Identification of constrained sequence elements across 239 primate genomes.

Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3-9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

Anesthesia for Ambulatory Pediatric Surgery in Sub-Saharan Africa: A Pilot Study in Burkina Faso.

BACKGROUND: Long surgical wait times and limited hospital capacity are common obstacles to surgical care in many countries in Sub-Saharan Africa (SSA). Introducing ambulatory surgery might contribute to a solution to these problems. The purpose of this study was to evaluate the safety and feasibility of introducing ambulatory surgery into a pediatric hospital in SSA. METHODS: This is a cross-sectional descriptive study that took place over 6 months. It includes all patients assigned to undergo ambulatory surgery in the Pediatric University Hospital in Ouagadougou, Burkina Faso. Eligibility criteria for the ambulatory surgery program included >1 year of age, American Society of Anesthesiologists (ASA) 1 status, surgery with a low risk of bleeding, lasting <90 minutes, and with an expectation of mild to moderate postoperative pain. The family had to live within 1 hour of the hospital and be available by telephone. RESULTS: During the study period, a total of 1250 patients underwent surgery, of whom 515 were elective cases; 115 of these met the criteria for ambulatory surgery; 103 patients, with an average age of 59.74 ± 41.57 months, actually underwent surgery. The principal indications for surgery were inguinal (62) and umbilical (47) hernias. All patients had general anesthesia with halothane. Sixty-five percent also received regional or local anesthesia consisting of caudal block in 79.23% or nerve block in 20.77%. The average duration of surgery was 33 ± 17.47 minutes. No intraoperative complications were noted. All the patients received acetaminophen and a nonsteroidal anti-inflammatory drug in the recovery room. Twelve (11.7%) patients had complications in recovery, principally nausea and vomiting. Eight (7.8%) patients were admitted to the hospital. CONCLUSIONS: No serious complications were associated with ambulatory surgery. Its introduction could possibly be a solution to improving pediatric surgical access in low-income countries.

Arrhythmia and thyroid dysfunction.

CONTEXT: Arrhythmia is a major cause of morbidity and mortality in Europe and in the United States. The aim of this review article was to assess the results of the prospective studies that evaluated the risk of arrhythmia in patients with overt and subclinical thyroid disease and discuss the management of this arrhythmia. EVIDENCE ACQUISITION: A literature search was carried out for reports published with the following terms: thyroid, hypothyroidism, hyperthyroidism, subclinical hyperthyroidism, subclinical hypothyroidism, levothyroxine, triiodothyronine, antithyroid drugs, radioiodine, deiodinase, atrial flutter, supraventricular arrhythmia, ventricular arrhythmia, ventricular tachycardia, ventricular fibrillation, torsade de pointes, amiodarone and atrial fibrillation. The investigation was restricted to reports published in English. EVIDENCE ANALYSIS: The outcome of this analysis suggests that patients with untreated overt clinical or subclinical thyroid dysfunction are at increased risk of arrhythmia. Hyperthyroidism increased atrial arrhythmia; however, hypothyroidism increased ventricular arrhythmia. CONCLUSION: The early recognition and effective treatment of thyroid dysfunction in patients with arrhythmia is mandatory because the long-term prognosis of arrhythmia may be improved with the appropriate treatment of thyroid dysfunction.

Bilateral vestibulopathy and disabling vertigo: A CARE case series.

INTRODUCTION: The term bilateral vestibulopathy (BV) was recently defined by the Bárány Society. Loss of otolith function was not included in their criteria. Although spontaneous progression to complete bilateral impairment of vestibular function is expected, it is unlikely that patients with advanced BV will continue to present episodes of intense vertigo. Here, following CARE case report guidelines, we report the case of patients meeting the criteria for BV and still disabled by vertigo. CASE SERIES: Three patients evaluated in our department meeting the Bárány criteria for definite BV but still complaining of disabling rotatory vertigo were included. All underwent clinical and instrumental vestibular examination. The observations are reported. CONCLUSION: In case of BV, the conservation of a stable otolithic reference frame could allow patients to optimize postural strategy. It would be useful to revisit a classification of BV by stages, by introducing an evaluation of otolithic function and postural control for possible subsequent vestibular implantation.

The landscape of tolerated genetic variation in humans and primates.

