DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88.

Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.

Multiple RNA- and DNA-binding proteins exhibit direct transfer of polynucleotides with implications for target-site search.

We previously demonstrated that the polycomb repressive complex 2 chromatin-modifying enzyme can directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1, heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally observed in single-molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated polynucleotides. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, proteins that bind both RNA and DNA might be capable of readily translocating between those ligands.

Finding the start site: redefining the human initiator element.

Transcription by RNA polymerase II (Pol II) is dictated in part by core promoter elements, which are DNA sequences flanking the transcription start site (TSS) that help direct the proper initiation of transcription. Taking advantage of recent advances in genome-wide sequencing approaches, Vo ngoc and colleagues (pp. 6-11) identified transcripts with focused sites of initiation and found that many were transcribed from promoters containing a new consensus sequence for the human initiator (Inr) core promoter element.

Association of hydroxyurea adherence with transcranial Doppler screenings in children with sickle cell disease.

BACKGROUND: National sickle cell disease (SCD) guidelines recommend oral hydroxyurea (HU) starting at 9 months of age, and annual transcranial Doppler (TCD) screenings to identify stroke risk in children aged 2-16 years. We examined prevalence and proportion of TCD screenings in North Carolina Medicaid enrollees to identify associations with sociodemographic factors and HU adherence over 3 years. STUDY DESIGN: We conducted a longitudinal study with children ages 2-16 years with SCD enrolled in NC Medicaid from years 2016-2019. Prevalence of TCD screening claims was calculated for 3 years, and proportion was calculated for 12, 24, and 36 months of Medicaid enrollment. Enrollee HU adherence was categorized using HU proportion of days covered. Multivariable Poisson regression assessed for TCD screening rates by HU adherence, controlling for age, sex, and rurality. RESULTS: The prevalence of annual TCD screening was between 39.5% and 40.1%. Of those with 12-month enrollment, 77.8% had no TCD claims, compared to 22.2% who had one or higher TCD claims. Inversely, in children with 36 months of enrollment, 36.7% had no TCD claims compared to 63.3% who had one or higher TCD claims. The proportion of children with two or higher TCD claims increased with longer enrollment (10.5% at 12 months, 33.7% at 24 months, and 52.6% at 36 months). Children with good HU adherence were 2.48 (p < .0001) times more likely to have TCD claims than children with poor HU adherence. CONCLUSION: While overall TCD screening prevalence was low, children with better HU adherence and longer Medicaid enrollment had more TCD screenings. Multilevel interventions are needed to engage healthcare providers and families to improve both evidence-based care and annual TCD screenings in children with SCD.

Emergency response to stormwater contamination: A framework for containment and treatment.

This paper presents a Stormwater Emergency Response Framework (SERF) for use in the containment and treatment of stormwater runoff following a hazardous material release. The framework consists of four high level process steps and a decision tree. These resources are intended to assist stormwater managers in fulfilling their emergency response responsibilities within the United States' National Incident Management System. Robust hydraulic and watershed modeling may take weeks to months to develop for a contaminated site, whereas decisions made in the initial hours can have a significant impact on limiting contamination spread. Many web resources are publicly available to assist responders in visualizing stormwater runoff flow paths. A case study provided in this paper also demonstrates how simple calculations may be utilized to estimate peak flows and storage volumes necessary to respond to precipitation events immediately. These calculations are useful for decision makers' allocation of containment and treatment resources within the impacted area. This includes where to deploy available resources to minimize contamination risks to downstream communities and where supplemental resources from outside partners are urgently needed.

Double-Stranded RNA Is a Novel Molecular Target in Osteomyelitis Pathogenesis: A Translational Avian Model for Human Bacterial Chondronecrosis with Osteomyelitis.

