Tissue-specific DNA methylation variability and its potential clinical value.

Complex diseases have multifactorial etiologies making actionable diagnostic biomarkers difficult to identify. Diagnostic research must expand beyond single or a handful of genetic or epigenetic targets for complex disease and explore a broader system of biological pathways. With the objective to develop a diagnostic tool designed to analyze a comprehensive network of epigenetic profiles in complex diseases, we used publicly available DNA methylation data from over 2,400 samples representing 20 cell types and various diseases. This tool, rather than detecting differentially methylated regions at specific genes, measures the intra-individual methylation variability within gene promoters to identify global shifts away from healthy regulatory states. To assess this new approach, we explored three distinct questions: 1) Are profiles of epigenetic variability tissue-specific? 2) Do diseased tissues exhibit altered epigenetic variability compared to normal tissue? 3) Can epigenetic variability be detected in complex disease? Unsupervised clustering established that global epigenetic variability in promoter regions is tissue-specific and promoter regions that are the most epigenetically stable in a specific tissue are associated with genes known to be essential for its function. Furthermore, analysis of epigenetic variability in these most stable regions distinguishes between diseased and normal tissue in multiple complex diseases. Finally, we demonstrate the clinical utility of this new tool in the assessment of a multifactorial condition, male infertility. We show that epigenetic variability in purified sperm is correlated with live birth outcomes in couples undergoing intrauterine insemination (IUI), a common fertility procedure. Men with the least epigenetically variable promoters were almost twice as likely to father a child than men with the greatest number of epigenetically variable promoters. Interestingly, no such difference was identified in men undergoing in vitro fertilization (IVF), another common fertility procedure, suggesting this as a treatment to overcome higher levels of epigenetic variability when trying to conceive.

Multiple Subthreshold GPCR Signals Combined by the G-Proteins Gαq and Gαs Activate the Caenorhabditis elegans Egg-Laying Muscles.

Individual neurons or muscle cells express many G-protein-coupled receptors (GPCRs) for neurotransmitters and neuropeptides, yet it remains unclear how cells integrate multiple GPCR signals that all must activate the same few G-proteins. We analyzed this issue in the Caenorhabditis elegans egg-laying system, where multiple GPCRs on muscle cells promote contraction and egg laying. We genetically manipulated individual GPCRs and G-proteins specifically in these muscle cells within intact animals and then measured egg laying and muscle calcium activity. Two serotonin GPCRs on the muscle cells, Gαq-coupled SER-1 and Gαs-coupled SER-7, together promote egg laying in response to serotonin. We found that signals produced by either SER-1/Gαq or SER-7/Gαs alone have little effect, but these two subthreshold signals combine to activate egg laying. We then transgenically expressed natural or designer GPCRs in the muscle cells and found that their subthreshold signals can also combine to induce muscle activity. However, artificially inducing strong signaling through just one of these GPCRs can be sufficient to induce egg laying. Knocking down Gαq and Gαs in the egg-laying muscle cells induced egg-laying defects that were stronger than those of a SER-1/SER-7 double knockout, indicating that additional endogenous GPCRs also activate the muscle cells. These results show that in the egg-laying muscles multiple GPCRs for serotonin and other signals each produce weak effects that individually do not result in strong behavioral outcomes. However, they combine to produce sufficient levels of Gαq and Gαs signaling to promote muscle activity and egg laying.SIGNIFICANCE STATEMENT How can neurons and other cells gather multiple independent pieces of information from the soup of chemical signals in their environment and compute an appropriate response? Most cells express >20 GPCRs that each receive one signal and transmit that information through three main types of G-proteins. We analyzed how this machinery generates responses by studying the egg-laying system of C. elegans, where serotonin and multiple other signals act through GPCRs on the egg-laying muscles to promote muscle activity and egg laying. We found that individual GPCRs within an intact animal each generate effects too weak to activate egg laying. However, combined signaling from multiple GPCR types reaches a threshold capable of activating the muscle cells.

What about lexical competition? Exploring the locus of lexical retrieval deficits in adults with developmental dyslexia.