Personalized genome sequencing has revealed millions of genetic differences between individuals, but our understanding of their clinical relevance remains largely incomplete. To systematically decipher the effects of human genetic variants, we obtained whole genome sequencing data for 809 individuals from 233 primate species, and identified 4.3 million common protein-altering variants with orthologs in human. We show that these variants can be inferred to have non-deleterious effects in human based on their presence at high allele frequencies in other primate populations. We use this resource to classify 6% of all possible human protein-altering variants as likely benign and impute the pathogenicity of the remaining 94% of variants with deep learning, achieving state-of-the-art accuracy for diagnosing pathogenic variants in patients with genetic diseases. One Sentence Summary: Deep learning classifier trained on 4.3 million common primate missense variants predicts variant pathogenicity in humans.

The landscape of tolerated genetic variation in humans and primates.

Personalized genome sequencing has revealed millions of genetic differences between individuals, but our understanding of their clinical relevance remains largely incomplete. To systematically decipher the effects of human genetic variants, we obtained whole-genome sequencing data for 809 individuals from 233 primate species and identified 4.3 million common protein-altering variants with orthologs in humans. We show that these variants can be inferred to have nondeleterious effects in humans based on their presence at high allele frequencies in other primate populations. We use this resource to classify 6% of all possible human protein-altering variants as likely benign and impute the pathogenicity of the remaining 94% of variants with deep learning, achieving state-of-the-art accuracy for diagnosing pathogenic variants in patients with genetic diseases.

Comparison of defibrillation probability of success curves for an endocardial lead configuration with and without an inactive epicardial patch.

OBJECTIVES: This study sought to assess the effect of passive "bystander" epicardial electrodes on defibrillation efficacy. BACKGROUND: We hypothesized that an inactive epicardial patch placed in an area of low potential gradient from an endocardial electrode shock might affect defibrillation efficacy through its effects on the shock field and the underlying potential gradient. METHODS: We studied the effects of an inactive 18-cm2 titanium mesh patch placed on the anterolateral left ventricular epicardium on the 50% probability of successful defibrillation. A biphasic shock with both phases 6 ms in duration was delivered between superior vena cava and right ventricular catheter electrodes 10 s after the electrical induction of ventricular fibrillation. Six dogs underwent an up/down defibrillation protocol randomized with or without the patch on the heart. RESULTS: Mean 50% (+/-) probability point for energy doubled with the conductive patch on the heart, from 8.0 +/- 3.2 to 16.8 +/- 7.0 J (p < 0.01), and leading-edge voltage increased from 334 +/- 64 to 477 +/- 98 V (p < 0.01). Mean 50% probability points for energy and leading-edge voltage were not significantly changed when the procedure was repeated using a nonconductive patch in another six dogs as a control group. In a saline-saturated foam model, measurements from electrodes placed around and under the patch revealed a 72% mean decrease in the potential gradient in the foam under the conductive patch. CONCLUSIONS: A passive defibrillator patch can markedly increase the energy requirements for defibrillation, probably by decreasing the potential gradient under the patch. These results suggest the use of caution when passive electrodes are present, for example, when a patient receives a nonthoracotomy defibrillator system while epicardial electrodes from a previously implanted system are left in place.

Choking under monitoring pressure: being watched by the experimenter reduces executive attention.

Performing more poorly given one's skill level ("choking") is likely in situations that offer an incentive if a certain outcome is achieved (outcome pressure) or when one is being watched by others-especially when one's performance is being evaluated (monitoring pressure). According to the choking literature, outcome pressure is associated with reduced executive control of attention, whereas monitoring pressure is associated with increased, yet counterproductive, attention to skill processes. Here, we show the first evidence that monitoring pressure-being watched by the experimenter-may lead individuals with higher working memory to choke on a classic measure of executive control-just the task effect thought to result from outcome pressure. Not only does this finding help refine our understanding of the processes underlying choking under monitoring pressure, but it also leads to a new look at classic audience effects, with an important implication for experimental psychology.

Influence of parasympathetic dysfunction and hyperinsulinemia on the hemodynamic response to an isometric exercise in non-insulin-dependent diabetic patients.

