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We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a . These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL + transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice., Competing Interests: AA, JD, KG, ZS, CR, ZH, BL, DO, JL, VI, LE, GG, GI, SY, HT No competing interests declared, (© 2024, Araujo, Dekker et al.)

Background: Published guidelines provide recommendations for risk stratification in pediatric Wolff-Parkinson-White (WPW). There are no data on provider concordance with these guidelines. We hypothesized that significant practice variation exists between pediatric cardiologists (PC) and electrophysiologists (EP)., Method: The records of all patients, age 8 to 21 years, with a new ECG diagnosis of WPW between 1/1/2013 and 12/31/2018, from a single center, were retrospectively reviewed. Subjects were categorized on the basis of symptoms and resting ECG findings as one of the following: asymptomatic intermittent WPW, asymptomatic persistent WPW, or symptomatic WPW. The performance and results of diagnostic testing, including Holter monitor, event monitor, exercise stress test (EST), and electrophysiology study (EPS), were recorded. The primary outcome was concordance with published guidelines. A secondary outcome was documentation of a discussion of sudden cardiac death (SCD) risk., Results: 615 patient encounters were analyzed in 231 patients with newly diagnosed WPW pattern on ECG (56% male; mean age at diagnosis 13.9 ± 2.5 years). EP were observed to have a significantly higher rate of guideline concordance than PC (95% vs. 71%, p < 0.001). There was significant practice variation between PC and EP in the documentation of a discussion of SCD risk: 96% in EP vs. 39% in PC (p < 0.001)., Conclusion: Significant practice variation exists in the non-invasive and invasive risk stratification of pediatric WPW, with lower concordance to published guidelines amongst PC, when compared to EP. This report highlights the need to promote awareness of current WPW guidelines in the pediatric cardiology community at large., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

One Health concept recognizes the inextricable interactions of diverse ecosystems and their subsequent effect on human, animal and plant health. Antimicrobial resistance (AMR) is a major One Health concern and is predicted to cause catastrophes if appropriate measures are not implemented. To understand the AMR landscape in a south Indian metropolitan city, metagenomic analysis of open drains was performed. The data suggests that in January 2022, macrolide class of antibiotics contributed the highest resistance of 40.1% in the city, followed by aminoglycoside- 24.4%, tetracycline- 11.3% and lincosamide- 6.7%. The 'mutations in the 23S rRNA gene conferring resistance to macrolide antibiotics' were the major contributor of resistance with a prevalence of 39.7%, followed by '16s rRNA with mutation conferring resistance to aminoglycoside antibiotics'- 22.2%, '16S rRNA with mutation conferring resistance to tetracycline derivatives'- 9.2%, and '23S rRNA with mutation conferring resistance to lincosamide antibiotics'- 6.7%. The most prevalent antimicrobial resistance gene (ARG) 'mutations in the 23S rRNA gene conferring resistance to macrolide antibiotics' was present in multiple pathogens including Escherichia coli, Campylobacter jejuni, Acinetobacter baumannii, Streptococcus pneumoniae, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Klebsiella pneumoniae and Helicobacter pylori. Most of the geographical locations in the city showed a similar landscape for AMR. Considering human mobility and anthropogenic activities, such an AMR landscape could be common across other regions too. The data indicates that pathogens are evolving and acquiring antibiotic resistance genes to evade antibiotics of multiple major drug classes in diverse hosts. The outcomes of the study are relevant not only in understanding the resistance landscape at a broader level but are also important for identifying the resistant drug classes, the mechanisms of gaining resistance and for developing new drugs that target specific pathways. This kind of surveillance protocol can be extended to regions in other developing countries to assess and combat the problem of antimicrobial resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Spinal muscular atrophy (SMA) is a neuromuscular, rare genetic disorder caused due to loss-of-function mutations in the survival motor neuron-1 ( SMN1 ) gene, leading to deficiency of the SMN protein. The severity of the disease phenotype is inversely proportional to the copy number of another gene, SMN2 , that differs from SMN1 by a few nucleotides. The current diagnostic methods for SMA include symptom-based diagnosis, biochemical methods like detection of serum creatine kinase, and molecular detection of disease-causing mutations using polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA), and exome or next-generation sequencing (NGS). Along with detection of the disease-causing mutation in the SMN1 gene, it is crucial to identify the copy number of the SMN2 gene, which is a disease modifier. Therapeutic options like gene therapy, antisense therapy, and small molecules are available for SMA, but, the costs are prohibitively high. This review discusses the prevalence, diagnosis, available therapeutic options for SMA, and their clinical trials in the Indian context, and highlights the need for measures to make indigenous diagnostic and therapeutic interventions.

The medical emergency of COVID-19 brought to the forefront mRNA vaccine technology where the mRNA vaccine candidates mRNA-1273 and BNT162b2 displayed superlative and more than 90% efficacy in protecting against SARS-CoV2 infections. Rare genetic disorders are rare individually, but collectively they are common and represent a medical emergency. In mRNA biotherapeutic technology, administration of a therapeutic protein-encoding mRNA-nanoparticle formulation allows for in vivo production of therapeutic proteins to functionally complement the protein functions lacking in rare disease patients. The platform nature of mRNA biotherapeutic technology propels rare disease drug discovery and, owing to the scalable and synthetic nature of mRNA manufacturing, empowers parallel product development using a universal production pipeline. This review focuses on the advantages of mRNA biotherapeutic technology over current therapies for rare diseases and provides summaries for the proof-of-concept preclinical studies performed to demonstrate the potential of mRNA biotherapeutic technology. Apart from preclinical studies, this review also spotlights the clinical trials currently being conducted for mRNA biotherapeutic candidates. Currently, seven mRNA biotherapeutic candidates have entered clinical trials for rare diseases, and of them, 3 candidates entered in the year 2023 alone. The rapid pace of clinical development promises a future where, as with mRNA vaccines for COVID-19, mRNA biotherapeutic technology would combat an emergency of rare genetic disorders.

Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STB bypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: The present study compares the treatment outcomes of only culprit vessel PCI and complete revascularization in patients with STEMI and multivessel disease (MVD) following thrombolysis., Methods: This was a single-center, prospective randomized study including a total of 108 patients presenting at a tertiary care center within 3-24 h post-thrombolysis and undergoing pharmacoinvasive PCI, and randomized into two groups: complete revascularization PCI group and culprit only PCI group. The primary outcomes were evaluated by cardiac mortality, repeat myocardial infarction (MI)/acute coronary syndrome (ACS) and refractory angina. The secondary outcomes include repeat revascularization and safety outcomes namely contrast induced nephropathy (CIN), cerebrovascular accident (CVA) and major bleeding were compared among both the groups at one year follow-up., Results: Complete revascularization PCI group and culprit only PCI group had 54 patients in each group. Left ventricular ejection fraction did not show significant difference at discharge (p = 1) but was significantly improved in complete revascularization PCI group (p = 0.001) at one year follow-up. Reduced number of outcomes with a significant difference in both the groups were seen for primary outcomes such as cardiac mortality (p = 0.01), repeat MI/ACS (p = 0.01) and refractory angina (p = 0.038) along with repeat revascularization (p = 0.001) at one year follow-up. Complete revascularization did not show any statistically significant difference for CIN (p = 0.567), CVA (p = 0.153) and major bleeding (p = 0.322) then culprit only revascularization group., Conclusion: In patients with STEMI and MVD, complete revascularization was found more favourable in terms of primary and secondary outcomes compared to culprit only revascularization., (Copyright © 2023. Published by Elsevier, a division of RELX India, Pvt. Ltd.)

Background: Guidelines for electrophysiology study (EPS) and catheter ablation in Wolff-Parkinson-White (WPW) are age based, but size may be a more relevant factor in determination of outcomes., Objectives: The goal of this study was to evaluate the association of patient weight with outcomes of catheter ablation for pediatric WPW., Methods: A multicenter retrospective cohort study was performed on children aged 1 to 21 years with WPW and first-time EPS from April 2016 to December 2019 recorded in the IMPACT (Improving Pediatric and Adult Congenital Treatment) registry, excluding those with congenital heart disease, cardiomyopathy, and >1 ablation target. A weight threshold of 30 kg was selected, representing 1 SD below the cohort mean. The primary outcome was major adverse events (MAEs); additional outcomes included deferred ablation, use of cryoablation, and ablation success., Results: A total of 4,456 subjects from 84 centers were evaluated, with 14% weighing <30 kg. Subjects weighing <30 kg were more likely to have preprocedural supraventricular tachycardia (45% vs 29%; P < 0.001) and less likely to have right septal accessory pathways (25% vs 33%; P < 0.001). MAEs were rare, although with higher incidence in the <30 kg cohort (0.3% vs 0.05%; P = 0.04). No difference was seen in likelihood of deferred ablation (9% vs 12%; P = 0.07) or use of cryoablation (11% vs 11%; P = 0.70). Success was higher in the <30 kg cohort: 95% vs 92% (P = 0.009). This effect persisted after adjusting for covariates (odds ratio: 1.6; 95% CI: 1.01-2.70; P = 0.046)., Conclusions: Weight <30 kg was associated with a small but elevated risk of MAEs. Rates of deferred ablation and cryoablation were similar. Adjusting for factors (including accessory pathway type and location), weight <30 kg remained an independent predictor of acute success., Competing Interests: Funding Support and Author Disclosures The specific analysis described in this paper was funded by the American College of Cardiology and the NCDR. The proposed project and paper were reviewed by the IMPACT Research and Publications Committee. The funding agencies had no role in the drafting of the manuscript or influencing its content. The views expressed in this paper represent those of the author(s), and do not necessarily represent the official views of the NCDR or its associated professional societies identified at CVQuality.ACC.org/NCDR. The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Sinus of valsalva aneurysm (SVA) with rupture is a rare cardiac anomaly which can be congenital or acquired with reported incidence of 0.46%-3.57% among Asians population., Aim of the Study: The aim of this study is to analyze 30 years of single institutional surgical experience in management of 216 cases with SVAs from 1992 till date., Methods: Age group was from 6 to 64 years (mean: 32.5 ± 11 years) with male to female ratio of 2.2:1. The aneurysms originated from right coronary sinus in 181 cases (83.79%), noncoronary sinus in 35 cases (15.74%) and ruptured into the right ventricle in 149 cases (68.98%), right atrium in 59 cases (27.31%). Bicameral approach was used in majority of the cases (n = 213, 98.61%). Aneurysms were repaired using Dacron patch in 173 cases (80.09%) and direct closure in 43 cases (19.9%). Associated ventricular septal defect was closed with Dacron patch in 123 cases (56.94%). Aortic valve was replaced in 21 cases (9.72%) and aortic valve repair was performed in 14 cases (6.48%) for associated Aortic regurgitation., Results: There were no perioperative hospital deaths. Follow-up was available in 204 patients (94.44%) ranging from 2 to 26 years (mean: 10 ± 5.6 years). Two deaths (0.92%) occurred during the postoperative follow-up period. The actual survival was 99.5% at 1 year, 99% at 5 and 10 years., Conclusion: Long term results of surgically repaired SVAs are good with low morbidity (3.24%) and mortality (0.92%) even when associated with major cardiac anomalies. Aortic valve repair and replacement both are equally feasible alternatives for management of moderate to severe aortic regurgitation with associated merits and demerits., (© 2022 Wiley Periodicals LLC.)

