Determination of Dosage Compensation of the Mammalian X Chromosome by RNA-seq is Dependent on Analytical Approach

Background An enduring question surrounding sex chromosome evolution is whether effective hemizygosity in the heterogametic sex leads inevitably to dosage compensation of sex-linked genes, and whether this compensation has been observed in a variety of organisms. Incongruence in the conclusions reached in some recent reports has been attributed to different high-throughput approaches to transcriptome analysis. However, recent reports each utilizing RNA-seq to gauge X-linked gene expression relative to autosomal gene expression also arrived at diametrically opposed conclusions regarding X chromosome dosage compensation in mammals. Results Here we analyze RNA-seq data from X-monosomic female human and mouse tissues, which are uncomplicated by genes that escape X-inactivation, as well as published RNA-seq data to describe relative X expression (RXE). We find that the determination of RXE is highly dependent upon a variety of computational, statistical and biological assumptions underlying RNA-seq analysis. Parameters implemented in short-read mapping programs, choice of reference genome annotation, expression data distribution, tissue source for RNA and RNA-seq library construction method have profound effects on comparing expression levels across chromosomes. Conclusions Our analysis shows that the high number of paralogous gene families on the mammalian X chromosome relative to autosomes contributes to the ambiguity in RXE calculations, RNA-seq analysis that takes into account that single- and multi-copy genes are compensated differently supports the conclusion that, in many somatic tissues, the mammalian X is up-regulated compared to the autosomes

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Evidence of Co-Transcriptional Regulation at the X-linked Imprinted Locus, X-linked Lymphocyte Regulated 3b

Genomic imprinting is defined as the mono-allelic expression of a gene based on parental origin. Eutherian mammals utilize a differentially methylated region, or DMR as an epigenetic mark to regulate transcription initiation events at all autosomal imprinted loci. However, an X-linked imprinted locus, X-linked lymphocyte regulated 3b, defies the classical mechanism of epigenetic regulation as no DMR has been identified. A recent finding at the Xlr3/4/5 locus shows that imprinted expression is only observed at the 3’ end of the Xlr3b primary transcript suggesting that paternal silencing is accomplished through a co-transcriptional mechanism. This study offers the first investigation into the potential imprinting mechanism by exploring the behavior of RNA Polymerase II (PolII) during transcription elongation. The observation that PolII is perturbed as it transits across the paternal copy of Xlr3b is further supported by H3K36me3 enrichment at the precise location of the stall, suggesting it too may play a role in the imprinting mechanism. The chromatin environment at the locus is not maintained by known allele specific histone modifications and histone methyltransferases that influence proper transcription elongation do not seem to play a role in the imprinting mechanism either. This work establishes a model where maternally biased gene expression is accomplished by severely impeding PolII at the paternal copy of Xlr3b during transcription elongation

Biological management of selected weeds of wheat through co-application of allelopathic rhizobacteria and sorghum extract

Traditional methods of weeds management have caused serious environmental and health concerns. Therefore, development of alternate strategies for effective management of weeds is becoming indispensable for sustainable agriculture. In this study, the comparative effectiveness of chemical and bio-herbicides for the management of weeds in wheat has been assessed under laboratory and field conditions. Two effective allelopathic rhizobacteria (6 K and 6) were selected from initial screening experiment which had abilities to suppress the growth of selective weeds as well as had potential to improve the growth of wheat. Based on 16S ribosomal RNA (16S rRNA) gene sequencing, the selected allelopathic rhizobacteria were identified as Pseudomonas fluorescens strain 6 K and Bacillus sp. strain 6. Further, sorghum allelopathic water extract was also used in combination with selected allelopathic rhizobacteria as a bio-herbicide. Five treatments used for the laboratory and field experiments were control (T1: without herbicide), chemical herbicide (T2: mesosulfuron methyl + idosulfuron methyl; Atlantis® 6WG), sorghum allelopathic extract (T3), consortium of two different allelopathic rhizobacteria (T4) and combined application of allelopathic extract of sorghum and consortium of two allelopathic rhizobacteria (T5 = T3 + T4). Results of laboratory experiment showed that T5 significantly suppressed the seed germination percentage of four selected weeds i.e., Anagallis arvensis L., Phalaris minor Retz., Cynodon dactylon L. and Melilotus indicus L. and the same treatment (T5) also significantly improved seed germination of wheat as compared to all other treatments. Further evaluation under field condition showed that T5 significantly decreased the weed density and total weed biomass at 15, 30 and 45 days after sowing (DAS) of all weeds as compared to T1 (control). Field trial results also indicated that T5 significantly increased the wheat growth traits including the biological yield (73%) and grain yield (53%) as compared to T1. Likewise, the economic analysis revealed that T5 improved the net benefits with a higher marginal rate of return than all other treatments. Our findings indicated that combined application of allelopathic rhizobacterial consortium and allelopathic extract of sorghum remained more effective for controlling weeds and improving the growth and yield traits of wheat as compared to their sole application. Therefore, co-application of allelopathic rhizobacterial consortium and sorghum allelopathic water extract could offer an economically viable lever for the biological management of weeds of wheat for sustainable production

