REVEILLE2 thermosensitive splicing: a molecular basis for the integration of nocturnal temperature information by the Arabidopsis circadian clock.

Cold stress is one of the major environmental factors that limit growth and yield of plants. However, it is still not fully understood how plants account for daily temperature fluctuations, nor how these temperature changes are integrated with other regulatory systems such as the circadian clock. We demonstrate that REVEILLE2 undergoes alternative splicing after chilling that increases accumulation of a transcript isoform encoding a MYB-like transcription factor. We explore the biological function of REVEILLE2 in Arabidopsis thaliana using a combination of molecular genetics, transcriptomics, and physiology. Disruption of REVEILLE2 alternative splicing alters regulatory gene expression, impairs circadian timing, and improves photosynthetic capacity. Changes in nuclear gene expression are particularly apparent in the initial hours following chilling, with chloroplast gene expression subsequently upregulated. The response of REVEILLE2 to chilling extends our understanding of plants immediate response to cooling. We propose that the circadian component REVEILLE2 restricts plants responses to nocturnal reductions in temperature, thereby enabling appropriate responses to daily environmental changes.

Broadband infrared absorber based on a sputter deposited hydrogenated carbon multilayer enhancing MEMS-based CMOS thermopile performance.

Based on pulsed DC sputter deposition of hydrogenated carbon, an absorber optical coating with maximized broadband infrared absorptance is reported. Enhanced broadband (2.5-20 µm) infrared absorptance (>90%) with reduced infrared reflection is achieved by combining a low-absorptance antireflective (hydrogenated carbon) overcoat with a broadband-absorptance carbon underlayer (nonhydrogenated). The infrared optical absorptance of sputter deposited carbon with incorporated hydrogen is reduced. As such, hydrogen flow optimization to minimize reflection loss, maximize broadband absorptance, and achieve stress balance is described. Application to complementary metal-oxide-semiconductor (CMOS) produced microelectromechanical systems (MEMS) thermopile device wafers is described. A 220% increase in thermopile output voltage is demonstrated, in agreement with modeled prediction.

A novel technique using chronic infusion of small extracellular vesicles from gestational diabetes mellitus causes glucose intolerance in pregnant mice.

Small extracellular vesicles (sEVs) play a central role in cell-to-cell communication in normal physiology and in disease, including gestational diabetes mellitus (GDM). The goal of the present study was to test the hypothesis that chronic administration of sEVs isolated from GDM causes glucose intolerance in healthy pregnant mice. Small EVs were isolated from plasma between 24 and 28 weeks gestation from healthy pregnant women (controls) and GDM, and infused intravenously for 4 days in late pregnant mice using a mini-osmotic pump. Subsequently in vivo glucose tolerance was assessed, and muscle and adipose tissue insulin sensitivity and islet glucose stimulated insulin secretion (GSIS) were determined in vitro. Mice infused with sEVs from GDM developed glucose intolerance. Administration of sEVs from controls, but not sEVs from GDM women, stimulated islet GSIS and increased fasting insulin levels in pregnant mice. Neither infusion of sEVs from controls nor from GDM women affected muscle insulin sensitivity, placental insulin or mTOR signaling, placental and fetal weight. Moreover, these results were not associated with immunomodulatory effects as human sEVs did not activate mouse T cells in vitro. We suggest that circulating sEVs regulate maternal glucose homeostasis in pregnancy and may contribute to the attenuated islet insulin secretion and more pronounced glucose intolerance in GDM as compared with healthy pregnancy.

Rapid and Dynamic Alternative Splicing Impacts the Arabidopsis Cold Response Transcriptome.

Plants have adapted to tolerate and survive constantly changing environmental conditions by reprogramming gene expression The dynamics of the contribution of alternative splicing (AS) to stress responses are unknown. RNA-sequencing of a time-series of Arabidopsis thaliana plants exposed to cold determines the timing of significant AS changes. This shows a massive and rapid AS response with coincident waves of transcriptional and AS activity occurring in the first few hours of temperature reduction and further AS throughout the cold. In particular, hundreds of genes showed changes in expression due to rapidly occurring AS in response to cold ("early AS" genes); these included numerous novel cold-responsive transcription factors and splicing factors/RNA binding proteins regulated only by AS. The speed and sensitivity to small temperature changes of AS of some of these genes suggest that fine-tuning expression via AS pathways contributes to the thermo-plasticity of expression. Four early AS splicing regulatory genes have been shown previously to be required for freezing tolerance and acclimation; we provide evidence of a fifth gene, U2B"-LIKE Such factors likely drive cascades of AS of downstream genes that, alongside transcription, modulate transcriptome reprogramming that together govern the physiological and survival responses of plants to low temperature.

Regulation of glucose homeostasis by small extracellular vesicles in normal pregnancy and in gestational diabetes.