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene ( CASQ2 ), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2 -K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known., Methods: A K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators., Results: K180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness., Conclusions: CASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

Background Insertable cardiac monitors (ICMs) are effective in the detection of paroxysmal arrhythmias. In 2014, the first miniaturized ICM was introduced with a less invasive implant technique. The impact of this technology on ICM use in pediatric patients has not been evaluated. We hypothesized an increase in annual pediatric ICM implants starting in 2014 attributable to device miniaturization. Methods and Results A retrospective observational study was conducted using administrative claims from MarketScan Medicaid and commercial insurance claims databases. Use of ICM between January 2013 and December 2018 was measured (normalized to the total enrolled population ≤18 years) and compared with balancing measures (Holter ambulatory monitors, cardiac event monitors, encounters with syncope diagnosis, implantation of implantable cardioverter-defibrillator/pacemaker). Secondary analyses included evaluations of subsequent interventions and complications. The study cohort included 33 532 185 individual subjects, of which 769 (0.002%) underwent ICM implantation. Subjects who underwent ICM implantation were 52% male sex, with a median age of 16 years (interquartile range, 10-17 years). A history of syncope was present in 71%, palpitations in 43%, and congenital heart disease in 28%. Following release of the miniaturized ICM, use of ICMs increased from 5 procedures per million enrollees in 2013 to 11 per million between 2015 and 2018 ( P <0.001), while balancing measures remained static. Of 394 subjects with ≥1 year of follow-up after implantation, interventions included catheter ablation in 24 (6%), pacemaker implantation in 15 (4%), and implantable cardioverter-defibrillator implantation in 7 (2%). Conclusions Introduction of the miniaturized ICM was followed by a rapid increase in pediatric use. The effects on outcomes and value deserve further attention.

Behavioral phenotyping has been gaining prominence due to the increased use of transgenic animal models of neurological disorders. Repeated testing in the same cohort of animals can reduce the overall number of animals used and is desired especially when animal numbers are difficult to obtain as well as for studies involving within-subject design such as drug treatments or aging. This review aims to provide researchers with a comprehensive overview of the carryover effects when subjecting the same set of animals to the same behavioral test. We have focused on three behavioral domains of testing: anxiety, cognition and depression. Based on a review of the literature and our own experiences as a neurobehavioral core facility, we have found that manipulating inter-test interval, environmental contextual cues and stimuli can mitigate the carryover effects to a large extent, although there are certain tests that still show strong residual effects. In addition, the effects of strain on carryover effects from repeated testing are also discussed in this review., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Malignant brain tumors are among the most intractable cancers, including malignancies such as glioblastoma, diffuse midline glioma, medulloblastoma, and ependymoma. Unfortunately, treatment options for these brain tumors have been inadequate and complex, leading to poor prognoses and creating a need for new treatment modalities. Aberrant epigenetics define these types of tumors, with underlying changes in DNA methylation, histone modifications, chromatin structure and noncoding RNAs. Epigenetic-targeted therapies are an alternative that have the potential to reverse the epigenetic deregulation underpinning brain malignancies. Various drugs targeting epigenetic regulators have shown promise in preclinical and clinical testing. In this review, we highlight some of the recent emerging epigenetic targeted therapies for brain tumors being evaluated in the discovery phase and in clinical trials., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Elevated levels of circulating immune complexes are associated with autoimmunity and with worse prognoses in cancer. Here, we examined the effects of well-defined, soluble immune complexes (ICs) on human peripheral T cells. We demonstrate that IgG-ICs inhibit the proliferation and differentiation of a subset of naïve T cells but stimulate the division of another naïve-like T cell subset. Phenotypic analysis by multi-parameter flow cytometry and RNA-Seq were used to characterize the inhibited and stimulated T cells revealing that the inhibited subset presented immature features resembling those of recent thymic emigrants and non-activated naïve T cells, whereas the stimulated subset exhibited transcriptional features indicative of a more differentiated, early memory progenitor with a naïve-like phenotype. Furthermore, we show that while IgG1-ICs do not profoundly inhibit the proliferation of memory T cells, IgG1-ICs suppress the production of granzyme-β and perforin in cytotoxic memory T cells. Our findings reveal how ICs can link humoral immunity and T cell function., Competing Interests: WC is employed by Juno Therapeutics a Bristol-Myers Squibb Company; HS is employed by Genentech; and MD is employed by Incyte Corporation. None of the work reported here is supported by or affiliated with these organizations. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Charab, Rosenberger, Shivram, Mirazee, Donkor, Shekhar, Gjuka, Khoo, Kim, Iyer and Georgiou.)