OBJECTIVE: Individuals with dyslexia do not only show deficits with reading but are also less accurate in naming pictures. This has mainly been linked to prevalent phonological deficits. However, deficits in lexical retrieval of picture names could also be due to increased lexical-semantic competition. The present study tested whether adults with dyslexia (AwDs) are more affected by a competitive lexical-semantic context than control participants. METHOD: Twenty-seven AwD and 34 control participants completed the blocked-cyclic picture-naming paradigm and the Hayling sentence completion task. RESULTS: In the blocked-cyclic naming task, AwDs showed a larger semantic interference effect than controls in terms of errors, especially producing competitor errors. In the Hayling sentence completion task, AwDs made more errors than controls when asked to complete sentences with semantically unrelated words, that is, in the competitive condition. They especially produced semantically related words or antonyms to target words. CONCLUSIONS: We found that AwDs experience difficulties with resolving lexical competition that go beyond their phonological deficits. Future studies will need to establish the mechanisms behind the increased lexical competition that AwDs exhibit. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

The neural G protein Gαo tagged with GFP at an internal loop is functional in Caenorhabditis elegans.

Gαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here, we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to compliment the already well-developed genetic analysis of Gαo signaling.

Double triage to identify poorly annotated genes in maize: The missing link in community curation.

The sophistication of gene prediction algorithms and the abundance of RNA-based evidence for the maize genome may suggest that manual curation of gene models is no longer necessary. However, quality metrics generated by the MAKER-P gene annotation pipeline identified 17,225 of 130,330 (13%) protein-coding transcripts in the B73 Reference Genome V4 gene set with models of low concordance to available biological evidence. Working with eight graduate students, we used the Apollo annotation editor to curate 86 transcript models flagged by quality metrics and a complimentary method using the Gramene gene tree visualizer. All of the triaged models had significant errors-including missing or extra exons, non-canonical splice sites, and incorrect UTRs. A correct transcript model existed for about 60% of genes (or transcripts) flagged by quality metrics; we attribute this to the convention of elevating the transcript with the longest coding sequence (CDS) to the canonical, or first, position. The remaining 40% of flagged genes resulted in novel annotations and represent a manual curation space of about 10% of the maize genome (~4,000 protein-coding genes). MAKER-P metrics have a specificity of 100%, and a sensitivity of 85%; the gene tree visualizer has a specificity of 100%. Together with the Apollo graphical editor, our double triage provides an infrastructure to support the community curation of eukaryotic genomes by scientists, students, and potentially even citizen scientists.

Serotonin and neuropeptides are both released by the HSN command neuron to initiate Caenorhabditis elegans egg laying.

Neurons typically release both a small-molecule neurotransmitter and one or more neuropeptides, but how these two types of signal from the same neuron might act together remains largely obscure. For example, serotonergic neurons in mammalian brain express the neuropeptide Substance P, but it is unclear how this co-released neuropeptide might modulate serotonin signaling. We studied this issue in C. elegans, in which all serotonergic neurons express the neuropeptide NLP-3. The serotonergic Hermaphrodite Specific Neurons (HSNs) are command motor neurons within the egg-laying circuit which have been shown to release serotonin to initiate egg-laying behavior. We found that egg-laying defects in animals lacking serotonin were far milder than in animals lacking HSNs, suggesting that HSNs must release other signal(s) in addition to serotonin to stimulate egg laying. While null mutants for nlp-3 had only mild egg-laying defects, animals lacking both serotonin and NLP-3 had severe defects, similar to those of animals lacking HSNs. Optogenetic activation of HSNs induced egg laying in wild-type animals, and in mutant animals lacking either serotonin or NLP-3, but failed to induce egg laying in animals lacking both. We recorded calcium activity in the egg-laying muscles of animals lacking either serotonin, NLP-3, or both. The single mutants, and to a greater extent the double mutant, showed muscle activity that was uncoordinated and unable to expel eggs. Specifically, the vm2 muscles cells, which are direct postsynaptic targets of the HSN, failed to contract simultaneously with other egg-laying muscle cells. Our results show that the HSN neurons use serotonin and the neuropeptide NLP-3 as partially redundant co-transmitters that together stimulate and coordinate activity of the target cells onto which they are released.

Mechanisms of Local Stress Sensing in Multifunctional Polymer Films Using Fluorescent Tetrapod Nanocrystals.

