Search

Perineuronal nets (PNNs) are densely packed extracellular matrices that cover the cell body of fast-spiking inhibitory neurons. PNNs stabilize synapses inhibiting synaptic plasticity. Here we show that synaptic terminals of fast-spiking interneurons localize to holes in the PNNs in the adult mouse somatosensory cortex. Approximately 95% of holes in the PNNs contain synapses and astrocytic processes expressing Kir4.1, glutamate and GABA transporters. Hence, holes in the PNNs contain tripartite synapses. In the adult mouse brain, PNN degradation causes an expanded astrocytic coverage of the neuronal somata without altering the axon terminals. The loss of PNNs impairs astrocytic transmitter and potassium uptake, resulting in the spillage of glutamate into the extrasynaptic space. Our data show that PNNs and astrocytes cooperate to contain synaptically released signals in physiological conditions. Their combined action is altered in mouse models of Alzheimer’s disease and epilepsy where PNNs are disrupted.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivofunction is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2gene in adult cells is likely to arise at the RNA level.

Transient receptor potential vanilloid family member 4 (TRPV4) transcript and protein expression increased in the urinary bladder and lumbosacral dorsal root ganglia of transgenic mice with chronic urothelial overexpression of nerve growth factor (NGF-OE). We evaluated the functional role of TRPV4 in bladder function with open-outlet cystometry, void spot assays, and natural voiding (Urovoid) assays with the TRPV4 antagonist HC-067047 (1 μM) or vehicle in NGF-OE and littermate wild-type (WT) mice. Blockade of TRPV4 at the level of the urinary bladder significantly (P≤ 0.01) increased the intercontraction interval (2.2-fold) and void volume (2.6-fold) and decreased nonvoiding contractions (3.0-fold) in NGF-OE mice, with lesser effects (1.3-fold increase in the intercontraction interval and 1.3-fold increase in the void volume) in WT mice. Similar effects of TRPV4 blockade on bladder function in NGF-OE mice were demonstrated with natural voiding assays. Intravesical administration of HC-067047 (1 µM) significantly (P≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in littermate WT mice. Blockade of urinary bladder TRPV4 or intravesical infusion of brefeldin A significantly (P≤ 0.01) reduced (2-fold) luminal ATP release from the urinary bladder in NGF-OE and littermate WT mice. The results of the present study suggest that TRPV4 contributes to luminal ATP release from the urinary bladder and increased voiding frequency and pelvic sensitivity in NGF-OE mice.

The etiology of interstitial cystitis/bladder pain syndrome (IC/BPS) is unknown but likely multifactorial. IC/BPS symptoms can be exacerbated by psychological stress, but underlying mechanisms remain to be defined. TRPV1 channels, expressed on nerve fibers, have been implicated in bladder dysfunction and colonic hypersensitivity with stress in rodents. Histamine/H1R activation of TRPV1+nerves increases bladder afferent fiber sensitivity to distension. TRPV1 channels are also expressed on mast cells, previously implicated in contributing to IC/BPS etiology and symptoms. We have examined the contribution of TRPV1 and mast cells to bladder dysfunction after repeated variate stress (RVS). RVS increased (p ≤ 0.05) serum and fecal corticosterone expression and induced anxiety-like behavior in wild type (WT) mice. Intravesical instillation of the selective TRPV1 antagonist capsazepine (CPZ) rescued RVS-induced bladder dysfunction in WT mice. Trpv1knockout (KO) mice did not increase voiding frequency with RVS and did not exhibit increased serum corticosterone expression despite exhibiting anxiety-like behavior. Mast cell deficient mice (B6.Cg-Kitw-sh) failed to demonstrate RVS-induced increased voiding frequency or serum corticosterone expression whereas control (no stress) mast cell deficient mice had similar functional bladder capacity to WT mice. TRPV1 protein expression was significantly increased in the rostral lumbar (L1-L2) spinal cord and dorsal root ganglia in WT mice exposed to RVS but no changes were observed in lumbosacral (L6-S1) spinal segments or DRG. These studies demonstrated TRPV1 and mast cell involvement in RVS-induced increased voiding frequency and suggest that TRPV1 and mast cells may be useful targets to mitigate stress-induced urinary bladder dysfunction.

