter inducing expression in vasculature tissues. Moreover, mRNA levels of AtSAH7 were acutely increased under selenite treatment in response to oxidative stress. We confirmed the aforementioned interaction in vivo, in silico and in planta. Following a bimolecular fluorescent complementation approach, we determined that the subcellular localization of the AtSAH7 and the AtSAH7/AtSBP1 interaction occur in the ER. Our results indicate the participation of AtSAH7 in a biochemical network regulated by selenite, possibly associated with responses to ROS production.

Investigation of the interaction of DAD1-LIKE LIPASE 3 (DALL3) with Selenium Binding Protein 1 (SBP1) in Arabidopsis thaliana.

Phospholipase PLA1-Iγ2 or otherwise DAD1-LIKE LIPASE 3 (DALL3) is a member of class I phospholipases and has a role in JA biosynthesis. AtDALL3 was previously identified in a yeast two-hybrid screening as an interacting protein of the Arabidopsis Selenium Binding Protein 1 (SBP1). In this work, we have studied AtDALL3 as an interacting partner of the Arabidopsis Selenium Binding Protein 1 (SBP1). Phylogenetic analysis showed that DALL3 appears in the PLA1-Igamma1, 2 group, paired with PLA1-Igammma1. The highest level of expression of AtDALL3 was observed in 10-day-old roots and in flowers, while constitutive levels were maintained in seedlings, cotyledons, shoots and leaves. In response to abiotic stress, DALL3 was shown to participate in the network of genes regulated by cadmium, selenite and selenate compounds. DALL3 promoter driven GUS assays revealed that the expression patterns defined were overlapping with the patterns reported for AtSBP1 gene, indicating that DALL3 and SBP1 transcripts co-localize. Furthermore, quantitative GUS assays showed that these compounds elicited changes in activity in specific cells files, indicating the differential response of DALL3 promoter. GFP::DALL3 studies by confocal microscopy demonstrated the localization of DALL3 in the plastids of the root apex, the plastids of the central root and the apex of emerging lateral root primordia. Additionally, we confirmed by yeast two hybrid assays the physical interaction of DALL3 with SBP1 and defined a minimal SBP1 fragment that DALL3 binds to. Finally, by employing bimolecular fluorescent complementation we demonstrated the in planta interaction of the two proteins.

A High-Performance System for Robust Stain Normalization of Whole-Slide Images in Histopathology.

Stain normalization is an important processing task for computer-aided diagnosis (CAD) systems in modern digital pathology. This task reduces the color and intensity variations present in stained images from different laboratories. Consequently, stain normalization typically increases the prediction accuracy of CAD systems. However, there are computational challenges that this normalization step must overcome, especially for real-time applications: the memory and run-time bottlenecks associated with the processing of images in high resolution, e.g., 40X. Moreover, stain normalization can be sensitive to the quality of the input images, e.g., when they contain stain spots or dirt. In this case, the algorithm may fail to accurately estimate the stain vectors. We present a high-performance system for stain normalization using a state-of-the-art unsupervised method based on stain-vector estimation. Using a highly-optimized normalization engine, our architecture enables high-speed and large-scale processing of high-resolution whole-slide images. This optimized engine integrates an automated thresholding technique to determine the useful pixels and uses a novel pixel-sampling method that significantly reduces the processing time of the normalization algorithm. We demonstrate the performance of our architecture using measurements from images of different sizes and scanner formats that belong to four different datasets. The results show that our optimizations achieve up to 58x speedup compared to a baseline implementation. We also prove the scalability of our system by showing that the processing time scales almost linearly with the amount of tissue pixels present in the image. Furthermore, we show that the output of the normalization algorithm can be adversely affected when the input images include artifacts. To address this issue, we enhance the stain normalization pipeline by introducing a parameter cross-checking technique that automatically detects the distortion of the algorithm's critical parameters. To assess the robustness of the proposed method we employ a machine learning (ML) pipeline that classifies images for detection of prostate cancer. The results show that the enhanced normalization algorithm increases the classification accuracy of the ML pipeline in the presence of poor-quality input images. For an exemplary ML pipeline, our new method increases the accuracy on an unseen dataset from 0.79 to 0.87.