The mechanisms underpinning maternal metabolic adaptations to a healthy pregnancy and in gestational diabetes mellitus (GDM) remain poorly understood. We hypothesized that small extracellular vesicles (sEVs) isolated from healthy pregnant women promote islet glucose-stimulated insulin secretion (GSIS) and peripheral insulin resistance in nonpregnant mice and that sEVs from GDM women fail to stimulate insulin secretion and cause exacerbated insulin resistance. Small EVs were isolated from plasma of nonpregnant, healthy pregnant, and GDM women at 24-28 weeks of gestation. We developed a novel approach in nonpregnant mice involving a mini-osmotic pump for continuous 4-day jugular venous infusion of sEVs and determined their effects on glucose tolerance in vivo and islets and skeletal muscle in vitro. Fasting insulin was elevated in mice infused with pregnant sEVs as compared to sEVs from nonpregnant and GDM women. Mice infused with sEVs from GDM women developed glucose intolerance. GSIS was increased in mice infused with healthy pregnancy sEVs compared to mice receiving nonpregnant sEVs. GSIS and muscle basal insulin signaling, and insulin responsiveness were attenuated in mice infused with GDM sEVs. sEVs represent a novel mechanism regulating maternal glucose homeostasis in pregnancy and we speculate that altered sEV content contributes to the development of GDM.

Afatinib and radiotherapy, with or without temozolomide, in patients with newly diagnosed glioblastoma: results of a phase I trial.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. Amplification or overexpression of the epidermal growth factor receptor gene, part of the ErbB family, occur in approximately 40% and 60% of patients with GBM, respectively. We present data from a dose-finding study of the ErbB inhibitor afatinib in combination with radiotherapy (RT), with or without temozolomide (TMZ), in patients with GBM. METHODS: This was a phase I, open-label, 3 + 3 dose-escalation trial in patients with newly-diagnosed, histologically-confirmed grade 4 malignant glioma and proven O6-methylguanine-DNA methyltransferase gene promoter methylation status. The primary endpoint was the maximum tolerated dose (MTD) of continuous daily afatinib when given in combination with RT, with (regimen M) or without (regimen U) concomitant TMZ treatment. RESULTS: Fifty-five patients were enrolled; 36 received ≥ 1 dose of trial medication (regimen M, n = 20, regimen U, n = 16). Afatinib was discontinued by all patients during the study. Reasons for afatinib discontinuation (regimen M/U) included disease progression (45%/50%), dose-limiting toxicity (10%/0%), and other adverse events (AEs; 35%/38%). The most frequently reported AEs with either regimen were diarrhea and rash, with no new safety signals identified. The MTD was determined as afatinib 30 mg in combination with daily TMZ and RT, and afatinib 40 mg in combination with RT alone. CONCLUSIONS: This study identified the MTD for afatinib in combination with RT, with and without TMZ, in patients with GBM. Further studies of afatinib in patients with GBM are warranted and should be based on appropriate biomarker-based preselection. TRIAL REGISTRATION: NCT00977431 (first posted September 15, 2009).

Artificial Metalloenzymes: Recent Developments and Innovations in Bioinorganic Catalysis.

Cellular life is orchestrated by the biochemical components of cells that include nucleic acids, lipids, carbohydrates, proteins, and cofactors such as metabolites and metals, all of which coalesce and function synchronously within the cell. Metalloenzymes allow for such complex chemical processes, as they catalyze a myriad of biochemical reactions both efficiently and selectively, where the metal cofactor provides additional functionality to promote reactivity not readily achieved in their absence. While the past 60 years have yielded considerable insight on how enzymes catalyze these reactions, a need to engineer and develop artificial metalloenzymes has been driven not only by industrial and therapeutic needs, but also by innate human curiosity. The design of miniature enzymes, both rationally and through serendipity, using both organic and inorganic building blocks has been explored by many scientists over the years and significant progress has been made. Herein, recent developments over the past 5 years in areas that have not been recently reviewed are summarized, and prospects for future research in these areas are addressed.

Comparative cohort study of volumetric modulated arc therapy for squamous cell cancer of unknown primary in the head and neck-Involved neck only versus mucosal irradiation.