Nanoscale stress-sensing can be used across fields ranging from detection of incipient cracks in structural mechanics to monitoring forces in biological tissues. We demonstrate how tetrapod quantum dots (tQDs) embedded in block copolymers act as sensors of tensile/compressive stress. Remarkably, tQDs can detect their own composite dispersion and mechanical properties with a switch in optomechanical response when tQDs are in direct contact. Using experimental characterizations, atomistic simulations and finite-element analyses, we show that under tensile stress, densely packed tQDs exhibit a photoluminescence peak shifted to higher energies ("blue-shift") due to volumetric compressive stress in their core; loosely packed tQDs exhibit a peak shifted to lower energies ("red-shift") from tensile stress in the core. The stress shifts result from the tQD's unique branched morphology in which the CdS arms act as antennas that amplify the stress in the CdSe core. Our nanocomposites exhibit excellent cyclability and scalability with no degraded properties of the host polymer. Colloidal tQDs allow sensing in many materials to potentially enable autoresponsive, smart structural nanocomposites that self-predict impending fracture.

Influence of three-dimensional nanoparticle branching on the Young's modulus of nanocomposites: Effect of interface orientation.

With the availability of nanoparticles with controlled size and shape, there has been renewed interest in the mechanical properties of polymer/nanoparticle blends. Despite the large number of theoretical studies, the effect of branching for nanofillers tens of nanometers in size on the elastic stiffness of these composite materials has received limited attention. Here, we examine the Young's modulus of nanocomposites based on a common block copolymer (BCP) blended with linear nanorods and nanoscale tetrapod Quantum Dots (tQDs), in electrospun fibers and thin films. We use a phenomenological lattice spring model (LSM) as a guide in understanding the changes in the Young's modulus of such composites as a function of filler shape. Reasonable agreement is achieved between the LSM and the experimental results for both nanoparticle shapes--with only a few key physical assumptions in both films and fibers--providing insight into the design of new nanocomposites and assisting in the development of a qualitative mechanistic understanding of their properties. The tQDs impart the greatest improvements, enhancing the Young's modulus by a factor of 2.5 at 20 wt.%. This is 1.5 times higher than identical composites containing nanorods. An unexpected finding from the simulations is that both the orientation of the nanoscale filler and the orientation of X-type covalent bonds at the nanoparticle-ligand interface are important for optimizing the mechanical properties of the nanocomposites. The tQD provides an orientational optimization of the interfacial and filler bonds arising from its three-dimensional branched shape unseen before in nanocomposites with inorganic nanofillers.

Agreeing to disagree: deaf and hearing children's awareness of subject-verb number agreement.

This study investigated deaf adolescents' implicit and explicit awareness of subject-verb number agreement. In Experiment 1, a self-paced reading task, the reading times of deaf and hearing children (matched for reading and chronological age, mean=8;3 and 13;10 years) increased when sentences contained disagreeing subject-verb number markers. However, deaf adolescents' slowing occurred later in the sentence than it did in both groups of hearing children. The same deaf adolescents were unable to detect and correct subject-verb agreement errors in Experiment 2, whereas both groups of hearing children performed well on this task. Thus, deaf adolescents demonstrated implicit awareness of agreement in the absence of explicit knowledge. Moreover, this nascent awareness was below that expected on the basis of their (substantially delayed) reading ability. Therefore, grammatical difficulties could be a significant impediment to deaf children's literacy. Future research should examine whether this is a result of late or incomplete learning of English, bilingualism, or another factor.

Tetrapod nanocrystals as fluorescent stress probes of electrospun nanocomposites.

A nanoscale, visible-light, self-sensing stress probe would be highly desirable in a variety of biological, imaging, and materials engineering applications, especially a device that does not alter the mechanical properties of the material it seeks to probe. Here we present the CdSe-CdS tetrapod quantum dot, incorporated into polymer matrices via electrospinning, as an in situ luminescent stress probe for the mechanical properties of polymer fibers. The mechanooptical sensing performance is enhanced with increasing nanocrystal concentration while causing minimal change in the mechanical properties even up to 20 wt % incorporation. The tetrapod nanoprobe is elastic and recoverable and undergoes no permanent change in sensing ability even upon many cycles of loading to failure. Direct comparisons to side-by-side traditional mechanical tests further validate the tetrapod as a luminescent stress probe. The tetrapod fluorescence stress-strain curve shape matches well with uniaxial stress-strain curves measured mechanically at all filler concentrations reported.