Novel interactions of Selenium Binding Protein family with the PICOT containing proteins AtGRXS14 and AtGRXS16 in Arabidopsis thaliana.

During abiotic stress the primary symptom of phytotoxicity can be ROS production which is strictly regulated by ROS scavenging pathways involving enzymatic and non-enzymatic antioxidants. Furthermore, ROS are well-described secondary messengers of cellular processes, while during the course of evolution, plants have accomplished high degree of control over ROS and used them as signalling molecules. Glutaredoxins (GRXs) are small and ubiquitous glutathione (GSH) -or thioredoxin reductase (TR)-dependent oxidoreductases belonging to the thioredoxin (TRX) superfamily which are conserved in most eukaryotes and prokaryotes. In Arabidopsis thaliana GRXs are subdivided into four classes playing a central role in oxidative stress responses and physiological functions. In this work, we describe a novel interaction of AtGRXS14 with the Selenium Binding Protein 1 (AtSBP1), a protein proposed to be integrated in a regulatory network that senses alterations in cellular redox state and acts towards its restoration. We further show that SBP protein family interacts with AtGRXS16 that also contains a PICOT domain, like AtGRXS14.

Temporal correlation detection using computational phase-change memory.

Conventional computers based on the von Neumann architecture perform computation by repeatedly transferring data between their physically separated processing and memory units. As computation becomes increasingly data centric and the scalability limits in terms of performance and power are being reached, alternative computing paradigms with collocated computation and storage are actively being sought. A fascinating such approach is that of computational memory where the physics of nanoscale memory devices are used to perform certain computational tasks within the memory unit in a non-von Neumann manner. We present an experimental demonstration using one million phase change memory devices organized to perform a high-level computational primitive by exploiting the crystallization dynamics. Its result is imprinted in the conductance states of the memory devices. The results of using such a computational memory for processing real-world data sets show that this co-existence of computation and storage at the nanometer scale could enable ultra-dense, low-power, and massively-parallel computing systems.

Contribution to the prediction of the fold code: application to immunoglobulin and flavodoxin cases.

BACKGROUND: Folding nucleus of globular proteins formation starts by the mutual interaction of a group of hydrophobic amino acids whose close contacts allow subsequent formation and stability of the 3D structure. These early steps can be predicted by simulation of the folding process through a Monte Carlo (MC) coarse grain model in a discrete space. We previously defined MIRs (Most Interacting Residues), as the set of residues presenting a large number of non-covalent neighbour interactions during such simulation. MIRs are good candidates to define the minimal number of residues giving rise to a given fold instead of another one, although their proportion is rather high, typically [15-20]% of the sequences. Having in mind experiments with two sequences of very high levels of sequence identity (up to 90%) but different folds, we combined the MIR method, which takes sequence as single input, with the "fuzzy oil drop" (FOD) model that requires a 3D structure, in order to estimate the residues coding for the fold. FOD assumes that a globular protein follows an idealised 3D Gaussian distribution of hydrophobicity density, with the maximum in the centre and minima at the surface of the "drop". If the actual local density of hydrophobicity around a given amino acid is as high as the ideal one, then this amino acid is assigned to the core of the globular protein, and it is assumed to follow the FOD model. Therefore one obtains a distribution of the amino acids of a protein according to their agreement or rejection with the FOD model. RESULTS: We compared and combined MIR and FOD methods to define the minimal nucleus, or keystone, of two populated folds: immunoglobulin-like (Ig) and flavodoxins (Flav). The combination of these two approaches defines some positions both predicted as a MIR and assigned as accordant with the FOD model. It is shown here that for these two folds, the intersection of the predicted sets of residues significantly differs from random selection. It reduces the number of selected residues by each individual method and allows a reasonable agreement with experimentally determined key residues coding for the particular fold. In addition, the intersection of the two methods significantly increases the specificity of the prediction, providing a robust set of residues that constitute the folding nucleus.

Validation of the Greek OSD-6 quality of life questionnaire in children undergoing polysomnography.