Chronic cerebral hypoperfusion is associated with vascular dementia (VaD). Cerebral hypoperfusion may initiate complex molecular and cellular inflammatory pathways that contribute to long-term cognitive impairment and memory loss. Here we used a bilateral common carotid artery stenosis (BCAS) mouse model of VaD to investigate its effect on the innate immune response-particularly the inflammasome signaling pathway. Comprehensive analyses revealed that chronic cerebral hypoperfusion induces a complex temporal expression and activation of inflammasome components and their downstream products (IL-1β and IL-18) in different brain regions, and promotes activation of apoptotic and pyroptotic cell death pathways. Polarized glial-cell activation, white-matter lesion formation and hippocampal neuronal loss also occurred in a spatiotemporal manner. Moreover, in AIM2 knockout mice we observed attenuated inflammasome-mediated production of proinflammatory cytokines, apoptosis, and pyroptosis, as well as resistance to chronic microglial activation, myelin breakdown, hippocampal neuronal loss, and behavioral and cognitive deficits following BCAS. Hence, we have demonstrated that activation of the AIM2 inflammasome substantially contributes to the pathophysiology of chronic cerebral hypoperfusion-induced brain injury and may therefore represent a promising therapeutic target for attenuating cognitive impairment in VaD., (© 2020. The Author(s), under exclusive licence to Springer Nature Limited.)

[Figure: see text].

The incorporation of histone variants into nucleosomes has important functional consequences in all aspects of eukaryotic chromatin biology. H2A.Z is a conserved histone variant found in all eukaryotes from yeast to mammals. Recent studies in yeast have shed light on the questions of where and how nucleosomes containing this variant are situated at promoters and in relation to genes, and what its specificity implies with regard to transcription. In yeast, H2A.Z appears to be primarily incorporated into the first nucleosome in the direction of transcription initiation, either of an mRNA transcript or a divergently transcribed upstream antisense non-coding RNA. This specificity of H2A.Z is due in part to the localization at promoters of SWR1, the ATP-dependent chromatin remodeler that incorporates H2A.Z into nucleosomes. Replacement of H2A.Z with canonical H2A is dependent on the function of the transcription pre-initiation complex. The recent studies summarized in this review reveal that the directionality of H2A.Z occupancy in relation to transcription thus reflects a balance of incorporation and eviction activities, which likely have varying contributions at distinct sets of genes across the genome.

Background: Low tube potential-high tube current computed tomography (CT) imaging allows reduction in iodine-based contrast dose and may extend the benefit of routine contrast-enhanced CT exams to patients at risk of nephrotoxicity., Purpose: To determine the ability of an iodine contrast reduction algorithm to maintain diagnostic image quality for contrast-enhanced abdominal CT., Material and Methods: CT exams with iodine contrast reduction were prescribed for patients at risk for renal dysfunction. The iodine contrast reduction algorithm combines weight-based contrast volume reduction with patient width-based low tube potential selection and bolus-tracking. Control exams with routine iodine dose were selected based on weight, width, and scan protocol. Three radiologists evaluated image quality and diagnostic confidence using a 4-point scale (<2 acceptable). Another radiologist assessed contrast reduction indications and measured portal vein and liver contrast-to-noise ratios., Results: Forty-six contrast reduction algorithm and control exams were compared (mean creatinine 1.6 vs. 1.2 mg/dL, P  ≤   0.0001). Thirty-nine contrast reduction patients had an eGFR <60 mL/min/1.73m 2 and 15 had single or transplanted kidney. Mean iodine contrast dose was lower in the contrast reduction group (20.9 vs. 39.4 g/mL, P  <   0.0001). Diagnostic confidence was rated as acceptable in 95% (131/138) of contrast reduction and 100% of control exams (1.18-1.28 vs. 1.02-1.13, respectively; P  >   0.06). Liver attenuation and contrast-to-noise ratio (CNR) were similar ( P  =   0.08), but portal vein attenuation and CNR were lower with contrast-reduction (mean 176 vs. 198 HU, P  =   0.02; 13 vs. 16, P  =   0.0002)., Conclusion: This size-based contrast reduction algorithm using low kV and bolus tracking reduced iodine contrast dose by 50%, while achieving acceptable image quality in 95% of exams.

Transcription start sites (TSS) in eukaryotes are characterized by a nucleosome-depleted region (NDR), which appears to be flanked upstream and downstream by strongly positioned nucleosomes incorporating the histone variant H2A.Z. H2A.Z associates with both active and repressed TSS and is important for priming genes for rapid transcriptional activation. However, the determinants of H2A.Z occupancy at specific nucleosomes and its relationship to transcription initiation remain unclear. To further elucidate the specificity of H2A.Z, we determined its genomic localization at single nucleosome resolution, as well as the localization of its chromatin remodelers Swr1 and Ino80. By analyzing H2A.Z occupancy in conjunction with RNA expression data that captures promoter-derived antisense initiation, we find that H2A.Z's bimodal incorporation on either side of the NDR is not a general feature of TSS, but is specifically a marker for bidirectional transcription, such that the upstream flanking -1 H2A.Z-containing nucleosome is more appropriately considered as a +1 H2A.Z nucleosome for antisense transcription. The localization of H2A.Z almost exclusively at the +1 nucleosome suggests that a transcription-initiation dependent process could contribute to its specific incorporation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

13- Cis -retinoic acid (RA) is typically used in postremission maintenance therapy in patients with neuroblastoma. However, side effects and recurrence are often observed. We investigated the use of miRNAs as a strategy to replace RA as promoters of differentiation. miR-124 was identified as the top candidate in a functional screen. Genomic target analysis indicated that repression of a network of transcription factors (TF) could be mediating most of miR-124's effect in driving differentiation. To advance miR-124 mimic use in therapy and better define its mechanism of action, a high-throughput siRNA morphologic screen focusing on its TF targets was conducted and ELF4 was identified as a leading candidate for miR-124 repression. By altering its expression levels, we showed that ELF4 maintains neuroblastoma in an undifferentiated state and promotes proliferation. Moreover, ELF4 transgenic expression was able to counteract the neurogenic effect of miR-124 in neuroblastoma cells. With RNA sequencing, we established the main role of ELF4 to be regulation of cell-cycle progression, specifically through the DREAM complex. Interestingly, several cell-cycle genes activated by ELF4 are repressed by miR-124, suggesting that they might form a TF-miRNA regulatory loop. Finally, we showed that high ELF4 expression is often observed in neuroblastomas and is associated with poor survival. IMPLICATIONS: miR-124 induces neuroblastoma differentiation partially through the downregulation of TF ELF4 , which drives neuroblastoma proliferation and its undifferentiated phenotype., (©2019 American Association for Cancer Research.)