The handgrip test has long been used as a test for investigating cardiac autonomic neuropathy in diabetic patients. However, the factors involved in the hemodynamic response to the handgrip test have not been thoroughly studied. The aim of this study was to investigate blood pressure (BP) and heart rate (HR) responses to an isometric test in non-insulin-dependent diabetics (NIDDs) and to correlate the results with vagal function evaluated by three standardized tests and with plasma insulin levels. Fifty-five NIDDs, 35 of whom had one to three abnormal parasympathetic tests (PS+), were compared with 10 healthy control subjects. Fasting and postprandial plasma insulin levels were significantly higher in the PS+ than in the PS- patients. Resting HR correlated significantly with log fasting and postprandial insulin. In PS+ NIDDs, resting HR was significantly higher than in PS- patients. Age-matched comparisons also showed that resting systolic BP was significantly higher in PS+ patients than in controls. In PS- patients, the mean acceleration of HR was significantly higher than in the control group from the second to the fifth minute, and the BP response was also higher than in controls. These data suggest that (1) sympathetic response to an isometric exercise is increased in PS- NIDDs; (2) cardiac parasympathetic dysfunction is associated with a more severe insulin resistance; and (3) the subsequent higher plasma insulin level may contribute to the increase in resting HR and BP through sympathetic activation while limiting the hemodynamic response to an isometric exercise through its vasodilative effect.

Real-time three-dimensional intracardiac echocardiography.

Using catheter-mounted 2-D array transducers, we have obtained real-time 3-D intracardiac ultrasound (US) images. We have constructed several transducers with 64 channels inside a 12 French catheter lumen operating at 5 MHz. The transducer configuration may be side-scanning or beveled, with respect to the long axis of the catheter lumen. We have also included six electrodes to acquire simultaneous electrocardiograms. Using an open-chest sheep model, we inserted the catheter into the cardiac chambers to study the utility of in vivo intracardiac 3-D scanning. Images obtained include a cardiac four-chamber view, mitral valve, pulmonic valve, tricuspid valve, interatrial septum, interventricular septum and ventricular volumes. We have also imaged two electrophysiological interventional devices in the right atrium, performed an in vitro ablation study, and viewed the pulmonary veins in vitro.

Efficient electrode spacing for examining spatial organization during ventricular fibrillation.

Spatial organization has been observed during episodes of ventricular fibrillation (VF) by recording epicardial unipolar electrograms on a grid of electrodes. In such studies, the choice of spacing between electrodes is an important decision, affecting the resolution and the size of the domain to be studied. A basic tenet of sampling theory, the Nyquist criterion, states that an electrode spacing smaller than half the smallest significant wavelength is required to capture the important details of a spatially sampled process. In this paper, we suggest a method to choose a practical interelectrode spacing by examining wavenumber power spectra of high-resolution VF data recorded from a square 11 x 11 array of electrodes spaced 0.28 mm apart. The plaque was sutured on the epicardium near the left ventricular apex in seven anesthetized pigs. VF was induced with ac simulation. Unipolar extracellular electrograms were simultaneously recorded from each channel for 2 s after the induction of VF. Each signal was sampled in time at 1000 Hz. Wavenumber power spectra were calculated for 100 ms segments using the zero-delay wavenumber spectrum method, for a total of 140 power spectra. All spectra had dominant peaks at the origin and fell off rapidly with increasing wavenumber (decreasing wavelength). In all the spectra, every wavelength shorter than 1.4 mm contributed insignificant power. Furthermore, in 134 of 140 spectra (96%), insignificant power levels were associated with every wavelength shorter than 2.8 mm. These results suggest that, for unipolar extracellular electrodes, an intersensor spacing on the order of 1 mm is appropriate to study organization during early VF.

Efficacy of signal-averaged electrocardiography in the young orthotopic heart transplant patient to detect allograft rejection.

Endomyocardial biopsy is the gold standard survey for cardiac graft rejection. Signal-averaged electrocardiography (SAECG) identifies slowly conducting, diseased myocardium. We sought to determine whether SAECG is a sensitive, noninvasive transplant surveillance method in the young.Ninety-four SAECGs recorded prior to biopsy in 20 young transplant (OHT) patients and those from 15 healthy age-matched controls (CTL) were analyzed. In the OHT group, 56 no-rejection (NOREJ) (ISHLT grades 0 or 1 A) and 37 acute rejection (REJ) (ISHLT grades IB, 2, and 3A) SAECGs were compared, SAECGs were filtered at 40-255 Hz. Total QRS duration (QRSd), duration of terminal low amplitude of QRS under 40 microV (LAS), and root mean square amplitude of terminal 40 msec of QRS (RMS40) were compared.SAECGs were significantly different in CTL vs NOREJ but not in NOREJ vs REJ: QRSd, 81.7 +/- 8, 107.2 +/- 18.4, and 112.3 +/- 21.6 msec, respectively; LAS, (18 +/- 5.8, 23.6 +/- 10.7, and 27 +/- 14.8 msec, respectively; and RMS40, (169.3 +/- 100.4, 68 +/- 48.8, and 57.5 +/- 45.6 microV, respectively. Children following OHT exhibited significant differences in the SAECG compared to controls. Differences between the NOREJ and REJ groups were negligible. Therefore, SAECG may not be effective in detecting OHT rejection in the young.