Through studying the localization of eIF2B we have identified cytoplasmic eIF2B bodies in mammalian cells. We provide evidence that the composition and function of these foci are regulated by the integrated stress response (ISR). ISR-inhibiting drugs modulate the dynamics of these eIF2B bodies, thereby providing insight into their function.

BackgroundHutchinson–Gilford progeria syndrome (HGPS) is an ultra-rare, fatal, segmental premature aging syndrome caused by the aberrant lamin A protein, progerin. The protein farnesyltransferase inhibitor, lonafarnib, ameliorates some aspects of cardiovascular and bone disease.MethodsWe performed a prospective longitudinal survey of plasma proteins in 24 children with HGPS (an estimated 10% of the world’s population at the time) at baseline and on lonafarnib therapy, compared with age- and gender-matched controls using a multi-analyte, microsphere-based immunofluorescent assay.ResultsThe mean levels for 23/66 (34.8%) proteins were significantly lower and 7/66 (10.6%) were significantly higher in HGPS samples compared with those in controls (P≤0.05). Six proteins whose concentrations were initially lower normalized with lonafarnib therapy: interleukins 1α, 7, and 13, beta-2 microglobulin, C-reactive protein, and myoglobin. Alpha-2 macroglobulin, a protease inhibitor associated with stroke, was elevated at baseline and subsequently normalized with lonafarnib therapy.ConclusionThis is the first study to employ a multi-analyte array platform in HGPS. Novel potential biomarkers identified in this study should be further validated by correlations with clinical disease status, especially proteins associated with cardiovascular disease and those that normalized with lonafarnib therapy.

BackgroundHutchinson-Gilford progeria syndrome (HGPS) is a fatal sporadic autosomal dominant premature ageing disease caused by single base mutations that optimise a cryptic splice site within exon 11 of the LMNAgene. The resultant disease-causing protein, progerin, acts as a dominant negative. Disease severity relies partly on progerin levels.Methods and resultsWe report a novel form of somatic mosaicism, where a child possessed two cell populations with different HGPS disease-producing mutations of the same nucleotide—one producing severe HGPS and one mild HGPS. The proband possessed an intermediate phenotype. The mosaicism was initially discovered when Sanger sequencing showed a c.1968+2T>A mutation in blood DNA and a c.1968+2T>C in DNA from cultured fibroblasts. Deep sequencing of DNA from the proband's blood revealed 4.7% c.1968+2T>C mutation, and 41.3% c.1968+2T>A mutation.ConclusionsWe hypothesise that the germline mutation was c.1968+2T>A, but a rescue event occurred during early development, where the somatic mutation from A to C at 1968+2 provided a selective advantage. This type of mosaicism where a partial phenotypic rescue event results from a second but milder disease-causing mutation in the same nucleotide has not been previously characterised for any disease.

Climate change is a critical scientific and social issue of our time, yet the topic is not frequently discussed by the general public. Additionally, misconceptions about the topic persist. One important aspect of climate change is the greenhouse effect. This activity adds a greenhouse gas discussion to an existing module about molecular shape and modeling. The module links the greenhouse effect to climate change. The activity has been used in several introductory chemistry settings. A discussion of student-generated answers and student-reported learning gains is presented.

This article describes the structure of the WomenHeart (WH) peer-led support groups and assesses participants' quality of life and social, emotional, and physical health. An online survey was administered to 50 WH groups. Participants reported low levels of stress, anxiety, and depression and high levels of social support and patient activation.