The energetic difference between synthesis of correct and incorrect base pairs accounts for highly accurate DNA replication.

To better understand the energetics of accurate DNA replication, we directly measured ΔG(o) for the incorporation of a nucleotide into elongating dsDNA in solution (ΔG(o)(incorporation)). Direct measurements of the energetic difference between synthesis of correct and incorrect base pairs found it to be much larger than previously believed (average ΔΔG(o)(incorporation) = 5.2 ± 1.34 kcal mol(-1)). Importantly, these direct measurements indicate that ΔΔG(o)(incorporation) alone can account for the energy required for highly accurate DNA replication. Evolutionarily, these results indicate that the earliest polymerases did not have to evolve sophisticated mechanisms to replicate nucleic acids; they may only have had to take advantage of the inherently more favorable ΔG(o) for polymerization of correct nucleotides. These results also provide a basis for understanding how polymerases replicate DNA (or RNA) with high fidelity.

Deaf and hearing children's plural noun spelling.

The present study examines deaf and hearing children's spelling of plural nouns. Severe literacy impairments are well documented in the deaf, which are believed to be a consequence of phonological awareness limitations. Fifty deaf (mean chronological age 13;10 years, mean reading age 7;5 years) and 50 reading-age-matched hearing children produced spellings of regular, semiregular, and irregular plural nouns in Experiment 1 and nonword plurals in Experiment 2. Deaf children performed reading-age appropriately on rule-based (regular and semiregular) plurals but were significantly less accurate at spelling irregular plurals. Spelling of plural nonwords and spelling error analyses revealed clear evidence for use of morphology. Deaf children used morphological generalization to a greater degree than their reading-age-matched hearing counterparts. Also, hearing children combined use of phonology and morphology to guide spelling, whereas deaf children appeared to use morphology without phonological mediation. Therefore, use of morphology in spelling can be independent of phonology and is available to the deaf despite limited experience with spoken language. Indeed, deaf children appear to be learning about morphology from the orthography. Education on more complex morphological generalization and exceptions may be highly beneficial not only for the deaf but also for other populations with phonological awareness limitations.

Spatially indirect emission in a luminescent nanocrystal molecule.

Recent advances in the synthesis of multicomponent nanocrystals have enabled the design of nanocrystal molecules with unique photophysical behavior and functionality. Here we demonstrate a highly luminescent nanocrystal molecule, the CdSe/CdS core/shell tetrapod, which is designed to have weak vibronic coupling between excited states and thereby violates Kasha's rule via emission from multiple excited levels. Using single particle photoluminescence spectroscopy, we show that in addition to the expected LUMO to HOMO radiative transition, a higher energy transition is allowed via spatially indirect recombination. The oscillator strength of this transition can be experimentally controlled, enabling control over carrier behavior and localization at the nanoscale.

Model evaluation and case series data.

We discuss aggregation of data from neuropsychological patients and the process of evaluating models using data from a series of patients. We argue that aggregation can be misleading but not aggregating can also result in information loss. The basis for combining data needs to be theoretically defined, and the particular method of aggregation depends on the theoretical question and characteristics of the data. We present examples, often drawn from our own research, to illustrate these points. We also argue that statistical models and formal methods of model selection are a useful way to test theoretical accounts using data from several patients in multiple-case studies or case series. Statistical models can often measure fit in a way that explicitly captures what a theory allows; the parameter values that result from model fitting often measure theoretically important dimensions and can lead to more constrained theories or new predictions; and model selection allows the strength of evidence for models to be quantified without forcing this into the artificial binary choice that characterizes hypothesis testing methods. Methods that aggregate and then formally model patient data, however, are not automatically preferred to other methods. Which method is preferred depends on the question to be addressed, characteristics of the data, and practical issues like availability of suitable patients, but case series, multiple-case studies, single-case studies, statistical models, and process models should be complementary methods when guided by theory development.

