Ultraconserved Enhancers Are Required for Normal Development.

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.

Progressive Loss of Function in a Limb Enhancer during Snake Evolution.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.

Author Correction: An atlas of dynamic chromatin landscapes in mouse fetal development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An atlas of dynamic chromatin landscapes in mouse fetal development.

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP-seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC-seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

Enhancer redundancy provides phenotypic robustness in mammalian development.

Distant-acting tissue-specific enhancers, which regulate gene expression, vastly outnumber protein-coding genes in mammalian genomes, but the functional importance of this regulatory complexity remains unclear. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Unexpectedly, none of the ten deletions of individual enhancers caused noticeable changes in limb morphology. By contrast, the removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain and heart) uncovered more than one thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes that provides an effective regulatory buffer to prevent deleterious phenotypic consequences upon the loss of individual enhancers.

Supervised enhancer prediction with epigenetic pattern recognition and targeted validation.

Enhancers are important non-coding elements, but they have traditionally been hard to characterize experimentally. The development of massively parallel assays allows the characterization of large numbers of enhancers for the first time. Here, we developed a framework using Drosophila STARR-seq to create shape-matching filters based on meta-profiles of epigenetic features. We integrated these features with supervised machine-learning algorithms to predict enhancers. We further demonstrated that our model could be transferred to predict enhancers in mammals. We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mice and transduction-based reporter assays in human cell lines (153 enhancers in total). The results confirmed that our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription factor binding patterns at predicted enhancers versus promoters. We demonstrated that these patterns enable the construction of a secondary model that effectively distinguishes enhancers and promoters.

Identification of a polymorphism within the Rosa multiflora muRdr1A gene linked to resistance to multiple races of Diplocarpon rosae W. in tetraploid garden roses (Rosa × hybrida).

KEY MESSAGE: A QTL for resistance to several races of black spot co-located with the known Rrd1 locus in Rosa. A polymorphism in muRdr1A linked to black spot resistance was identified and molecular markers were designed. Black spot, caused by Diplocarpon rosae, is one of the most serious foliar diseases of landscape roses that reduces the marketability and weakens the plants against winter survival. Genetic resistance to black spot (BS) exists and race-specific resistance is a good target to implement marker-assisted selection. High-density single nucleotide polymorphism-based genetic maps were created for the female parent of a tetraploid cross between 'CA60' and 'Singing in the Rain' using genotyping-by-sequencing following a two-way pseudo-testcross strategy. The female linkage map was generated based on 227 individuals and included 31 linkage groups, 1055 markers, with a length of 1980 cM. Race-specific resistance to four D. rosae races (5, 7, 10, 14) was evaluated using a detached leaf assay. BS resistance was also evaluated under natural infection in the field. Resistance to races 5, 10 and 14 of D. rosae and field resistance co-located on chromosome 1. A unique sequence of 32 bp in exon 4 of the muRdr1A gene was identified in 'CA60' that co-segregates with D. rosae resistance. Two diagnostic markers, a presence/absence marker and an INDEL marker, specific to this sequence were designed and validated in the mapping population and a backcross population derived from 'CA60.' Resistance to D. rosae race 7 mapped to a different location on chromosome 1.

Changes in light quality alter physiological responses of soybean to thiamethoxam.

MAIN CONCLUSION: The interaction between neighboring weed-induced far-red enriched light and thiamethoxam can significantly alter soybean seedling morphology, nodulation, isoflavone levels, UV-absorbing phenolics, and carbon and nitrogen content. Neonicotinoid insecticides that are widely used on major crop plants can enhance plant growth and yield. Although the underlying mechanism of this enhanced growth and yield is not clear, recent studies suggest that neonicotinoids such as thiamethoxam (TMX) may exert their effects at least in part via signals that involve salicylic acid (SA) and jasmonic acid (JA). In the current research, effects of TMX on morphological and physiological responses of soybean have been compared under far-red-depleted (FR-D) and far-red-enriched (FR-E) light reflected by neighboring weeds. TMX significantly enhanced shoot and root growth but did not prevent stem elongation under FR-E light. Also, TMX did not prevent reductions in shoot carbon content and shoot carbon to nitrogen ratio under FR-E light. Despite similarities between these TMX effects in soybean and those known for SA and JA in other plant species, TMX significantly enhanced root-nodule numbers per plant and levels of root isoflavones malonyl-daidzin and malonyl-genistin under FR-E light only. These results suggest that the combined effect of FR-E light and TMX triggers a mechanism that operates concomitantly to enhance root isoflavones and nodulation in soybean.