Unique Small RNA Signatures Uncovered in the Tammar Wallaby Genome

Background Small RNAs have proven to be essential regulatory molecules encoded within eukaryotic genomes. These short RNAs participate in a diverse array of cellular processes including gene regulation, chromatin dynamics and genome defense. The tammar wallaby, a marsupial mammal, is a powerful comparative model for studying the evolution of regulatory networks. As part of the genome sequencing initiative for the tammar, we have explored the evolution of each of the major classes of mammalian small RNAs in an Australian marsupial for the first time, including the first genome-scale analysis of the newest class of small RNAs, centromere repeat associated short interacting RNAs (crasiRNAs). Results Using next generation sequencing, we have characterized the major classes of small RNAs, micro (mi) RNAs, piwi interacting (pi) RNAs, and the centromere repeat associated short interacting (crasi) RNAs in the tammar. We examined each of these small RNA classes with respect to the newly assembled tammar wallaby genome for gene and repeat features, salient features that define their canonical sequences, and the constitution of both highly conserved and species-specific members. Using a combination of miRNA hairpin predictions and co-mapping with miRBase entries, we identified a highly conserved cluster of miRNA genes on the X chromosome in the tammar and a total of 94 other predicted miRNA producing genes. Mapping all miRNAs to the tammar genome and comparing target genes among tammar, mouse and human, we identified 163 conserved target genes. An additional nine genes were identified in tammar that do not have an orthologous miRNA target in human and likely represent novel miRNA-regulated genes in the tammar. A survey of the tammar gonadal piRNAs shows that these small RNAs are enriched in retroelements and carry members from both marsupial and tammar-specific repeat classes. Lastly, this study includes the first in-depth analyses of the newly discovered crasiRNAs. These small RNAs are derived largely from centromere-enriched retroelements, including a novel SINE. Conclusions This study encompasses the first analyses of the major classes of small RNAs for the newly completed tammar genome, validates preliminary annotations using deep sequencing and computational approaches, and provides a foundation for future work on tammar-specific as well as conserved, but previously unknown small RNA progenitors and targets identified herein. The characterization of new miRNA target genes and a unique profile for crasiRNAs has allowed for insight into multiple RNA mediated processes in the tammar, including gene regulation, species incompatibilities, centromere and chromosome function

The ectopy-triggering ganglionated plexuses in atrial fibrillation

Background Epicardial ganglionated plexus (GP) have an important role in the pathogenesis of atrial fibrillation (AF). The relationship between anatomical, histological and functional effects of GP is not well known. We previously described atrioventricular (AV) dissociating GP (AVD-GP) locations. In this study, we hypothesised that “ET-GP” are upstream triggers of atrial ectopy/AF and have different anatomical distribution to AVD-GP. Objectives We mapped and characterised ET-GP to understand their neural mechanism in AF and anatomical distribution in the left atrium (LA). Methods 26 patients with paroxysmal AF were recruited. All were paced in the LA with an ablation catheter. HFS (80 ms) was synchronised to each paced stimulus (after 20 ms delay) for delivery within the local atrial refractory period. HFS responses were tagged onto CARTO™ 3D LA geometry. All geometries were transformed onto one reference LA shell. A probability distribution atlas of ET-GP was created. This identified high/low ET-GP probability regions. Results 2302 sites were tested with HFS, identifying 579 (25%) ET-GP. 464 ET-GP were characterised, where 74 (16%) triggered ≥30s AF/AT. Median 97 (IQR 55) sites were tested, identifying 19 (20%) ET-GP per patient. >30% of ET-GP were in the roof, mid-anterior wall, around all PV ostia except in the right inferior PV (RIPV) in the posterior wall. Conclusion ET-GP can be identified by endocardial stimulation and their anatomical distribution, in contrast to AVD-GP, would be more likely to be affected by wide antral circumferential ablation. This may contribute to AF ablation outcomes