OBJECTIVE. The purpose of this study was to evaluate the prevalence and severity of pain reported during image-guided percutaneous biopsies and to identify factors associated with increased reported pain. MATERIALS AND METHODS. In this retrospective study, a database of adult patients who underwent CT- or ultrasound-guided percutaneous core needle biopsy between July 22, 2013, and February 1, 2018, was reviewed. Data collected included patient age and sex, biopsy site, biopsy type (lesion or parenchymal), needle gauge, number of passes, use of sedation, and whether it was the patient's first recorded biopsy. The maximum procedure-related pain reported on a 0-10 numeric rating scale was recorded. Multivariable logistic regression with generalized estimating equations was used to assess the association between covariates and patient-reported pain. RESULTS. A total of 13,344 biopsy procedures were performed in 10,474 patients. Patients reported no pain (0 of 10 scale) during 9765 (73.2%) procedures. Female sex, younger age at biopsy, undergoing IV sedation, and larger needle diameter were all associated with increases in patient-reported pain. Biopsies of renal allografts were the least likely to be painful, followed by hepatic allografts. CONCLUSION. Patients typically report mild or no pain from image-guided biopsy performed by radiologists. Younger patients and women report greater pain. This information can assist preprocedural counseling and reassurance of patients and may help them predict procedure-related patient needs.

Objective: To evaluate the ability of pediatric patients with known or suspected inflammatory bowel disease to ingest a new oral distending agent at CT or MR enterography (CTE/MRE), and to determine the impact on small bowel (SB) distension and diagnostic confidence., Materials and Methods: The study design is that of retrospective review of pediatric patients who underwent CTE or MRE from January 2014 to June 2016. Patients ingested low-concentration barium suspension or flavored beverage containing sorbitol and mannitol. The need for nasogastric tube (NGT) administration, amount ingested, emesis, distal extent of contrast, SB distension, terminal ileum (TI) transverse dimension, and diagnostic confidence in TI disease were assessed. Three radiologists each blindly reviewed a subset of the studies., Results: Of the total 591 scans in 504 patients, 316 scans used low-concentration barium suspension and 275 scans flavored beverage. Nearly all consumed the entire amount (97% vs. 96%). Low-concentration barium suspension exams required NGT more often (7% [23/316] vs. 1% [3/275]; p < 0.0003), and tended to have more emesis (3% [9/316] vs. 1% [3/275]; p = 0.13). Diagnostic confidence score was nearly identical (p = 0.94). Qualitative and quantitative analyses showed no difference in SB distension, except for distension of mid-ileum (flavored beverage > low-concentration barium suspension; p = 0.02). Flavored beverage exams demonstrated a slight increase in distal extent of luminal distension (p = 0.02)., Conclusions: A new flavored beverage distends small bowel as well as low-concentration barium suspension, with decreased requirement for NGT insertion and improved distal extent of luminal distension, and without any decrease in diagnostic confidence in the presence or the absence of TI disease.

Polycomb repressive complex 2 (PRC2) is a chromatin binding complex that represses gene expression by methylating histone H3 at K27 to establish repressed chromatin domains. PRC2 can either regulate genes directly through the methyltransferase activity of its component EZH2 or indirectly by regulating other gene regulators. Gene expression analysis of glioblastoma (GBM) cells lacking EZH2 showed that PRC2 regulates hundreds of interferon-stimulated genes (ISGs). We found that PRC2 directly represses several ISGs and also indirectly activates a distinct set of ISGs. Assessment of EZH2 binding proximal to miRNAs showed that PRC2 directly represses miRNAs encoded in the chromosome 14 imprinted DLK1-DIO3 locus. We found that repression of this locus by PRC2 occurs in immortalized GBM-derived cell lines as well as in primary bulk tumors from GBM and anaplastic astrocytoma patients. Through repression of these miRNAs and several other miRNAs, PRC2 activates a set of ISGs that are targeted by these miRNAs. This PRC2-miRNA-ISG network is likely to be important in regulating gene expression programs in GBM., Competing Interests: The authors have declared that no competing interests exist.

Recent advances, specifically cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC), offer advantages compared to the traditional therapeutic approach of systemic chemotherapy in the treatment of peritoneal carcinomatosis from mucinous appendiceal neoplasms (MAN). This review provides an up-to-date, comprehensive summary of the histologic classification of MAN, reviews common imaging findings of mucoceles and pseudomyxoma peritonei, and describes the radiologist's role in the multidisciplinary care team in quantifying disease and in helping select patients for definitive surgery.