Time to fall asleep (sleep latency) is a major determinant of sleep quality. Chronic, long sleep latency is a major characteristic of sleep-onset insomnia and/or delayed sleep phase syndrome. In this study we aimed to discover common polymorphisms that contribute to the genetics of sleep latency. We performed a meta-analysis of genome-wide association studies (GWAS) including 2 572 737 single nucleotide polymorphisms (SNPs) established in seven European cohorts including 4242 individuals. We found a cluster of three highly correlated variants (rs9900428, rs9907432 and rs7211029) in the RNA-binding protein fox-1 homolog 3 gene (RBFOX3) associated with sleep latency (P-values=5.77 × 10−08, 6.59 × 10−08and 9.17 × 10−08). These SNPs were replicated in up to 12 independent populations including 30 377 individuals (P-values=1.5 × 10−02, 7.0 × 10−03and 2.5 × 10−03; combined meta-analysis P-values=5.5 × 10−07, 5.4 × 10−07and 1.0 × 10−07). A functional prediction of RBFOX3 based on co-expression with other genes shows that this gene is predominantly expressed in brain (P-value=1.4 × 10−316) and the central nervous system (P-value=7.5 × 10−321). The predicted function of RBFOX3 based on co-expression analysis with other genes shows that this gene is significantly involved in the release cycle of neurotransmitters including gamma-aminobutyric acid and various monoamines (P-values<2.9 × 10−11) that are crucial in triggering the onset of sleep. To conclude, in this first large-scale GWAS of sleep latency we report a novel association of variants in RBFOX3 gene. Further, a functional prediction of RBFOX3 supports the involvement of RBFOX3 with sleep latency.

The article discusses the development of a use case and its influence on national cooperation through the development of standards in the health information technology (IT) market. It explains how a health problem with a user story can serve as a starting point for standards development and its endorsement of the solution by groups like the International Organization for Standardization (ISO) or Health Level Seven (HL7).

Cybersecurity is a broad and growing job field, encompassing many different job categories with different cognitive demands. Traditional, knowledge-based assessments may exclude candidates who are cognitively suited to performing cybersecurity work but who have not had the opportunity to learn the subject matter. Using the job categories included in the National Initiative for Cybersecurity Education (NICE) framework for the cybersecurity workforce, we propose a model for predicting cybersecurity aptitude beyond a general- intelligence approach. In addition to including general intelligence, the model is based on a classification of jobs as requiring real-time or deliberate performance, and proactive or reactive actions. We suggest that tasks, work roles, and people can be represented along the same set of axes to match job requirements to person attributes. These constructs can then be used to create assessments of potential for cybersecurity applicants, including one we propose, called the Cyber Aptitude and Talent Assessment (CATA).

The Pediatric Outcomes Data Collection Instrument (PODCI) is an outcomes assessment tool developed to allow measurement of health-related quality of life in children with disorders having musculoskeletal impact. The instrument was tested by Hunsaker and colleagues on a large population-based sample of children (n=5300), and partial results of that survey were published in 2002. Further publication of the findings did not occur. The PODCI was designed to collect data on age, sex, comorbidities, race and ethnicity, makeup of the household, and other demographic data that could have an impact on function and psychosocial issues. This retrospective study evaluated the impact of age, sex, and healthcomorbid conditions on the subscales of the PODCI.

The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.

Translation technology, including translation memory software, can make the work of professional translators more efficient and effective. Using a multi-disciplinary approach with converging methods, we evaluated translation memory software in the context of a work environment in which it was to be deployed. Our evaluation had three phases: (1) a contextual inquiry of translation practice, (2) a usability test of the candidate software package, and (3) a heuristic evaluation to evaluate the ways that the software package matched or did not match the needs identified in the contextual inquiry. From this, we derived a set of recommendations for incorporating translation technology into the organization’s processes. Suggestions included incorporating shared translation memory in the workflow and working with the software vendor to improve text handling for non-European languages. Problems with both the software under evaluation and standard workplace software highlighted the need for developers to evaluate language technology with non-Roman orthography.

We present two closely-related approaches to product evaluation in less-commonly taught languages with native speaker informants and test participants. The Afghan Language Aptitude Battery (ALAB) was designed to be used to identify promising Afghan candidates for English language instruction before they started training in Afghanistan. In evaluating the battery, however, we were limited to testing in the United States. We asked Afghan service members who were already enrolled in language courses to act as usability participants and solicited their input on the design of the tests, the quality of the translations, and on the battery’s suitability for use in Afghanistan. Our testing found that the students were able to accomplish the tests, though the elements were unfamiliar, but that the instructions required extensive revisions in order to make them understandable to a less-educated audience in Afghanistan. The difference in the outcomes of our two evaluations suggests that translation evaluation should be done separately from test evaluation.

