Distribution and intensity of constraint in mammalian genomic sequence

Comparisons of orthologous genomic DNA sequences can be used to characterize regions that have been subject to purifying selection and are enriched for functional elements. We here present the results of such an analysis on an alignment of sequences from 29 mammalian species. The alignment captures approximately 3.9 neutral substitutions per site and spans approximately 1.9 Mbp of the human genome. We identify constrained elements from 3 bp to over 1 kbp in length, covering approximately 5.5% of the human locus. Our estimate for the total amount of nonexonic constraint experienced by this locus is roughly twice that for exonic constraint. Constrained elements tend to cluster, and we identify large constrained regions that correspond well with known functional elements. While constraint density inversely correlates with mobile element density, we also show the presence of unambiguously constrained elements overlapping mammalian ancestral repeats. In addition, we describe a number of elements in this region that have undergone intense purifying selection throughout mammalian evolution, and we show that these important elements are more numerous than previously thought. These results were obtained with Genomic Evolutionary Rate Profiling (GERP), a statistically rigorous and biologically transparent framework for constrained element identification. GERP identifies regions at high resolution that exhibit nucleotide substitution deficits, and measures these deficits as "rejected substitutions". Rejected substitutions reflect the intensity of past purifying selection and are used to rank and characterize constrained elements. We anticipate that GERP and the types of analyses it facilitates will provide further insights and improved annotation for the human genome as mammalian genome sequence data become richer.     

Linkage refinement localises Sorsby fundus dystrophy between markers D22S275 and D22S278.

Sorsby fundus dystrophy is an autosomal dominant disorder which both clinically and histopathologically bears striking similarities to age related macular degeneration, one of the leading causes of blindness in the developed world. Recent studies have suggested a genetic localisation of the disease to chromosome 22q in a large genetic interval of approximately 25 cM. Independent genetic linkage analysis in a six generation British pedigree confirms linkage to the chromosome 22q region. A maximum two point lod score of 7.09 with no recombination was obtained with marker D22S280. Haplotype data positioned the disease between loci D22S275 and D22S278, thus significantly reducing the region on chromosome 22q where the gene is located.     

Comorbidity of asthma and anxiety and depression in Puerto Rican children

Studies have reported that childhood asthma is associated with internalizing disorders, but most of these studies have used global measures of depressive and anxiety symptoms. The Diagnostic Interview Schedule for Children was administered to a group of 1891 youth ages 4 to 17 and their caregivers in Puerto Rico to determine DSM-IV symptoms and diagnoses. Asthma diagnosis and having had an asthma attack were assessed by parental report. A diagnosis of asthma was associated with having any depressive disorder and one symptom of separation anxiety. An asthma attack was associated with any depressive disorder and any anxiety disorder and, more specifically, with separation anxiety disorder, major depressive disorder, and symptoms of depression, separation anxiety, and generalized anxiety. Possible explanations for the findings are discussed.     

In vitro degradation of silk fibroin

A significant need exists for long-term degradable biomaterials which can slowly and predictably transfer a load-bearing burden to developing biological tissue. In this study Bombyx mori silk fibroin yarns were incubated in 1mg/ml Protease XIV at 37 degrees C to create an in vitro model system of proteolytic degradation. Samples were harvested at designated time points up to 12 weeks and (1) prepared for scanning electron microscopy (SEM), (2) lyophilized and weighed, (3) mechanical properties determined using a servohydraulic Instron 8511, (4) dissolved and run on a SDS-PAGE gel, and (5) characterized with Fourier transform infrared spectroscopy. Control samples were incubated in phosphate-buffered saline. Fibroin was shown to proteolytically degrade with predictable rates of change in fibroin diameter, failure strength, cycles to failure, and mass. SEM indicated increasing fragmentation of individual fibroin filaments from protease-digested samples with time of exposure to the enzyme; particulate debris was present within 7 days of incubation. Gel electrophoresis indicated a decreasing amount of the silk 25 kDa light chain and a shift in the molecular weight of the heavy chain with increasing incubation time in protease. Results support that silk is a mechanically robust biomaterial with predictable long-term degradation characteristics.     