Objectives: To assess the impact of participation in an educational presentation on electrocardiogram (ECG) interpretation in children on pediatric practitioners' ability to accurately interpret ECGs., Study Design: Pediatric healthcare providers at a pediatric clinic with >65 000 visits/year were eligible to participate. A 1-hour ECG educational module that provided a systematic approach to ECG interpretation was presented to 8 providers who consented (6 pediatricians, 2 pediatric nurse practitioners). A test on 11 ECGs (normal, normal-variant, and abnormal ECGs) was given before and 2 weeks after the educational module. Outcomes included correct interpretation of each ECG as normal or abnormal and correct identification of specific ECG findings. Data analysis was descriptive and included χ 2 and Student t test., Results: Mean score (SD) for correct interpretation of ECGs as normal or abnormal improved from 35% (48%) (95% CI 25.0-45.4) to 77% (42%) (95% CI 68.3-86.2) after the ECG educational module (P < .001). Mean (SD) pretest score for correct identification in the normal ECG category improved from 45% (50%) (95% CI 28.9-61.1) to 68% (47%) (95% CI 52.3-82.7) (P= .003). In the abnormal ECG category, correct identification improved from 31% (47%) (95% CI 17.6-44.9) to 83% (5%) (95% CI 72.4-94.3) after the module (P < .001)., Conclusions: Education of pediatric practitioners on ECG interpretation significantly improves their ability to distinguish normal from abnormal and to identify specific abnormalities. Limitations included small sample size and short-term follow-up., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Gene expression can be regulated at multiple levels, but it is not known if and how there is broad coordination between regulation at the transcriptional and post-transcriptional levels. Transcription factors and chromatin regulate gene expression transcriptionally, whereas microRNAs (miRNAs) are small regulatory RNAs that function post-transcriptionally. Systematically identifying the post-transcriptional targets of miRNAs and the mechanism of transcriptional regulation of the same targets can shed light on regulatory networks connecting transcriptional and post-transcriptional control. We used individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) for the RNA-induced silencing complex (RISC) component AGO2 and global miRNA depletion to identify genes directly targeted by miRNAs. We found that Polycomb repressive complex 2 (PRC2) and its associated histone mark, H3K27me3, is enriched at hundreds of miRNA-repressed genes. We show that these genes are directly repressed by PRC2 and constitute a significant proportion of direct PRC2 targets. For just over half of the genes corepressed by PRC2 and miRNAs, PRC2 promotes their miRNA-mediated repression by increasing expression of the miRNAs that are likely to target them. miRNAs also repress the remainder of the PRC2 target genes, but independently of PRC2. Thus, miRNAs post-transcriptionally reinforce silencing of PRC2-repressed genes that are inefficiently repressed at the level of chromatin, by either forming a feed-forward regulatory network with PRC2 or repressing them independently of PRC2., (© 2019 Shivram et al.; Published by Cold Spring Harbor Laboratory Press.)

The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Nonspecific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published data sets. We show that mispriming can occur with as little as two bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity. We also provide a computational pipeline that identifies cDNA reads produced from RT mispriming, allowing users to filter them out from any aligned data set. Using this analysis pipeline, we identify thousands of mispriming events in a dozen published data sets from diverse technologies including short RNA-seq, total/mRNA-seq, HITS-CLIP, and GRO-seq. We further show how RT mispriming can lead to misinterpretation of data. In addition to providing a solution to computationally remove RT-misprimed reads, we also propose an experimental solution to completely avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq)., (© 2018 Shivram and Iyer; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

To detect functional somatic mutations in tumor samples, whole-exome sequencing (WES) is often used for its reliability and relative low cost. RNA-seq, while generally used to measure gene expression, can potentially also be used for identification of somatic mutations. However there has been little systematic evaluation of the utility of RNA-seq for identifying somatic mutations. Here, we develop and evaluate a pipeline for processing RNA-seq data from glioblastoma multiforme (GBM) tumors in order to identify somatic mutations. The pipeline entails the use of the STAR aligner 2-pass procedure jointly with MuTect2 from genome analysis toolkit (GATK) to detect somatic variants. Variants identified from RNA-seq data were evaluated by comparison against the COSMIC and dbSNP databases, and also compared to somatic variants identified by exome sequencing. We also estimated the putative functional impact of coding variants in the most frequently mutated genes in GBM. Interestingly, variants identified by RNA-seq alone showed better representation of GBM-related mutations cataloged by COSMIC. RNA-seq-only data substantially outperformed the ability of WES to reveal potentially new somatic mutations in known GBM-related pathways, and allowed us to build a high-quality set of somatic mutations common to exome and RNA-seq calls. Using RNA-seq data in parallel with WES data to detect somatic mutations in cancer genomes can thus broaden the scope of discoveries and lend additional support to somatic variants identified by exome sequencing alone., Competing Interests: The authors declare that they have no competing interests.

Pediatric cardiac tumors are rare and often benign with an incidence of approximately 0.03-0.32% and can be associated with genetic conditions. For example, approximately 3% of individuals with nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome, have a cardiac fibroma. NBCCS is also characterized by lamellar or early calcification of the falx, jaw keratocysts, palmar and/or plantar pits, and a predisposition for basal cell carcinomas. Given the management implications of NBCCS, including appropriate cancer screenings and precautions, prompt identification of affected individuals is critical. We report a case of a 6-year-old female presenting with ventricular tachycardia secondary to cardiac fibroma. After diagnosis of recurrent jaw keratocysts, she was clinically and molecularly diagnosed with NBCCS. Identification of a cardiac fibroma should prompt careful assessment of past medical and family history with consideration of a diagnosis of NBCCS.