Characteristics of emergency pages using a computer-based anesthesiology paging system in children and adults undergoing procedures at a tertiary care medical center.

BACKGROUND: In our large academic supervisory practice, attending anesthesiologists concomitantly care for multiple patients. To manage communications within the procedural environment, we use a proprietary electronic computer-based anesthesiology visual paging system. This system can send an emergency page that instantly alerts the attending anesthesiologist and other available personnel that immediate help is needed. We analyzed the characteristics of intraoperative emergency pages in children and adults. METHODS: We identified all emergency page activations between January 1, 2005 and July 31, 2010 in our main operating rooms. Electronic medical records were reviewed for rates and characteristics of pages such as primary etiology, performed interventions, and outcomes. RESULTS: During the study period, 258,135 anesthetics were performed (n = 32,103 children, younger than 18 years) and 370 emergency pages (n = 309 adults, n = 61 children) were recorded (1.4 per 1000 cases; 95% confidence interval, 1.3-1.6). Infants had the highest rates (9.4 per 1000; 95% confidence interval, 5.7-14.4) of emergency page activations (P < 0.001 compared with each other age group). In adults, the most frequent causes were hemodynamic (55%), and in children respiratory and airway (60.7%) events. CONCLUSION: Emergency pages were rare in patients older than 2 years. Infants were more likely than children 1 to 2 years of age to have emergency page activation, despite both groups being cared for by pediatric fellowship trained anesthesiologists.

Glucan affinity of starch synthase IIa determines binding of starch synthase I and starch-branching enzyme IIb to starch granules.

The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein-protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522→Arg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein-protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.

Preliminary findings of the relationship of lower heart rate variability with military sexual trauma and presumed posttraumatic stress disorder.

Decreased heart rate variability (HRV) occurs with physical and psychological disorders and is a predictor of cardiac and all-cause mortality. This study was the first of which we are aware to examine and report the relationship between military sexual trauma (MST) and HRV measures. In a historical cohort study of female veterans with (n = 27) and without (n = 99) MST who received Holter and electrocardiogram evaluation at a Veteran Affairs medical center during 2007-2010, we examined the relationship between MST and the standard deviation of all R-R intervals (SDNN) and the square root of the mean of the sum of the squares of differences between adjacent R-R intervals (RMSSD). Female veterans with MST were younger, p = .002, frequently had a probable posttraumatic stress disorder diagnosis, 80% versus 15%, p = < .0001, and had lower SDNN, p = .0001, and RMSSD, p = .001, than those without MST. The SDNN and RMSSD of a 25-year-old female veteran with MST were comparable to that of female veterans aged 69 to 81 years without MST. Further research is needed to evaluate relationships between MST and HRV measures.

Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions.

Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae(-) mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-(32)P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn(2+)-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.

Hyperspectral time series datasets of maize during the grain filling period.