OBJECTIVE: OSD-6 is a disease specific questionnaire for pediatric obstructive sleep apnea (OSA). The aims of this study were to validate OSD-6 in Greek language and correlate OSD-6 with polysomnography results. STUDY DESIGN: Prospective study. SETTING: Tertiary referral center. SUBJECTS AND METHODS: OSD-6 questionnaire was translated to Greek and back to English. A prospective study was conducted on children undergoing overnight polysomnography due to snoring and disrupted sleep. Test-retest evaluation was carried out. Internal consistency and test-retest reliability were evaluated. Validity was assessed by exploring correlations between OSD-6 scores and apnea-hypopnea index (AHI), and by comparing total scores of OSA and non-OSA groups. Responsiveness was assessed by comparing preoperative to postoperative total scores in OSA children who underwent adenotonsillectomy. RESULTS: Test-retest evaluation of 91 subjects showed good internal consistency (Cronbach's alpha 0.860 for test and 0.873 for retest) and reliability (Pearson's correlation coefficients between test and retest scores: 0.751-0.546; p<0.01). Total and domains' OSD-6 scores and AHI were significantly correlated (Spearman's correlation coefficients: 0.277-0.630; p<0.01), while children with OSA had higher total OSD-6 score than those without OSA (median (interquartile range): 16 (11) vs. 10 (7), respectively; p<0.01), indicating good validity. Postoperative OSD-6 scores were significantly lower than preoperative (2.84 ± 3.21 vs. 15.42 ± 6.48, respectively; p<0.001), suggesting good responsiveness. CONCLUSION: The Greek version of the OSD-6 questionnaire proved to be a valid instrument with satisfactory internal consistency, reliability, validity and responsiveness. Furthermore, in our study OSD-6 was significantly correlated to polysomnography results.

Tonsilloplasty versus tonsillectomy in children with sleep-disordered breathing: short- and long-term outcomes.

OBJECTIVES/HYPOTHESIS: Adenoidectomy and tonsillectomy (TE) is the standard treatment for obstructive sleep-disordered breathing (SDB) in children with adenotonsillar hypertrophy. Tonsilloplasty (TP) is a new surgical technique that includes partial TE. The purpose of this study was to assess the short- and long-term outcomes of TP compared to TE. STUDY DESIGN: A comparison study. METHODS: Children with SDB and tonsillar hypertrophy underwent TP or TE. The two groups were compared regarding immediate postoperative course and long-term effects. RESULTS: Fifty-one children (age, 6.3 ± 2.5 years) underwent TE, and 50 children (age, 5.9 ± 2.1 years) had TP. The TP group had significantly less intraoperative bleeding compared to the TE group (25.6 ± 8.2 vs. 38.3 ± 12.3 mL, P < .001). Subjects with TP were pain free earlier than children with TE (4.5 ± 0.4 vs. 7.7 ± 0.4 days, P < .001) and returned to a normal diet sooner (3.8 ± 0.2 vs. 7.1 ± 0.3 days, P < .001). By the 3rd to 4th postoperative night, upper airway obstruction was relieved in all participants. Six years postoperatively, 48 of 51 children in the TE group and 43 of 50 children in the TP group participated in a telephone survey. No significant differences were found between the two groups regarding the frequency of recurrent snoring (30.2% in TP vs. 25% in TE), apneas (4.7% vs. 0%), and upper airway infections per year (P > .05). CONCLUSIONS: TP is an alternative surgical method for treatment of SDB related to tonsillar hypertrophy with favorable postoperative course and comparable long-term results. LEVEL OF EVIDENCE: 2c.

Complete genome sequence of the dairy isolate Streptococcus macedonicus ACA-DC 198.

The species Streptococcus macedonicus is associated with the food environment, especially with fermented dairy products. Here we present the complete 2.1-Mb genome sequence of strain ACA-DC 198, which was isolated from naturally fermented Greek kasseri cheese.

GPCRs, G-proteins, effectors and their interactions: human-gpDB, a database employing visualization tools and data integration techniques.