Purpose: To identify reproducible CT imaging features of small bowel gastrointestinal stromal tumors (GIST) that are associated with biologic aggressiveness., Methods: Patients with histologically proven small bowel GISTs and CT enterography or abdominopelvic CT were included. Biologic aggressiveness was established based on initial histologic grading (very low risk to malignant), with "malignant" assigned if recurrence or metastases developed subsequently. CT exams were independently evaluated by three gastrointestinal radiologists for tumor size, growth pattern, enhancement, tumor borders, necrosis, calcification, ulceration, multiplicity, internal air or enteric contrast, nodal metastasis, liver metastasis, peritoneal metastasis, ascites, and draining vein size. Inter-observer variability and imaging features associated with high-grade and malignant small bowel GISTs were determined., Results: Of 78 patients with small bowel GISTs, 10/78 (13%) were high grade and 18/78 (23%) were malignant. There was moderate to substantial inter-observer agreement (Kappa > 0.4) for all findings except tumor border, ulceration, and nodal metastases. Tumor size, irregular or invasive tumor border, necrosis, liver metastasis, ascites, and iso-enhancement were associated with high-grade/malignant small bowel GISTs (p < 0.04). Internal air or enteric contrast and peritoneal metastases additionally predicted malignant behavior (p < 0.03). When imaging features predicting malignant small bowel GISTs were absent and size was ≤ 3 cm, 0% (0/16), 5% (1/19), and 5% (1/17) of patients had high grade, and 0% (0/16, 0/19, and 0/17) had malignant tumors for the three readers, respectively., Conclusion: Multiple, reproducibly identified, small bowel GIST imaging features suggest biologic aggressiveness. The absence of these imaging features may identify small tumors that can be followed in asymptomatic or high-risk patients.

Glioblastoma multiforme (GBM) can be clustered by gene expression into four main subtypes associated with prognosis and survival, but enhancers and other gene-regulatory elements have not yet been identified in primary tumors. Here, we profiled six histone modifications and CTCF binding as well as gene expression in primary gliomas and identified chromatin states that define distinct regulatory elements across the tumor genome. Enhancers in mesenchymal and classical tumor subtypes drove gene expression associated with cell migration and invasion, whereas enhancers in proneural tumors controlled genes associated with a less aggressive phenotype in GBM. We identified bivalent domains marked by activating and repressive chromatin modifications. Interestingly, the gene interaction network from common (subtype-independent) bivalent domains was highly enriched for homeobox genes and transcription factors and dominated by SHH and Wnt signaling pathways. This subtype-independent signature of early neural development may be indicative of poised dedifferentiation capacity in glioblastoma and could provide potential targets for therapy. Significance: Enhancers and bivalent domains in glioblastoma are regulated in a subtype-specific manner that resembles gene regulation in glioma stem cells. Cancer Res; 78(10); 2463-74. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Recombination activating gene 1 (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining [V(D)J] segment recombination, an essential process for antigen receptor expression and lymphocyte development. The BCL11A transcription factor is required for B cell and plasmacytoid dendritic cell (pDC) development, but its molecular function(s) in early B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds directly to the RAG1 promoter as well as directly to regulatory regions of transcription factors previously implicated in both B cell and pDC development to activate RAG1 and RAG2 gene transcription in pro- and pre-B cells. We employed BCL11A overexpression with recombination substrates to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination., (Copyright © 2017 American Society for Microbiology.)

Background: Histone acetylation and deacetylation are essential for gene regulation and have been implicated in the regulation of plant hormone responses. Many studies have indicated the role of histone acetylation in ethylene signaling; however, few studies have investigated how ethylene signaling regulates the genomic landscape of chromatin states. Recently, we found that ethylene can specifically elevate histone H3K14 acetylation and the non-canonical histone H3K23 acetylation in etiolated seedlings and the gene activation is positively associated with the elevation of H3K14Ac and H3K23Ac in response to ethylene. To assess the role of H3K9, H3K14, and H3K23 histone modifications in the ethylene response, we examined how ethylene regulates histone acetylation and the transcriptome at global level and in ethylene regulated genes both in wild type (Col-0) and ein2-5 seedlings., Results: Our results revealed that H3K9Ac, H3K14Ac, and H3K23Ac are preferentially enriched around the transcription start sites and are positively correlated with gene expression levels in Col-0 and ein2-5 seedlings both with and without ethylene treatment. In the absence of ethylene, no combinatorial effect of H3K9Ac, H3K14Ac, and H3K23Ac on gene expression was detected. In the presence of ethylene, however, combined enrichment of the three histone acetylation marks was associated with high gene expression levels, and this ethylene-induced change was EIN2 dependent. In addition, we found that ethylene-regulated genes are expressed at medium or high levels, and a group of ethylene regulated genes are marked by either one of H3K9Ac, H3K14Ac or H3K23Ac. In this group of genes, the levels of H3K9Ac were altered by ethylene, but in the absence of ethylene the levels of H3K9Ac and peak breadths are distinguished in up- and down- regulated genes. In the presence of ethylene, the changes in the peak breadths and levels of H3K14Ac and H3K23Ac are required for the alteration of gene expressions., Conclusions: Our study reveals that the plant hormone ethylene induces combinatorial effects of H3K9Ac, K14Ac and K23Ac histone acetylation in gene expression genome widely. Further, for a group of ethylene regulated genes, in the absence of ethylene the levels and the covered breadths of H3K9Ac are the preexist markers for distinguishing up- and down- regulated genes, the change in the peak breadths and levels of H3K14Ac and H3K23Ac are required for the alteration of gene expression in the presence of ethylene.

