Multiplex Microarrays in 96-Well Plates Photoactivated with 4-Azidotetrafluorobenzaldehyde for the Identification and Quantification of ß-Lactamase Genes and Their RNA Transcripts.

Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin-peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to ß-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria.

Measuring the predictability of life outcomes with a scientific mass collaboration.

How predictable are life trajectories? We investigated this question with a scientific mass collaboration using the common task method; 160 teams built predictive models for six life outcomes using data from the Fragile Families and Child Wellbeing Study, a high-quality birth cohort study. Despite using a rich dataset and applying machine-learning methods optimized for prediction, the best predictions were not very accurate and were only slightly better than those from a simple benchmark model. Within each outcome, prediction error was strongly associated with the family being predicted and weakly associated with the technique used to generate the prediction. Overall, these results suggest practical limits to the predictability of life outcomes in some settings and illustrate the value of mass collaborations in the social sciences.

The Nature and Clinical Significance of Atypical Mononuclear Cells in Infectious Mononucleosis Caused by the Epstein-Barr Virus in Children.

Atypical mononuclear cells (AM) appear in significant numbers in peripheral blood of patients with Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). We investigated the number and lineage-specific clusters of differentiation (CD) expression of atypical mononuclear cells in 110 children with IM using the anti-CD antibody microarray for panning leukocytes by their surface markers prior to morphology examination. The AM population consisted primarily of CD8+ T cells with a small fraction (0%-2% of all lymphocytes) of CD19+ B lymphocytes. AM amount in children with mononucleosis caused by primary EBV infection was significantly higher than for IM caused by EBV reactivation or other viruses and constituted 1%-53% of all peripheral blood mononuclear cells compared to 0%-11% and 0%-8%, respectively. Children failing to recover from classic IM associated with primary EBV infection within 6 months had significantly lower percentage of CD8+ AM compared to patients with normal recovery rate.

Possible role of small secreted peptides (SSPs) in immune signaling in bryophytes.

Plants utilize a plethora of peptide signals to regulate their immune response. Peptide ligands and their cognate receptors involved in immune signaling share common motifs among many species of vascular plants. However, the origin and evolution of immune peptides is still poorly understood. Here, we searched for genes encoding small secreted peptides in the genomes of three bryophyte lineages-mosses, liverworts and hornworts-that occupy a critical position in the study of land plant evolution. We found that bryophytes shared common predicted small secreted peptides (SSPs) with vascular plants. The number of SSPs is higher in the genomes of mosses than in both the liverwort Marchantia polymorpha and the hornwort Anthoceros sp. The synthetic peptide elicitors-AtPEP and StPEP-specific for vascular plants, triggered ROS production in the protonema of the moss Physcomitrella patens, suggesting the possibility of recognizing peptide ligands from angiosperms by moss receptors. Mass spectrometry analysis of the moss Physcomitrella patens, both the wild type and the Δcerk mutant secretomes, revealed peptides that specifically responded to chitosan treatment, suggesting their role in immune signaling.

The role of chloroplast protein remodeling in stress responses and shaping of the plant peptidome.

In addition to photosynthesis, chloroplasts perform a variety of important cellular functions in the plant cell, which can, for example, regulate plant responses to abiotic and biotic stress conditions. Under stress, intensive chloroplast protein remodeling and degradation can occur, releasing large numbers of endogenous peptides. These protein-derived peptides can be found intracellularly, but also in the plant secretome. Although the pathways of chloroplast protein degradation and the types of chloroplast proteases implicated in this process have received much attention, the role of the resulting peptides is less well understood. In this review we summarize the data on peptide generation processes during the remodeling of the chloroplast proteome under stress conditions and discuss the mechanisms leading to these changes. We also review the experimental evidence which supports the concept that peptides derived from chloroplast proteins can function as regulators of plant responses to (a)biotic stresses.

Phytohormone treatment induces generation of cryptic peptides with antimicrobial activity in the Moss Physcomitrella patens.