OBJECTIVES: Remotely sensed hyperspectral data are increasingly being used to assess crop development and growth throughout the growing season. Large datasets capturing key growth stages can be useful to researchers studying many physiological plant responses. A time series analysis of hyperspectral reflectance measurements taken during the grain filling period and published within a publicly accessible database are described herein. These datasets document the spectral reflectance pattern of the canopy within the visible and near-infrared portion of the electromagnetic spectrum during the late stages of the grain filling period as plants approach and reach physiological maturity. DATA DESCRIPTION: Included within the data repository are canopy-level hyperspectral datasets collected in 2017 and 2018. Data is included in its raw form, as well as with several manipulations to smooth and standardize the raw data. Data are released as comma separated value spreadsheets as well as Microsoft Excel open XLSX spreadsheets. These are accompanied by README text files which further describe the data and supplemental files that record hybrids used and plant phenology for each year of data collection.

Genome-wide fetalization of enhancer architecture in heart disease.

Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the regulatory landscape in heart disease is unclear. Here, we use RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from up to 26 healthy controls, 18 individuals with idiopathic dilated cardiomyopathy (DCM), and five fetal hearts. Healthy individuals have a highly reproducible epigenomic landscape, consisting of more than 33,000 predicted heart enhancers. In contrast, we observe reproducible disease-associated changes in activity at 6,850 predicted heart enhancers. Combined analysis of adult and fetal samples reveals that the heart disease epigenome and transcriptome both acquire fetal-like characteristics, with 3,400 individual enhancers sharing fetal regulatory properties. We also provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of DCM etiology.

Heterosis for carotenoid concentration and profile in maize hybrids.

Production of high-lutein maize grain is of particular interest as a value-added feed source to produce high-lutein eggs. In this paper, it is demonstrated that heterosis for total carotenoid concentration and for the ratio of lutein to zeaxanthin (L:Z ratio), or profile type, exists infrequently in yellow dent crosses. However, yellow dent inbred maize lines A619 and CG102, both possessing high-lutein profiles, produce F1 seed with a classic overdominant expression of lutein levels (i.e., 49 µg/g dry weight (DW) above the high-parent value). Reciprocal crosses of A619 and CG102 with one another and with two high-zeaxanthin (i.e., low lutein), high-carotenoid lines both suggest that the A619 and CG102 high-lutein phenotypes are achieved by different and complementary genotypes. The contribution of CG102 to the heterotic response was examined using a QTL-based approach that involved phenotyping the mapping population in a testcross to A619. Significant QTL were found at loci known to be involved in the carotenoid pathway but also at loci proximate to, but separate from, known carotenoid pathway steps. Exploiting an overdominant heterotic response for lutein and total carotenoids should be given strong consideration as a viable method of producing high-carotenoid hybrid maize lines.

Peer socialization of masculinity and femininity: differential effects of overt and relational forms of peer victimization.

Although peer influence has been implicated in recent theories of gender socialization, few investigations have tested whether children's gendered behaviours change over time as a function of peer experiences and whether some peer experiences may exacerbate, rather than dampen, gender non-conformity. Accordingly, the current study examined prospective links between specific forms of peer victimization and children's adherence to traditional gender roles. Peer reports of victimization and self-reports of engagement in stereotypically masculine and feminine activities were collected from 199 children (104 girls; 95 boys) in the Fall and Spring of their fifth-grade year. Multi-group path analysis was used to explore the relations between forms of victimization and masculinity and femininity for girls and boys. For girls, peer victimization predicted withdrawal from both feminine and masculine behaviours. For boys, physical, verbal, and general victimization predicted lower levels of feminine behaviours, but social exclusion forecast heightened engagement in traditionally feminine activities. These findings underscore how social experiences can amplify, as well as reduce, gender non-conformity.

Healthcare Team Vitality Instrument (HTVI): developing a tool assessing healthcare team functioning.