At least two distinct epigenetic mechanisms are correlated with high-frequency “switching” for aprt phenotypic expression in mouse embryonal carcinoma stem cells

A series of clones displaying high frequency switching phenotypes for expression of the adenine phosphoribosyltransferase (aprt) gene were previously isolated from the P19 mouse embryonal carcinoma stem cell line. Most clones contained only oneaprt allele. We report here the characterization of each of these clones with regards to enzymatic activity, mRNA steady state levels, DNA methylation, and chromatin conformation. When clones were selected for resistance to the purine analog 2,6-diaminopurine, which requires markedly reduced levels of APRT enzymatic activity, two distinct classes were observed. The first class was associated with reduced or undetectable levels ofaprt mRNA, hypermethylation of the 5 CpG island, and a closed chromatin conformation within this region. When clones of this class were selected for reacquisition of APRT enzymatic activity they were found to have increased mRNA levels, a hypomethylated CpG island, and an open chromatin conformation. In contrast, the second class of clones displayed wild-type levels of mRNA, CpG island hypomethylation, and an open chromatin conformation regardless of whether they were selected for the presence or absence of APRT enzymatic activity. The implications of these results for general mechanisms of epigenetic change in somatic cells and the possibility that expression of the mouseaprt gene may be developmentally regulated are discussed.     

Carbohydrate feeding and exercise: effect of beverage carbohydrate content

Summary The purpose of this study was to determine the effect of ingesting fluids of varying carbohydrate content upon sensory response, physiologic function, and exercise performance during 1.25 h of intermittent cycling in a warm environment (T _db=33.4°C). Twelve subjects (7 male, 5 female) completed four separate exercise sessions; each session consisted of three 20 min bouts of cycling at 65% , with each bout followed by 5 min rest. A timed cycling task (1200 pedal revolutions) completed each exercise session. Immediately prior to the first 20 min cycling bout and during each rest period, subjects consumed 2.5 ml·kg BW⁻¹ of water placebo (WP), or solutions of 6%, 8%, or 10% sucrose with electrolytes (20 mmol·l⁻¹ Na⁺, 3.2 mmol·l⁻¹ K⁺). Beverages were administered in double blind, counterbalanced order. Mean (±SE) times for the 1200 cycling task differed significantly: WP=13.62±0.33 min, *6%=13.03±0.24 min, 8%=13.30±0.25 min, 10%=13.57±0.22 min (*=different from WP and 10%,P<0.05). Compared to WP, ingestion of the CHO beverages resulted in higher plasma glucose and insulin concentrations, and higher RER values during the final 20 min of exercise (P<0.05). Markers of physiologic function and sensory perception changed similarly throughout exercise; no differences were observed among subjects in response to beverage treatments for changes in plasma concentrations of lactate, sodium, potassium, for changes in plasma volume, plasma osmolality, rectal temperature, heart rate, oxygen uptake, rating of perceived exertion, or for indices of gastrointestinal distress, perceived thirst, and overall beverage acceptance. Compared to ingestion of a water placebo, consumption of beverages containing 6% to 10% sucrose resulted in similar physiologic and sensory response, while ingestion of the 6% sucrose beverage resulted in significantly improved end-exercise performance following only 60 min of intermittent cycling exercise.     

Bronchiolitis obliterans syndrome (BOS), bronchiolitis obliterans organizing pneumonia (BOOP), and other late-onset noninfectious pulmonary complications following allogeneic hematopoietic stem cell transplantation.

Pulmonary dysfunction is a significant complication following allogeneic hematopoietic stem cell transplantation (HSCT), and is associated with significant morbidity and mortality. Effective antimicrobial prophylaxis and treatment strategies have increased the incidence of noninfectious lung injury, which can occur in the early posttransplant period or in the months and years that follow. Late-onset noninfectious pulmonary complications are frequently encountered, but diagnostic criteria and terminology for these disorders can be confusing and therapeutic approaches are suboptimal. As a consequence, inaccurate diagnosis of these conditions may hamper the appropriate data collection, enrollment into clinical trials, and appropriate patient care. The purpose of this review is to clarify the pathogenesis and diagnostic criteria of representative conditions, such as bronchiolitis obliterans syndrome and bronchiolitis obliterans organizing pneumonia, and to discuss the appropriate diagnostic strategies and treatment options.     

Two kinds of resting discharge in cat muscle spindles.