OBJECTIVES: Target volumes for irradiation remain ill-defined for squamous cell cancer of unknown primary in the head and neck (SCCUP). The aim of this study was to compare involved neck only (INO) radiotherapy (RT) with irradiating involved neck plus potential mucosal primary sites and contralateral neck (MUC) in patients diagnosed and treated with modern diagnostics and techniques. DESIGN: This is a retrospective cohort study. Patients with a diagnosis of SCCUP with unilateral neck disease were included. RESULTS: Thirty patients were identified. All underwent FDG PET-CT. 47% of patients had HPV-positive SCC. 20 patients received RT to INO, 10 patients to MUC, all with volumetric modulated arc therapy (VMAT). A significantly lower dose for each organ at risk was delivered in INO-treated patients, with mean dose to contralateral parotid gland 57% less. The proportion of patients with late grade 2 or worse xerostomia was higher in MUC patients. The incidence of grade 2-3 mucositis (89% vs 45%) and grade 3 or worse dysphagia (50% vs 10%) was higher in MUC patients. Median follow-up was 31 months. No mucosal primaries emerged. Progression-free survival at 2 years was 74.7% for INO patients, 70% in the MUC group. Overall survival at 2 years was 79.7% in the INO group and 70% in the MUC patients. CONCLUSION: INO radiotherapy for patients with SCCUP of the head and neck is a safe treatment strategy resulting in clinically significant lower RT doses to OARS. Acute and late toxicities are reduced without detriment to patient survival.

A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing.

Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.

Global spatial analysis of Arabidopsis natural variants implicates 5'UTR splicing of LATE ELONGATED HYPOCOTYL in responses to temperature.

How plants perceive and respond to temperature remains an important question in the plant sciences. Temperature perception and signal transduction may occur through temperature-sensitive intramolecular folding of primary mRNA transcripts. Recent studies suggested a role for retention of the first intron in the 5'UTR of the clock component LATE ELONGATED HYPOCOTYL (LHY) in response to changes in temperature. Here, we identified a set of haplotypes in the LHY 5'UTR, examined their global spatial distribution, and obtained evidence that haplotype can affect temperature-dependent splicing of LHY transcripts. Correlations of haplotype spatial distributions with global bioclimatic variables and altitude point to associations with annual mean temperature and temperature fluctuation. Relatively rare relict type accessions correlate with lower mean temperature and greater temperature fluctuation and the spatial distribution of other haplotypes may be informative of evolutionary processes driving colonization of ecosystems. We propose that haplotypes may possess distinct 5'UTR pre-mRNA folding thermodynamics and/or specific biological stabilities based around the binding of trans-acting RNA splicing factors, a consequence of which is scalable splicing sensitivity of a central clock component that is likely tuned to specific temperature environments.

How does temperature affect splicing events? Isoform switching of splicing factors regulates splicing of LATE ELONGATED HYPOCOTYL (LHY).

One of the ways in which plants can respond to temperature is via alternative splicing (AS). Previous work showed that temperature changes affected the splicing of several circadian clock gene transcripts. Here, we investigated the role of RNA-binding splicing factors (SFs) in temperature-sensitive AS of the clock gene LATE ELONGATED HYPOCOTYL (LHY). We characterized, in wild type plants, temperature-associated isoform switching and expression patterns for SF transcripts from a high-resolution temperature and time series RNA-seq experiment. In addition, we employed quantitative RT-PCR of SF mutant plants to explore the role of the SFs in cooling-associated AS of LHY. We show that the splicing and expression of several SFs responds sufficiently, rapidly, and sensitively to temperature changes to contribute to the splicing of the 5'UTR of LHY. Moreover, the choice of splice site in LHY was altered in some SF mutants. The splicing of the 5'UTR region of LHY has characteristics of a molecular thermostat, where the ratio of transcript isoforms is sensitive to temperature changes as modest as 2 °C and is scalable over a wide dynamic range of temperature. Our work provides novel insight into SF-mediated coupling of the perception of temperature to post-transcriptional regulation of the clock.

How pervasive is mind wandering, really?

Recent claims that people spend 30-50% of their waking lives mind wandering (Killingsworth & Gilbert, 2010; Kane et al., 2007) have become widely accepted and frequently cited. While acknowledging attention to be inconstant and wavering, and mind wandering to be ubiquitous, we argue and present evidence that such simple quantitative estimates are misleading and potentially meaningless without serious qualification. Mind-wandering estimates requiring dichotomous judgments of inner experience rely on questionable assumptions about how such judgments are made, and the resulting data do not permit straightforward interpretation. We present evidence that estimates of daily-life mind wandering vary dramatically depending on the response options provided. Offering participants a range of options in estimating task engagement yielded variable mind-wandering estimates, from approximately 60% to 10%, depending on assumptions made about how observers make introspective judgments about their mind-wandering experiences and how they understand what it means to be on- or off-task.

Design of Artificial Glycosidases: Metallopeptides that Remove H†Antigen from Human Erythrocytes.