G-protein coupled receptors (GPCRs) are a major family of membrane receptors in eukaryotic cells. They play a crucial role in the communication of a cell with the environment. Ligands bind to GPCRs on the outside of the cell, activating them by causing a conformational change, and allowing them to bind to G-proteins. Through their interaction with G-proteins, several effector molecules are activated leading to many kinds of cellular and physiological responses. The great importance of GPCRs and their corresponding signal transduction pathways is indicated by the fact that they take part in many diverse disease processes and that a large part of efforts towards drug development today is focused on them. We present Human-gpDB, a database which currently holds information about 713 human GPCRs, 36 human G-proteins and 99 human effectors. The collection of information about the interactions between these molecules was done manually and the current version of Human-gpDB holds information for about 1663 connections between GPCRs and G-proteins and 1618 connections between G-proteins and effectors. Major advantages of Human-gpDB are the integration of several external data sources and the support of advanced visualization techniques. Human-gpDB is a simple, yet a powerful tool for researchers in the life sciences field as it integrates an up-to-date, carefully curated collection of human GPCRs, G-proteins, effectors and their interactions. The database may be a reference guide for medical and pharmaceutical research, especially in the areas of understanding human diseases and chemical and drug discovery. Database URLs: http://schneider.embl.de/human_gpdb; http://bioinformatics.biol.uoa.gr/human_gpdb/

Prediction of stability upon point mutation in the context of the folding nucleus.

Proteins come in all shapes and sizes. Although it is possible to predict with reasonable success their structure from their sequence, the process of folding a chain of amino acids into its tertiary structure remains partially understood. This article addresses several characteristics pertaining to protein folding. The development of the Most Interacting Residues (MIR) algorithm, which dynamically simulates the early folding events, permits a reasonable ab initio prediction of the deeply buried critical residues involved in the formation of the protein core. The analysis of MIR positions with respect to protein 3D topology, in particular, to fragments called Tightened End Fragments (TEF) that might be good candidate for autonomous folding units, suggests that they are also essential for defining core stability. To validate this hypothesis, this study measures the sensitivity of MIR residues to point mutations. It is performed on a set of 385 proteins from a database that contains stability data calculated with five different algorithms. Tools have been developed to help the analysis and a consensus of the five methods is proposed. It results that positions predicted both as a MIR and a minimum of stability for the consensus are good candidates for the folding nucleus, and consequently their mutations may be hazardous.

Quantitative prediction of critical amino acid positions for protein folding.

The MIR algorithm provides an ab initio prediction of a protein's core residues. An improved version, the MIR2, is presented and validated on 3203 proteins from PDB. Structures are decomposed in Closed Loops, their limits constituting the observed core residues. They are predicted by MIR2 with an accuracy approaching 80%.

Sequence analyses reveal that a TPR-DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR-DP domains and prokaryotic GerD proteins.

The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR-DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR-DP domains.

SPROUTS: a database for the evaluation of protein stability upon point mutation.

SPROUTS (Structural Prediction for pRotein fOlding UTility System) is a new database that provides access to various structural data sets and integrated functionalities not yet available to the community. The originality of the SPROUTS database is the ability to gain access to a variety of structural analyses at one place and with a strong interaction between them. SPROUTS currently combines data pertaining to 429 structures that capture representative folds and results related to the prediction of critical residues expected to belong to the folding nucleus: the MIR (Most Interacting Residues), the description of the structures in terms of modular fragments: the TEF (Tightened End Fragments), and the calculation at each position of the free energy change gradient upon mutation by one of the 19 amino acids. All database results can be displayed and downloaded in textual files and Excel spreadsheets and visualized on the protein structure. SPROUTS is a unique resource to access as well as visualize state-of-the-art characteristics of protein folding and analyse the effect of point mutations on protein structure. It is available at http://bioinformatics.eas.asu.edu/sprouts.html.

T-cell recognition of HLA-DQ2-bound gluten peptides can be influenced by an N-terminal proline at p-1.