BACKGROUND: Cryptic peptides (cryptides) are small bioactive molecules generated via degradation of functionally active proteins. Only a few examples of plant cryptides playing an important role in plant defense have been reported to date, hence our knowledge about cryptic signals hidden in protein structure remains very limited. Moreover, little is known about how stress conditions influence the size of endogenous peptide pools, and which of these peptides themselves have biological functions is currently unclear. RESULTS: Here, we used mass spectrometry to comprehensively analyze the endogenous peptide pools generated from functionally active proteins inside the cell and in the secretome from the model plant Physcomitrella patens. Overall, we identified approximately 4,000 intracellular and approximately 500 secreted peptides. We found that the secretome and cellular peptidomes did not show significant overlap and that respective protein precursors have very different protein degradation patterns. We showed that treatment with the plant stress hormone methyl jasmonate induced specific proteolysis of new functional proteins and the release of bioactive peptides having an antimicrobial activity and capable to elicit the expression of plant defense genes. Finally, we showed that the inhibition of protease activity during methyl jasmonate treatment decreased the secretome antimicrobial potential, suggesting an important role of peptides released from proteins in immune response. CONCLUSIONS: Using mass-spectrometry, in vitro experiments and bioinformatics analysis, we found that methyl jasmonate acid induces significant changes in the peptide pools and that some of the resulting peptides possess antimicrobial and regulatory activities. Moreover, our study provides a list of peptides for further study of potential plant cryptides.

Salicylic acid influences the protease activity and posttranslation modifications of the secreted peptides in the moss Physcomitrella patens.

Plant secretome comprises dozens of secreted proteins. However, little is known about the composition of the whole secreted peptide pools and the proteases responsible for the generation of the peptide pools. The majority of studies focus on target detection and characterization of specific plant peptide hormones. In this study, we performed a comprehensive analysis of the whole extracellular peptidome, using moss Physcomitrella patens as a model. Hundreds of modified and unmodified endogenous peptides that originated from functional and nonfunctional protein precursors were identified. The plant proteases responsible for shaping the pool of endogenous peptides were predicted. Salicylic acid (SA) influenced peptide production in the secretome. The proteasome activity was altered upon SA treatment, thereby influencing the composition of the peptide pools. These results shed more light on the role of proteases and posttranslational modification in the "active management" of the extracellular peptide pool in response to stress conditions. It also identifies a list of potential peptide hormones in the moss secretome for further analysis.

The Role of Peptide Signals Hidden in the Structure of Functional Proteins in Plant Immune Responses.

Plants have evolved a sophisticated innate immune system to cope with a diverse range of phytopathogens and insect herbivores. Plasma-membrane-localized pattern recognition receptors (PRRs), such as receptor-like kinases (RLK), recognize special signals, pathogen- or damage-associated molecular patterns (PAMPs or DAMPs), and trigger immune responses. A growing body of evidence shows that many peptides hidden in both plant and pathogen functional protein sequences belong to the group of such immune signals. However, the origin, evolution, and release mechanisms of peptide sequences from functional and nonfunctional protein precursors, known as cryptic peptides, are largely unknown. Various special proteases, such as metacaspase or subtilisin-like proteases, are involved in the release of such peptides upon activation during defense responses. In this review, we discuss the roles of cryptic peptide sequences hidden in the structure of functional proteins in plant defense and plant-pathogen interactions.

Tales of resilience: voices from detention and imprisonment in Russia.