Catalysts that promote carbohydrate degradation have a wide range of potential applications, but the use of either enzyme glycosidases or small-molecule catalysts in biological systems raises significant challenges. Herein, we demonstrate a novel strategy for the design of synthetic agents that mimic natural glycosidases and address current problems for biological use. This strategy is illustrated by application to the development of potential blood substitutes for the rare Bombay blood type that is characterized by a deficiency of H2 antigen. Metallopeptides with 16 to 20 amino acids were constructed as artificial fucosidases that exhibit selective carbohydrate cleavage reactivity toward l-fucose over d-glucose. Selective fucose cleavage from the H2-antigen saccharide enables efficient removal of H2 antigen from erythrocytes and thereby accomplishes the conversion of regular human type-O blood into a potential blood substitute for the rare Bombay blood type.

"You're on ten, where can you go from there?" Tufnel problems in repeated experiential judgments.

We describe a set of Tufnel problems that arise when repeated use of a fixed-point scale precipitates failures to assess a full range of subjective experiences. As empirical evidence, participants in Study 1 periodically reported their depth of mind wandering on either 5- or 7-point Likert scales during a sustained attention task. The proportion of participants providing maximum scale ratings increased quickly over time-on-task and did so more quickly for the 5-point than for the 7-point group. Participants in Study 2 completed the same task using a 10-point scale before indicating whether and where they could have used a scale extended to "11" during the task. Slightly more than 20% of participants reported needing a scale extension. This Need for 11 was associated with differences in both reports of mind wandering depth and task performance. We conclude that Tufnel problems warrant methodological consideration and reflect interesting constraints on human judgment.

Is the cognitive reflection test a measure of both reflection and intuition?

The Cognitive Reflection Test (CRT) is one of the most widely used tools to assess individual differences in intuitive-analytic cognitive styles. The CRT is of broad interest because each of its items reliably cues a highly available and superficially appropriate but incorrect response, conventionally deemed the "intuitive" response. To do well on the CRT, participants must reflect on and question the intuitive responses. The CRT score typically employed is the sum of correct responses, assumed to indicate greater "reflectiveness" (i.e., CRT-Reflective scoring). Some recent researchers have, however, inverted the rationale of the CRT by summing the number of intuitive incorrect responses, creating a putative measure of intuitiveness (i.e., CRT-Intuitive). We address the feasibility and validity of this strategy by considering the problem of the structural dependency of these measures derived from the CRT and by assessing their respective associations with self-report measures of intuitive-analytic cognitive styles: the Faith in Intuition and Need for Cognition scales. Our results indicated that, to the extent that the dependency problem can be addressed, the CRT-Reflective but not the CRT-Intuitive measure predicts intuitive-analytic cognitive styles. These results provide evidence that the CRT is a valid measure of reflective but not of intuitive thinking.

AtRTD - a comprehensive reference transcript dataset resource for accurate quantification of transcript-specific expression in Arabidopsis thaliana.

RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.

Alternative splicing mediates responses of the Arabidopsis circadian clock to temperature changes.

Alternative splicing plays crucial roles by influencing the diversity of the transcriptome and proteome and regulating protein structure/function and gene expression. It is widespread in plants, and alteration of the levels of splicing factors leads to a wide variety of growth and developmental phenotypes. The circadian clock is a complex piece of cellular machinery that can regulate physiology and behavior to anticipate predictable environmental changes on a revolving planet. We have performed a system-wide analysis of alternative splicing in clock components in Arabidopsis thaliana plants acclimated to different steady state temperatures or undergoing temperature transitions. This revealed extensive alternative splicing in clock genes and dynamic changes in alternatively spliced transcripts. Several of these changes, notably those affecting the circadian clock genes late elongated hypocotyl (LHY) and pseudo response regulator7, are temperature-dependent and contribute markedly to functionally important changes in clock gene expression in temperature transitions by producing nonfunctional transcripts and/or inducing nonsense-mediated decay. Temperature effects on alternative splicing contribute to a decline in LHY transcript abundance on cooling, but LHY promoter strength is not affected. We propose that temperature-associated alternative splicing is an additional mechanism involved in the operation and regulation of the plant circadian clock.

Media multitasking and failures of attention in everyday life.

Using a series of online self-report measures, we examine media multitasking, a particularly pervasive form of multitasking, and its relations to three aspects of everyday attention: (1) failures of attention and cognitive errors (2) mind wandering, and (3) attentional control with an emphasis on attentional switching and distractibility. We observed a positive correlation between levels of media multitasking and self-reports of attentional failures, as well as with reports of both spontaneous and deliberate mind wandering. No correlation was observed between media multitasking and self-reported memory failures, lending credence to the hypothesis that media multitasking may be specifically related to problems of inattention, rather than cognitive errors in general. Furthermore, media multitasking was not related with self-reports of difficulties in attention switching or distractibility. We offer a plausible causal structural model assessing both direct and indirect effects among media multitasking, attentional failures, mind wandering, and cognitive errors, with the heuristic goal of constraining and motivating theories of the effects of media multitasking on inattention.