AIM: This paper is a report of a study conducted to refine, shorten and validate the Healthcare Team Vitality Instrument. BACKGROUND: The Healthcare Team Vitality Instrument was developed to assess team vitality of nurses as well as other licensed and unlicensed personnel working as part of healthcare teams in inpatient hospital units. This instrument was necessary for two reasons. First, other commonly used instruments assess characteristics of Registered Nurses or perceptions about and characteristics of the organizations in which they work, but not these factors in combination with critical factors of interdisciplinary team functioning and collaboration. Second, a short tool for repeated, regular measurement of team vitality was needed to track the impact of changes to improve work environments. METHOD: Revisions to the Healthcare Team Vitality Instrument occurred in two phases. Phase 1 entailed collecting preliminary data and conducting cognitive interviews to refine the initial items. During Phase 2, the factor structure of the Healthcare Team Vitality Instrument was identified and a brief form developed and validated. Data were collected in 2006 and 2007. FINDINGS: Exploratory factor analyses suggested a four-factor solution with the following dimensions: (1) support structures, (2) engagement and empowerment, (3) patient care transitions and (4) team communication. CONCLUSION: The Healthcare Team Vitality Instrument can contribute both to better management practices and advancing knowledge to promote retention of nurses, and to some extent other healthcare professionals, as well as efforts to transform the acute healthcare work environment.

Clinical features and radiographic findings in cats with eosinophilic, neutrophilic, and mixed airway inflammation (2011-2018).

BACKGROUND: Idiopathic inflammatory airway disease (IAD) in cats often is described as asthmatic (eosinophilic) or bronchitic (neutrophilic), but this designation requires collection of airway fluid and it fails to consider cats with mixed airway inflammation. OBJECTIVE: To identify clinical features that would differentiate inflammatory disease types. ANIMALS: Forty-nine cats with nonspecific airway inflammation identified by bronchoscopic bronchoalveolar lavage (BAL) between 2011 and 2018 were evaluated. METHODS: This is a retrospective study. Cats were categorized by BAL differential cytology as having eosinophilic (eosinophils >20% with neutrophils <14%, or eosinophils >50%), mixed (eosinophils 20%-50% and neutrophils >14% or discordant inflammation from 2 BAL sites), or neutrophilic (neutrophils >14% and eosinophils <20%) inflammation. Type and duration of presenting complaints, signalment, body condition score, respiratory rate, CBC results, bronchoscopy, BAL results (% recovery, total nucleated cell count, differential cell count), and radiographic findings were compared among groups. RESULTS: Idiopathic IAD was diagnosed in 49 cats, with BAL eosinophilic inflammation in 23, mixed inflammation in 14, and neutrophilic inflammation in 12. Cough was the predominant presenting complaint with no difference in duration of signs among groups (median, 5.5 months). Respiratory rate and effort also did not differ. Cats with eosinophilic inflammation were significantly younger (4.4 ± 3.3 years) than those with neutrophilic (8.0 ±5.6 years) or mixed inflammation (7.5 ± 4.0 years; P = .03). Results of CBC and interpretation of radiographic findings did not differ among groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Substantial overlap exists in clinical and radiographic findings in cats with various forms of idiopathic airway inflammation.

Coaching Strategies Used to Support Interprofessional Teams in 3 Primary Care Centers.

PURPOSE/AIMS: Clinical nurse specialists and other advanced practice registered nurses use healthcare team coaching to foster interprofessional practice and enhance healthcare quality. Although coaching has been shown to support positive changes in healthcare, little is known about how coaching strategies are used in practice. The purpose of this study was to describe the strategies used by an experienced healthcare team coach tasked with advancing interprofessional care and teamwork in primary care clinics. METHODS: This qualitative description study was part of a larger project that included an objective to increase interprofessional practice in 3 primary care clinics in the midwestern United States. Data drawn from 35 audio-recorded and transcribed coaching telephone calls were analyzed using content analysis. RESULTS: Twelve coaching strategies were identified and divided into the following groups: (a) enhancing team development, (b) affirming the work of the team, (c) facilitating progress, (d) providing resources, and (e) connecting work to theoretical frameworks. CONCLUSIONS: The coaching strategies described in this study can inform the work of clinical nurse specialists and other advanced practice registered nurses charged with advancing interprofessional collaborative practice. Future research is recommended to examine the efficacy of strategies and develop a comprehensive model of healthcare team coaching.