Cellular stress can globally inhibit translation initiation, and glucose removal from yeast causes one of the most dramatic effects in terms of rapidity and scale. Here we show that the same rapid inhibition occurs during yeast growth as glucose levels diminish. We characterize this novel regulation showing that it involves alterations within the 48S preinitiation complex. In particular, the interaction between eIF4A and eIF4G is destabilized, leading to a temporary stabilization of the eIF3–eIF4G interaction on the 48S complex. Under such conditions, specific mRNAs that are important for the adaptation to the new conditions must continue to be translated. We have determined which mRNAs remain translated early after glucose starvation. These experiments enable us to provide a physiological context for this translational regulation by ascribing defined functions that are translationally maintained or up-regulated. Overrepresented in this class of mRNA are those involved in carbohydrate metabolism, including several mRNAs from the pentose phosphate pathway. Our data support a hypothesis that a concerted preemptive activation of the pentose phosphate pathway, which targets both mRNA transcription and translation, is important for the transition from fermentative to respiratory growth in yeast.

The mechanism and consequences of the translational inhibition caused by glucose depletion in yeast are characterized. eIF4A is lost from the preinitiation complex, and the pentose phosphate pathway is translationally up-regulated, allowing an efficient transition to the new conditions.

We wished to identify a major transcript that is upregulated during in vivopancreatic islet neogenesis and examine the expression of the gene in beta and ductal cells.

Usability of an interface is an emergent property of the system and the user; it does not exist independently of either one. For this reason, characteristics of the user which affect his or her performance on a task can affect the apparent usability of the interface in a usability study. We propose and investigate, using a Wikipedia information-seeking task, a model relating spatial abilities and performance measures for system usability. In the context of World Wide Web (WWW) site usability, we found that spatial visualization ability and system experience predicted system effectiveness measures, while spatial orientation ability, spatial visualization ability, and general computer experience predicted system efficiency measures. We suggest possible extensions and further tests of this model.

Recycling of eIF2-GDP to the GTP-bound form constitutes a core essential, regulated step in eukaryotic translation. This reaction is mediated by eIF2B, a heteropentameric factor with important links to human disease. eIF2 in the GTP-bound form binds to methionyl initiator tRNA to form a ternary complex, and the levels of this ternary complex can be a critical determinant of the rate of protein synthesis. Here we show that eIF2B serves as the target for translation inhibition by various fusel alcohols in yeast. Fusel alcohols are endpoint metabolites from amino acid catabolism, which signal nitrogen scarcity. We show that the inhibition of eIF2B leads to reduced ternary complex levels and that different eIF2B subunit mutants alter fusel alcohol sensitivity. A DNA tiling array strategy was developed that overcame difficulties in the identification of these mutants where the phenotypic distinctions were too subtle for classical complementation cloning. Fusel alcohols also lead to eIF2α dephosphorylation in a Sit4p-dependent manner. In yeast, eIF2B occupies a large cytoplasmic body where guanine nucleotide exchange on eIF2 can occur and be regulated. Fusel alcohols impact on both the movement and dynamics of this 2B body. Overall, these results confirm that the guanine nucleotide exchange factor, eIF2B, is targeted by fusel alcohols. Moreover, they highlight a potential connection between the movement or integrity of the 2B body and eIF2B regulation.

This study highlights a connection between the eIF2B body and the regulation of translation initiation as a response to stress in Saccharomyces cerevisiae. Fusel alcohols are involved in signaling nitrogen scarcity to the cell and they inhibit protein synthesis by preventing the movement of the eIF2B body throughout the cell.

Spatial Visualization Ability is a determinant of performance efficiency on website navigation tasks. It is unclear, however, why this is the case; though the spatial metaphor for web “navigation” is popular, it is not accurate. We put forth a hypothesis that strategy use is another important component of web navigation efficiency that relates to Spatial Visualization Ability. To investigate this hypothesis, we designed a series of experiments using numeric indicators instead of verbal labels in model websites. We then characterized the strategies that participants used on each website into categories based on their resemblance to prototype strategies. We found that, though strategy choice does not seem to depend on Spatial Visualization Ability, precise implementation does seem to depend to some degree on Spatial Visualization Ability.