A MULTICHANNEL FIBER-OPTIC MAPPING SYSTEM FOR INTRAMURAL RECORDING OF CARDIAC ACTION POTENTIALS.

Measuring cardiac action potentials at many sites within the ventricular wall is important for understanding cardiac arrhythmias; however, recording in the depth of the heart wall presents many difficulties. We have developed a multichannel optical mapping system for recording cardiac action potentials transmurally. Each channel uses a single small-diameter optical fiber to transmit and collect light from the cardiac tissue. Excitation light is supplied by low-power green lasers. Wavelength separation is performed with a dichroic mirror, and fluorescence is detected with a photodiode. We have recorded action potentials with an unfiltered signal-to-noise ratio (SNR) as high as 60:1 and a temporally filtered SNR as high as 200:1. The collection of fluorescence is optimized so that low excitation light intensity can be used, which increases the available recording time. Channels are modular and compact, and the system can be easily expanded to include additional channels, ratiometry or dual-dye mapping. In addition, the system is highly flexible and can be used for virtually any experiment from single cell recording to surface and transmural mapping of the whole heart.

Chronic asymptomatic contralateral wrist scapholunate dissociation.

BACKGROUND: The authors report a series of six patients who presented with scapholunate dissociation, with no significant contralateral antecedent trauma and no rheumatoid arthritis or congenital ligamentous laxity. Such patients may present with pain or a snapping/popping sensation in one wrist, undergo radiographs of both wrists, and are discovered to have scapholunate gapping bilaterally. The literature contains few reports of this condition, and this series of six is a relatively large one for this infrequently reported condition. METHODS: The six charts were reviewed retrospectively; each patient was asked to return for follow-up and radiographic examination and each participated in a telephone questionnaire about pain, activity changes, new treatments, and exacerbation of wrist problems. The average follow-up was 39 months, with a range of 6 months to 13.5 years. RESULTS: One patient with severe unilateral instability and persistent pain underwent soft-tissue surgical repair (Blatt reconstruction); another demonstrated unilateral dorsal intercalated segment instability with moderate pain symptoms but declined surgical reconstruction. The other 10 wrists, despite radiographically demonstrated widened scapholunate angles and rotatory subluxation of the scaphoid, had mild or no pain and no dorsal intercalated segment instability deformity. CONCLUSIONS: The evolution of the bilateral form of scapholunate dissociation seems to be benign unless dorsal intercalated segment instability deformity is present, which may then rapidly progress to degenerative arthritis and scapholunate advanced collapse wrist. Severe or minor repetitive trauma, inflammation, infection, tumors, and congenital ligamentous laxity have been etiologically implicated in scapholunate dissociation. The natural history of scapholunate dissociation involves volar rotation of the scaphoid and dorsal rotation of the lunate, progressing to malalignment and eventual arthrosis between the scaphoid and radius, the capitate and lunate, and the lunate and hamate bones.

Marginal adaptation of class II resin composite restorations using incremental and bulk placement techniques: an ESEM study.

This in vitro study compared marginal gap formation in class II resin composite restorations. Forty caries-free extracted molars were prepared in a standardized manner for class II restoration by one of four methods: bulk- or incrementally-placed light-activated resin composite (Amelogen), and bulk- or incrementally-placed chemically activated composite (Rapidfill). The restored teeth, after finishing and polishing, and thermocycling, were examined using environmental scanning electron microscopy. Marginal gap measurements at predetermined facial and lingual margin sites showed no significant differences between the two sites within any of the groups. Both the light- and the chemically-activated restorations showed no significant differences in mean marginal gap sizes whether they were placed by incremental or bulk techniques. Amelogen restorations placed by both methods had significantly larger margin gaps than those of each of the Rapidfill groups (P<0.05). Thus, although method of placement of a given material had no significant effect on the quality of marginal adaptation, both of the chemically activated resin composite restorations produced significantly smaller marginal gaps than both the bulk- and incrementally-placed light-activated composites.

Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene.

The transformer (tra) gene regulates female somatic sexual differentiation and has no known function in males. It gives rise to two sizes of RNA, one non-sex-specific and one female-specific. These two RNAs are shown to be present throughout the life cycle, and related by the use of alternative first intron splice acceptor sites. The non-sex-specific RNA has a 73 base first intron, while that in the female-specific RNA is 248 bases. The non-sex-specific RNA has no long open reading frame, while the female-specific RNA has a single long open reading frame beginning at the first AUG. Substitution of a heat shock promoter for the tra promoter still leads to female-specific differentiation of otherwise tra-females. We suggest a mechanism by which Sex-lethal controls itself and tra.

Studies on cytosolic guanylate cyclase from human placenta.

We have purified the soluble form of guanylate cyclase from human placenta greater than 2400-fold. The enzyme shared several characteristics with the enzyme purified from other sources including molecular mass and subunit composition, activation by divalent cations, inhibition by ATP and Michaelis constants. The enzyme, however, had a lower absorption maximum in the Soret region (417 +/- 1 nm) than the enzyme from other sources and was activated only one-fifth as much by nitric oxide as the bovine lung enzyme. It appears that the heme prosthetic group in the human placental enzyme may be hexa-coordinate and in the bovine lung enzyme the heme group may be penta-coordinate.

The effect of cardiac compression on defibrillation efficacy and the upper limit of vulnerability.

INTRODUCTION: We determined the effects of decreasing the ventricular blood volume and altering cardiac geometry on defibrillation, the upper limit of vulnerability (ULV), and the relationship between them. METHODS AND RESULTS: In six pigs, fibrillation/defibrillation trials were performed with a left ventricular apex patch to a superior vena cava catheter electrode configuration and a biphasic waveform. Thirty trials each were performed on a compressed versus noncompressed (normal) heart. Compression was achieved using direct mechanical ventricular actuation. Dose-response curves were constructed, and the 50% probability points (ED50) were compared for leading edge voltage (LEV), leading edge current (LEI), and total energy (TE). In another 12 pigs, triplicate defibrillation thresholds (DFTs) and ULVs were determined for each heart state. The T wave was scanned with shocks in 10-msec steps for determining the ULV. Compression resulted in decreased ED50s for LEV (delta = 138 +/- 77 V, P < 0.05, mean +/- SD), LEI (delta = 1.57 +/- 0.7 A, P < 0.05), and TE (delta = 4.9 +/- 3.6 J, P < 0.05) compared to normal. In the second study, compression significantly reduced DFT (P < 0.02) and ULV (P < 0.02) for LEV, LEI, and TE compared to normal. The ULV tended to be lower than the DFT for the normal heart state (delta = 23 +/- 46 V LEV: P = NS). However, the ULV was significantly greater than the DFT for the compressed heart state (delta = 19 +/- 25 V LEV; P < 0.03). CONCLUSIONS: Shock delivery during cardiac compression improves defibrillation efficacy. Additionally, cardiac compression decreases both DFT and ULV, which supports the ULV hypothesis of defibrillation. Finally, maintaining the heart's geometric and volumetric state during ULV testing in paced rhythm and DFT testing in ventricular fibrillation moves the ULV higher than the DFT-the position predicted by the ULV hypothesis for defibrillation.

Nitric oxide and cGMP analogs activate transcription from AP-1-responsive promoters in mammalian cells.

Nitric oxide (NO) increases cytosolic guanylate cyclase activity and thereby activates the cGMP signal transduction pathway. The cAMP and Ca2+/phospholipid signal transduction pathways activate transcription factors that bind to the cAMP response element (CRE) and phorbol ester response element (TRE), respectively. Little is known about transcriptional regulation of gene expression by NO/cGMP. In transient and stable transfection experiments and in microinjection studies we found that three different NO-releasing agents and two membrane-permeable cGMP analogs activated TRE-regulated but not CRE-regulated reporter genes in rodent fibroblast and epithelial cell lines. Activation of TRE-regulated genes by NO-releasing agents and cGMP analogs appeared to be mediated by the AP-1 (Jun/Fos) transcription factor complex because we observed increased DNA binding of AP-1 and increased junB and c-fos mRNA in cells treated with these agents. The mechanism of gene activation by NO/cGMP was distinct from that used by phorbol esters and cAMP because it was not associated with c-jun mRNA induction and was not observed with CRE-containing promoters.