Regulation of influenza RNA polymerase activity and the switch between replication and transcription by the concentrations of the vRNA 5' end, the cap source, and the polymerase.

The influenza RNA-dependent RNA polymerase (RdRp) both replicates the flu's RNA genome and transcribes its mRNA. Replication occurs de novo; however, initiation of transcription requires a 7-methylguanosine 5'-capped primer that is "snatched" from host mRNA via endonuclease and cap binding functions of the influenza polymerase. A key question is how the virus regulates the relative amounts of transcription and replication. We found that the concentration of a capped cellular mRNA, the concentration of the 5' end of the viral RNA, and the concentration of RdRp all regulate the relative amounts of replication versus transcription. The host mRNA, from which the RdRp snatches its capped primer, acts to upregulate transcription and repress replication. Elevated concentrations of the RdRp itself switch the influenza polymerase toward replication, likely through an oligomerization of the polymerase. The 5' end of the vRNA template both activates replication and inhibits transcription of the vRNA template, thereby indicating that RdRp contains an allosteric binding site for the 5' end of the vRNA template. These data provide insights into the regulation of RdRp throughout the viral life cycle and how it synthesizes the appropriate amounts of viral mRNA and replication products (vRNA and cRNA).

Luminescent nanocrystal stress gauge.

Microscale mechanical forces can determine important outcomes ranging from the site of material fracture to stem cell fate. However, local stresses in a vast majority of systems cannot be measured due to the limitations of current techniques. In this work, we present the design and implementation of the CdSe-CdS core-shell tetrapod nanocrystal, a local stress sensor with bright luminescence readout. We calibrate the tetrapod luminescence response to stress and use the luminescence signal to report the spatial distribution of local stresses in single polyester fibers under uniaxial strain. The bright stress-dependent emission of the tetrapod, its nanoscale size, and its colloidal nature provide a unique tool that may be incorporated into a variety of micromechanical systems including materials and biological samples to quantify local stresses with high spatial resolution.

Atomic resolution structures of Escherichia coli and Bacillus anthracis malate synthase A: comparison with isoform G and implications for structure-based drug discovery.

Enzymes of the glyoxylate shunt are important for the virulence of pathogenic organisms such as Mycobacterium tuberculosis and Candida albicans. Two isoforms have been identified for malate synthase, the second enzyme in the pathway. Isoform A, found in fungi and plants, comprises approximately 530 residues, whereas isoform G, found only in bacteria, is larger by approximately 200 residues. Crystal structures of malate synthase isoform G from Escherichia coli and Mycobacterium tuberculosis were previously determined at moderate resolution. Here we describe crystal structures of E. coli malate synthase A (MSA) in the apo form (1.04 A resolution) and in complex with acetyl-coenzyme A and a competitive inhibitor, possibly pyruvate or oxalate (1.40 A resolution). In addition, a crystal structure for Bacillus anthracis MSA at 1.70 A resolution is reported. The increase in size between isoforms A and G can be attributed primarily to an inserted alpha/beta domain that may have regulatory function. Upon binding of inhibitor or substrate, several active site loops in MSA undergo large conformational changes. However, in the substrate bound form, the active sites of isoforms A and G from E. coli are nearly identical. Considering that inhibitors bind with very similar affinities to both isoforms, MSA is as an excellent platform for high-resolution structural studies and drug discovery efforts.

Localizing the deficit in a case of jargonaphasia.

We report the case of a neologistic jargonaphasic and ask whether her target-related and abstruse neologisms are the result of a single deficit, which affects some items more severely than others, or two deficits: one to lexical access and the other to phonological encoding. We analyse both correct/incorrect performance and errors and apply both traditional and formal methods (maximum-likelihood estimation and model selection). All evidence points to a single deficit at the level of phonological encoding. Further characteristics are used to constrain the locus still further. V.S. does not show the type of length effect expected of a memory component, nor the pattern of errors associated with an articulatory deficit. We conclude that her neologistic errors can result from a single deficit at a level of phonological encoding that immediately follows lexical access where segments are represented in terms of their features. We do not conclude, however, that this is the only possible locus that will produce phonological errors in aphasia, or, indeed, jargonaphasia.