Background and Purpose: Evaluating chronic sequelae of optic neuritis, such as optic neuropathy with or without optic nerve atrophy, can be challenging on whole brain MRI. This study evaluated the utility of dedicated coronal contrast-enhanced fat-suppressed FLAIR (CE-FS-FLAIR) MR imaging to detect optic neuropathy and optic nerve atrophy., Materials and Methods: Over 4.5 years, a 3 mm coronal CE-FS-FLAIR sequence at 1.5T was added to the routine brain MRIs of 124 consecutive patients, 102 of whom had suspected or known demyelinating disease. Retrospective record reviews confirmed that 28 of these 102 had documented onset of optic neuritis >4 weeks prior to the brain MRI. These 28 were compared to the other 22 ("controls") of the 124 patients who lacked a history of demyelinating disease or visual symptoms. Using coronal CE-FS-FLAIR, two neuroradiologists separately graded each optic nerve (n = 50 patients, 100 total nerves) as either negative, equivocal, or positive for optic neuropathy or atrophy. The scoring was later repeated., Results: The mean time from acute optic neuritis onset to MRI was 4.1 ± 4.6 years (range 34 days-17.4 years). Per individual nerve grading, the range of sensitivity, specificity, and accuracy of coronal CE-FS-FLAIR in detecting optic neuropathy was 71.4-77.1%, 93.8-95.4%, and 85.5-89.0%, respectively, with strong interobserver (k = 0.667 - 0.678, p < 0.0001), and intraobserver (k = 0.706 - 0.763, p < 0.0001) agreement. For optic atrophy, interobserver agreement was moderate (k = 0.437 - 0.484, p < 0.0001), while intraobserver agreement was moderate-strong (k = 0.491 - 0.596, p < 0.0001)., Conclusion: Coronal CE-FS-FLAIR is quite specific in detecting optic neuropathy years after the onset of acute optic neuritis, but is less useful in detecting optic nerve atrophy.

Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca 2+ imbalance by depressing RyR2 channel activity during excitation-contraction coupling, resulting in random bursts of Ca 2+ release probably due to Ca 2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2 A4860G+/- cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower., Competing Interests: The authors declare no conflict of interest.

Research into regulation of the differentiation of stem cells is critical to understanding early developmental decisions and later development growth. The transcription factor ARID3A previously was shown to be critical for trophectoderm and hematopoetic development. Expression of ARID3A increases during embryonic differentiation, but the underlying reason remained unclear. Here we show that Arid3a null embryonic stem (ES) cells maintain an undifferentiated gene expression pattern and form teratomas in immune-compromised mice. However, Arid3a null ES cells differentiated in vitro into embryoid bodies (EBs) significantly faster than control ES cells, and the majority forming large cystic embryoid EBs. Analysis of gene expression during this transition indicated that Arid3a nulls differentiated spontaneously into mesoderm and neuroectoderm lineages. While young ARID3A-deficient mice showed no gross tissue morphology, proliferative and structural abnormalities were observed in the kidneys of older null mice. Together these data suggest that ARID3A is not only required hematopoiesis, but is critical for early mesoderm differentiation.

Background: Bystander cardiopulmonary resuscitation (CPR) rates are low. Our study objective was to encourage Philadelphia high school students to develop CPR/AED (automated external defibrillator) training programs and to assess their efficacy. The focus was on developing innovative ways to learn the skills of CPR/AED use, increasing willingness to respond in an emergency, and retention of effective psychomotor resuscitation skills., Methods and Results: Health education classes in 15 Philadelphia School District high schools were selected, with one Control and one Study Class per school. Both completed CPR/AED pre- and post-tests to assess cognitive knowledge and psychomotor skills. After pre-tests, both were taught CPR skills and AED use by their health teacher. Study Classes developed innovative programs to learn, teach, and retain CPR/AED skills. The study culminated with Study Classes competing in multiple CPR/AED skills events at the CPR/AED Olympic event. Outcomes included post-tests, Mock Code, and presentation scores. All students' cognitive and psychomotor skills improved with standard classroom education (p<0.001). Competition with other schools at the CPR/AED Olympics and the development of their own student-directed education programs resulted in remarkable retention of psychomotor skill scores in the Study Class (88%) vs the Control Class (79%) (p<0.001). Olympic participants averaged 93.1% on the Mock Code with 10 of 12 schools ≥94%., Conclusion: Students who developed creative and novel methods of teaching and learning resuscitation skills showed outstanding application of these skills in a Mock Code with remarkable psychomotor skill retention, potentially empowering a new generation of effectively trained CPR bystanders., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Most reverse transcriptase (RT) enzymes belong to a single protein family of ancient evolutionary origin. These polymerases are inherently error prone, owing to their lack of a proofreading (3'- 5' exonuclease) domain. To determine if the lack of proofreading is a historical coincidence or a functional limitation of reverse transcription, we attempted to evolve a high-fidelity, thermostable DNA polymerase to use RNA templates efficiently. The evolutionarily distinct reverse transcription xenopolymerase (RTX) actively proofreads on DNA and RNA templates, which greatly improves RT fidelity. In addition, RTX enables applications such as single-enzyme reverse transcription-polymerase chain reaction and direct RNA sequencing without complementary DNA isolation. The creation of RTX confirms that proofreading is compatible with reverse transcription., (Copyright © 2016, American Association for the Advancement of Science.)

A new paradigm has emerged in recent years characterizing transcription initiation as a bidirectional process encompassing a larger proportion of the genome than previously thought. Past concepts of coding genes thinly scattered among a vast background of transcriptionally inert noncoding DNA have been abandoned. A richer picture has taken shape, integrating transcription of coding genes, enhancer RNAs (eRNAs), and various other noncoding transcriptional events. In this review we give an overview of recent studies detailing the mechanisms of RNA polymerase II (RNA Pol II)-based transcriptional initiation and discuss the ways in which transcriptional direction is established as well as its functional implications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell-cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.