His resynchronization versus biventricular pacing in patients with heart failure and left bundle branch block

Background His bundle pacing is a new method for delivering cardiac resynchronization therapy (CRT). Objectives The authors performed a head-to-head, high-precision, acute crossover comparison between His bundle pacing and conventional biventricular CRT, measuring effects on ventricular activation and acute hemodynamic function. Methods Patients with heart failure and left bundle branch block referred for conventional biventricular CRT were recruited. Using noninvasive epicardial electrocardiographic imaging, the authors identified patients in whom His bundle pacing shortened left ventricular activation time. In these patients, the authors compared the hemodynamic effects of His bundle pacing against biventricular pacing using a high-multiple repeated alternation protocol to minimize the effect of noise, as well as comparing effects on ventricular activation. Results In 18 of 23 patients, left ventricular activation time was significantly shortened by His bundle pacing. Seventeen patients had a complete electromechanical dataset. In them, His bundle pacing was more effective at delivering ventricular resynchronization than biventricular pacing: greater reduction in QRS duration (−18.6 ms; 95% confidence interval [CI]: −31.6 to −5.7 ms; p = 0.007), left ventricular activation time (−26 ms; 95% CI: −41 to −21 ms; p = 0.002), and left ventricular dyssynchrony index (−11.2 ms; 95% CI: −16.8 to −5.6 ms; p < 0.001). His bundle pacing also produced a greater acute hemodynamic response (4.6 mm Hg; 95% CI: 0.2 to 9.1 mm Hg; p = 0.04). The incremental activation time reduction with His bundle pacing over biventricular pacing correlated with the incremental hemodynamic improvement with His bundle pacing over biventricular pacing (R = 0.70; p = 0.04). Conclusions His resynchronization delivers better ventricular resynchronization, and greater improvement in hemodynamic parameters, than biventricular pacing

Visualizing Localized Reentry With Ultra-High Density Mapping in Iatrogenic Atrial Tachycardia Beware Pseudo-Reentry

Background—The activation pattern of localized reentry (LR) in atrial tachycardia remains incompletely understood. We used the ultra–high density Rhythmia mapping system to study activation patterns in LR. Methods and Results—LR was suggested by small rotatory activations (carousels) containing the full spectrum of the color-coded map. Twenty-three left-sided atrial tachycardias were mapped in 15 patients (age: 64±11 years). 16 253±9192 points were displayed per map, collected over 26±14 minutes. A total of 50 carousels were identified (median 2; quartiles 1–3 per map), although this represented LR in only n=7 out of 50 (14%): here, rotation occurred around a small area of scar (<0.03 mV; 12±6 mm diameter). In LR, electrograms along the carousel encompassed the full tachycardia cycle length, and surrounding activation moved away from the carousel in all directions. Ablating fractionated electrograms (117±18 ms; 44±13% of tachycardia cycle length) within the carousel interrupted the tachycardia in every LR case. All remaining carousels were pseudo-reentrant (n=43/50 [86%]) occurring in areas of wavefront collision (n=21; median 0.5; quartiles 0–2 per map) or as artifact because of annotation of noise or interpolation in areas of incomplete mapping (n=22; median 1, quartiles 0–2 per map). Pseudo-reentrant carousels were incorrectly ablated in 5 cases having been misinterpreted as LR. Conclusions—The activation pattern of LR is of small stable rotational activations (carousels), and this drove 30% (7/23) of our postablation atrial tachycardias. However, this appearance is most often pseudo-reentrant and must be differentiated by interpretation of electrograms in the candidate circuit and activation in the wider surrounding region