As the exposure to state violence could have long-term negative consequences on the survivors, we analyzed which optics and measures could be used regardless of the vulnerabilities of the individual, their social status and institutional context. We examine the ways in which law enforcement system challenges individuals and measures used to practice resilience.We use the concept of resilience within torturing environment to achieve our goal. Various actors of Russian law enforcement and penitentiary systems - detainees and pris-oners, their family members, human rights activists, state agents etc. - participated in semi-structured in-depth interviews, which we coded basing on grounded theory.Measures employed by survivors to exer-cise resilience involve regaining bodily control (both by exercise and self-harm), having pro-jections for future (by threats of legal prose-cution or publicity), exploiting bureaucracy to one's advantage, controlling information flow (by bluffing), controlling material evidence, employing allies and preserving the meaning of "normal self ".We believe that our findings can have prac-tical applications, e.g. for preventing some of the negative consequences of torture by train-ing vulnerable individuals to employ resilient strategies. Describing the way to interpret the power imbalances inherent to the torture en-vironment might also be helpful for appre-ciating even the smallest acts, including the choice not to act.

Multiplex Digital Quantification of ß-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips.

Digital quantification based on counting of individual molecules is a promising approach for different biomedical applications due to its enhanced sensitivity. Here, we present a method for the digital detection of nucleic acids (DNA and RNA) on silicon microchips based on the counting of gold nanoparticles (GNPs) in DNA duplexes by scanning electron microscopy (SEM). Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microchips. Then biotin is revealed by a streptavidin-GNP conjugate followed by the detection of GNPs. Sharp images of each nanoparticle allow the visualization of hybridization results on a single-molecule level. The technique was shown to provide highly sensitive quantification of both short oligonucleotide and long double-strand DNA sequences up to 800 bp. The lowest limit of detection of 0.04 pM was determined for short 19-mer oligonucleotide. The method's applicability was demonstrated for the multiplex quantification of several ß-lactamase genes responsible for the development of bacterial resistance against ß-lactam antibiotics. Determination of nucleic acids is effective for both specific DNA in lysates and mRNA in transcripts. The method is also characterized by high selectivity for single-nucleotide polymorphism discrimination. The proposed principle of digital quantification is a perspective for studying the mechanisms of bacterial antibiotic resistance and bacterial response to drugs.

Humans in the Loop: Incorporating Expert and Crowd-Sourced Knowledge for Predictions Using Survey Data.

Survey data sets are often wider than they are long. This high ratio of variables to observations raises concerns about overfitting during prediction, making informed variable selection important. Recent applications in computer science have sought to incorporate human knowledge into machine-learning methods to address these problems. The authors implement such a "human-in-the-loop" approach in the Fragile Families Challenge. The authors use surveys to elicit knowledge from experts and laypeople about the importance of different variables to different outcomes. This strategy offers the option to subset the data before prediction or to incorporate human knowledge as scores in prediction models, or both together. The authors find that human intervention is not obviously helpful. Human-informed subsetting reduces predictive performance, and considered alone, approaches incorporating scores perform marginally worse than approaches that do not. However, incorporating human knowledge may still improve predictive performance, and future research should consider new ways of doing so.

Alternative splicing shapes transcriptome but not proteome diversity in Physcomitrella patens.

Alternative splicing (AS) can significantly impact the transcriptome and proteome of a eukaryotic cell. Here, using transcriptome and proteome profiling data, we analyzed AS in two life forms of the model moss Physcomitrella patens, namely protonemata and gametophores, as well as in protoplasts. We identified 12 043 genes subject to alternative splicing and analyzed the extent to which AS contributes to proteome diversity. We could distinguish a few examples that unambiguously indicated the presence of two or more splice isoforms from the same locus at the proteomic level. Our results indicate that alternative isoforms have a small effect on proteome diversity. We also revealed that mRNAs and pre-mRNAs have thousands of complementary binding sites for long non-coding RNAs (lncRNAs) that may lead to potential interactions in transcriptome. This finding points to an additional level of gene expression and AS regulation by non-coding transcripts in Physcomitrella patens. Among the differentially expressed and spliced genes we found serine/arginine-rich (SR) genes, which are known to regulate AS in cells. We found that treatment with abscisic (ABA) and methyl jasmonic acids (MeJA) led to an isoform-specific response and suggested that ABA in gametophores and MeJA in protoplasts regulate AS and the transcription of SR genes.