Vincristine sulfate liposomes injection (VSLI) is a liposomal formulation of vincristine encapsulated in sphingosomes composed of sphinogomyelin and cholesterol (58/42; mol/mol). The pharmacokinetics and urinary excretion of VSLI were evaluated in 12 patients with metastatic melanoma after single‐dose (2.0 mg/m2every 2 weeks = 1 cycle) and multiple‐dose (cycle 3, pharmacokinetics only) administrations (intravenous infusion over 1 hour). After VSLI infusion, total (released and encapsulated) vincristine concentrations in plasma remained relatively constant for 3 to 12 hours and thereafter declined, with interpatient variability seen in the rate of decline resulting in monoexponential or biexponential profiles. The area under the plasma concentration‐time curve from time zero to infinity of total vincristine in plasma ranged from 4933 to 40495 h·ng/mL and total clearance ranged from 131 to 445 mL/h. The volume of distribution at steady state was 2650 ± 731 mL, indicating VSLI was mainly confined within the plasma. The released vincristine concentrations in plasma were below the level of quantitation in 95% of samples. The pharmacokinetic parameters were similar between cycles 1 and 3, and trough plasma levels of total vincristine were below the level of quantitation of 1 ng/mL. Approximately 8% of the injected dose was excreted in the urine as unchanged vincristine (7%) or N‐desformylvincristine (0.8%). Overall, VSLI exhibited a longer circulation half‐life and higher area under the plasma concentration‐time curve compared to conventional vincristine, whereas its route of elimination remained unchanged.

Both glucocorticoid (GC) administration and brief occlusion of the main pancreatic duct result in an increase in total islet mass. Consequently, it was questioned whether these 2 stimuli would produce similar islet growth, indicating commonality in the mechanism of expansion. To test this, we assessed the effects on morphology after single and dual stimulation of the pancreas.

1. Florida red tides produce profound neurotoxicity that is evidenced by massive fish kills, neurotoxic shellfish poisoning, and respiratory distress. Red tides vary in potency, potency that is not totally governed by toxin concentration. The purpose of the study was to understand the variable potency of red tides by evaluating the potential for other natural pharmacological agents which could modulate or otherwise reduce the potency of these lethal environmental events. 2. A synaptosome binding preparation with 3-fold higher specific brevetoxin binding was developed to detect small changes in toxin binding in the presence of potential antagonists. Rodent brain labeled in vitro with tritiated brevetoxin shows high specific binding in the cerebellum as evidenced by autoradiography. Synaptosome binding assays employing cerebellum-derived synaptosomes illustrate 3-fold increased specific binding. 3. A new polyether natural product from Florida's red tide dinoflagellate Karenia brevis, has been isolated and characterized. Brevenal, as the nontoxic natural product is known, competes with tritiated brevetoxin for site 5 associated with the voltage-sensitive sodium channel (VSSC). Brevenal displacement of specific brevetoxin binding is purely competitive in nature. 4. Brevenal, obtained from either laboratory cultures or field collections during a red tide, protects fish from the neurotoxic effects of brevetoxin exposure. 5. Brevenal may serve as a model compound for the development of therapeutics to prevent or reverse intoxication in red tide exposures.

We examined the course of maternal depressive symptoms and children's attachment security at 36 months in a large sample of mother–child pairs from 10 sites across the country participating in the NICHD Study of Early Child Care (N = 1077). Maternal depressive symptoms predicted higher rates of insecure attachment. Women who reported intermittent symptoms across the first 36 months had preschoolers who were more likely to be classified as insecure C or D; women with chronic symptoms were more likely to have preschoolers who were classified as insecure D. Symptoms reported only during the first 15 months were not associated with elevated rates of later insecurity. After controlling for potentially confounding demographic variables, maternal sensitivity (observed at 6, 15, 24, and 36 months) did not meaningfully account for links between attachment security and patterns of depressive symptoms. However, the course and timing of maternal depressive symptoms interacted with maternal sensitivity to predict insecurity. Women with late, intermittent, or chronic symptoms who were also low in sensitivity were more likely to have preschoolers who were insecure, in contrast to symptomatic women who were high in sensitivity. These data have implications for understanding the combined impact of maternal depressive symptoms and maternal sensitivity on children's socioemotional development.