Recent research has implicated a large number of gluten-derived peptides in the pathogenesis of celiac disease, a preponderantly HLA-DQ2-associated disorder. Current evidence indicates that the core of some of those peptides is ten amino acids long, while HLA class II normally accommodates nine amino acids in the binding groove. We have now investigated this in detail, using gluten-specific T-cell clones, HLA-DQ2-specific peptide-binding assays and molecular modelling. T-cell recognition of both a gamma-gliadin peptide and a low-molecular-weight glutenin peptide was found to be strictly dependent on a ten-amino acids-long peptide. Subsequent peptide-binding studies indicated that the glutenin peptide bound in a conventional p1/p9 register, with an additional proline at p-1. Testing of substitution analogues demonstrated that the nature of the amino acid at p-1 strongly influenced T-cell recognition of the peptide. Moreover, molecular modelling confirmed that the glutenin peptide binds in a p1/p9 register, and that the proline at p-1 points upward towards the T-cell receptor. Database searches indicate that a large number of potential T-cell stimulatory gluten peptides with an additional proline at relative position p-1 exist, suggesting that the recognition of other gluten peptides may depend on this proline as well. This knowledge may be of importance for the identification of additional T-cell stimulatory gluten peptides and the design of a peptide-based, tolerance-inducing therapy.

Universal positions in globular proteins.

The description of globular protein structures as an ensemble of contiguous 'closed loops' or 'tightened end fragments' reveals fold elements crucial for the formation of stable structures and for navigating the very process of protein folding. These are the ends of the loops, which are spatially close to each other but are situated apart in the polypeptide chain by 25-30 residues. They also correlate with the locations of highly conserved hydrophobic residues (referred to as topohydrophobic), in a structural alignment of the members of a protein family. This study analysed these positions in 111 representatives of different protein folds, and then carried out dynamic Monte Carlo simulations of the first steps of the folding process, aimed at predicting the origins of the assembling folds. The simulations demonstrated that there is an obvious trend for certain sets of residues, named 'mostly interacting residues', to be buried at the early stages of the folding process. Location of these residues at the loop ends and correlation with topohydrophobic positions are demonstrated, thereby giving a route to simulations of the protein folding process.

Analysis of fragments induced by simulated lattice protein folding.

The folding process of a set of 42 proteins, representative of the various folds, has been simulated by means of a Monte Carlo method on a discrete lattice, using two different potentials of mean force. Multiple compact fragments of contiguous residues are formed in the simulation, stable in composition, but not in geometry. During time, the number of fragments decreases until one final compact globular state is reached. We focused on the early steps of the folding in order to evidence the maximum number of fragments, provided they are sufficiently stable in sequence. A correlation has been established between these proto fragments and regular secondary-structure elements, whatever their nature, alpha helices or beta strands. Quantitatively, this is revealed by an overall mean one-residue quality factor of nearly 60%, which is better for proteins mainly composed of alpha helices. The correspondence between the number of fragments and the number of secondary-structure elements is of 77% and the regions separating successive fragments are mainly located in loops. Besides, hydrophobic clusters deduced from HCA correspond to fragments with an equivalent accuracy. These results suggest that folding pathways do not contain structurally static intermediate. However, since the beginning of folding, most residues that will later form one given secondary structure are kept close in space by being involved in the same fragment. This aggregation may be a way to accelerate the formation of the native state and enforces the key role played by hydrophobic residues in the formation of the fragments, thus in the folding process itself.

2,4-Diamino-5-(1-naphthyl)-3,5-diaza-1-azoniaspiro[5.5]undeca-1,3-diene chloride.

The title salt, C(18)H(22)N(5)(+).Cl(-), is a member of a new series of lipophilic 4,6-diamino spiro-s-triazines which are potent inhibitors of dihydrofolate reductase. The protonated triazine ring deviates from planarity, whereas the cyclohexane ring adopts a chair conformation. A rather unusual hydrogen-bonding scheme exists in the crystal. There is a centrosymmetric arrangement involving two amino groups and two triazine ring N atoms, with graph-set R(2)(2)(8) and an N.N distance of 3.098 (3) A, flanked by two additional R(3)(2)(8) systems, involving two amino groups, a triazine ring N atom and a Cl(-) anion, with N.Cl distances in the range 3.179 (2)-3.278 (2) A. Furthermore, the Cl(-) anion, the protonated triazine ring N atom and an amino group form a hydrogen-bonding system with graph-set R(2)(1)(6).