1. The behavior of primary endings of cat soleus muscle spindles was studied during shortening steps carried out at different muscle lengths. 2. Spindles were of two kinds: one, silent spindles, whose afferents fell silent after the shortening, at least over part of the range of lengths tested. The second, spontaneous spindles, resumed firing at all lengths. 3. For silent spindles, the duration of the silent period, measured at lengths where they did recover a resting rate, depended directly on muscle length and became shorter at longer lengths. This is what would be expected if the slack introduced in the spindle by the shortening step was removed more rapidly at longer lengths by the higher passive tension. For spontaneous spindles, on the other hand, the duration of the silent period after the shortening was largely independent of muscle length and depended on the spindle's rate of firing immediately before the shortening. 4. At intermediate lengths the discharge of slack spontaneous spindles remained unaffected by an isometric muscle contraction. It was therefore not possible to produce a pause in the discharge, behavior normally taken as typical of spindles. The discharge could be interrupted by the contraction if this was combined with a large shortening movement. 5. It is proposed that when intrafusal fibers are slackened by a shortening step, the resting discharge in spontaneous spindles is generated by a maintained depolarization of the annulospiral ending resulting from extension of the terminal coils by forces from within the receptor. A shortening contraction compresses the spirals to interrupt the discharge. The sensory endings of silent spindles remain below threshold until the spirals have been opened out sufficiently by external stretch.     

The dendritic architecture of the medial terminal nucleus of the accessory optic system in the rat,rabbit, and cat

Summary The neurons of the medial terminal nucleus (MTN) of the accessory optic system (AOS) have been studied in the rat, rabbit and cat in Golgi-Cox and Golgi-Kopsch impregnated brain sections. The present anatomical findings permit a division of the MTN of these species into dorsal and ventral components (MTNd, MTNv), in agreement with other investigations. The MTNd contains predominantly linear-bipolar and linear-multipolar shaped neurons with cell bodies that measure in the range of 25–50 m. These neurons have 2 to 4 primary dendrites which, along with their smaller dendritic branches, are oriented in the plane of the long axis of the MTN (i.e. from ventromedial to dorsolateral). These linear-bipolar and linear-multipolar cells represent 70–80% of the neurons of the MTNd as seen in the Golgi impregnated sections. The remaining 20–30% of the MTNd neurons are nearly all multipolar in shape with somata measuring in the range of 15–25 m. An occasional multipolar neuron is larger, has a soma that measures around 30–60 m and has dendrites which extend outward from the cell body to cover large areas of the MTNd. There was considerable extension of the dendrites of MTNd neurons into the MTNv; however, the dendrites of MTNd neurons were not observed extending into the adjacent substantia nigra (SN) or ventral tegmental area (VTA) of Tsai (1925). Conversely, the dendrites of neurons in the neighboring SN and VTA course along the borders of the MTN but only occasionally extend into the MTN. The neuron population of the MTNv consists almost entirely of small-multipolar shaped cells with somata measuring from 15–25 m and dendritic trees resembling those described for multipolar cells of the MTNd. A small number of neurons of the ventral division are medium-multipolar in shape with cell bodies that measure approximately 30–60 m. Typically, these cells have several dendrites which extend ventrally within the MTNv and one or more dendrites that extend either across the MTNv or dorsally into the MTNd. Only a few linear-bipolar and linear-multipolar neurons were observed in the MTNv. The present findings are discussed in relation to anatomical, physiological, and histochemical studies on the MTN.Abbreviations to Figures CP Cerebral Peduncle - DTN Dorsal Terminal Nucleus - LG Lateral Geniculate Nucleus - LP Lateral Posterior Nucleus - MG Medial Geniculate Nucleus - ML Medial Lemniscus - MTNd Medial Terminal Nucleus, dorsal division - MTNv Medial Terminal Nucleus, ventral division - NTO Nucleus of the Optic Tract - PA Anterior Pretectal Nucleus - pn Nucleus Paranigralis - PP Posterior Pretectal Nucleus - Pul Pulvinar - PO Olivary Pretectal Nucleus - RN Red Nucleus - SGS Stratum Griseum Superficiale, Superior Colliculus - SN Substantia Nigra Supported by USPHS research grant EYO3642 from the National Eye Institute