Osteomyelitis remains a serious inflammatory bone disease that affects millions of individuals worldwide and for which there is no effective treatment. Despite scientific evidence that Staphylococcus bacteria are the most common causative species for human bacterial chondronecrosis with osteomyelitis (BCO), much remains to be understood about the underlying virulence mechanisms. Herein, we show increased levels of double-stranded RNA (dsRNA) in infected bone in a Staphylococcus-induced chicken BCO model and in human osteomyelitis samples. Administration of synthetic [poly(I:C)] or genetic (Alu) dsRNA induces human osteoblast cell death. Similarly, infection with Staphylococcus isolated from chicken BCO induces dsRNA accumulation and cell death in human osteoblast cell cultures. Both dsRNA administration and Staphylococcus infection activate NACHT, LRR and PYD domains-containing protein (NLRP)3 inflammasome and increase IL18 and IL1B gene expression in human osteoblasts. Pharmacologic inhibition with Ac-YVAD-cmk of caspase 1, a critical component of the NLRP3 inflammasome, prevents DICER1 dysregulation- and dsRNA-induced osteoblast cell death. NLRP3 inflammasome and its components are also activated in bone from BCO chickens and humans with osteomyelitis, compared with their healthy counterparts. These findings provide a rationale for the use of chicken BCO as a human-relevant spontaneous animal model for osteomyelitis and identify dsRNA as a new treatment target for this debilitating bone pathogenesis.

miR-206 knockout shows it is critical for myogenesis and directly regulates newly identified target mRNAs.

The muscle specific miRNA, miR-206, is important for the process of myogenesis; however, studying the function of miR-206 in muscle development and differentiation still proves challenging because the complement of mRNA targets it regulates remains undefined. In addition, miR-206 shares close sequence similarity to miR-1, another muscle specific miRNA, making it hard to study the impact of miR-206 alone in cell culture models. Here we used CRISPR/Cas9 technology to knockout miR-206 in C2C12 muscle cells. We show that knocking out miR-206 significantly impairs and delays differentiation and myotube formation, revealing that miR-206 alone is important for myogenesis. In addition, we use an experimental affinity purification technique to identify new mRNA targets of miR-206 in C2C12 cells. We identified over one hundred mRNAs as putative miR-206 targets. Functional experiments on six of these targets indicate that Adam19, Bgn, Cbx5, Smarce1, and Spg20 are direct miR-206 targets in C2C12 cells. Our data show a unique and important role for miR-206 in myogenesis.

Monitoring transcriptional activity by RNA polymerase II in vitro using single molecule co-localization.

RNA polymerase II (Pol II) transcribes eukaryotic mRNA genes. To initiate transcription, pre-initiation complexes (PICs) containing Pol II and general transcription factors (GTFs) form on the core promoters of target genes. In cells this process is regulated by transcriptional activators, co-activators, and chromatin modifying complexes. Reconstituted in vitro transcription systems are important tools for studying the enzymology and fundamental steps in the transcription reaction. In these systems, studying transcription can be complex due to the heterogeneous mixture of transcriptionally active and inactive complexes that assemble at promoters. Accordingly, we developed a technique to use single molecule microscopy to resolve this heterogeneity and distinguish transcriptionally active complexes from inactive complexes. This system uses fluorescently-labeled promoter DNA and a minimal reconstituted transcription system consisting of purified human Pol II and GTFs. Here we describe the materials, methods, and analysis required to study Pol II transcription at the single molecule level. The flexibility of our single molecule method allows for adaptation to answer diverse mechanistic questions about transcription that would otherwise be difficult to study using ensemble assays.

The limits of endoscopic endonasal approaches in young children: a review.

INTRODUCTION: The endoscopic endonasal approach (EEA) provides visualization of four deep surgical corridors (transcribiform, transtubercular, transsellar, and transclival) with superior illumination and specialized deep-reaching instruments, as compared to microscopic techniques. Several studies have evaluated EEAs in children but do not stratify for the very young of age, whose particularly small nares and developmental anatomy may limit endonasal instrumentation. METHODOLOGY: A comprehensive review of EEAs in infants and children to age 4 was performed to determine the limitations in this age group. RESULTS: Eighteen studies were identified describing this approach for pediatric patients and the surgical caveats and limitations were reviewed. In very small children, CSF leaks, meningioencephaloceles, tumors of the anterior skull base, and lesions at the rostral cervical spine have been successfully treated endonasally. While newer studies advocate using 2.7-mm diameter (18-cm length) lenses, 4-mm diameter rigid lenses have been used without technical difficulty. The youngest patient in whom an EEA was used was a 6-week-old for a dermoid resection. Some have advocated that due to the small nares, approaches via bilateral entry are optimal for multiple instruments, however, others, including authors of a series of 28 repaired CSF leaks demonstrate successful single nare access. DISCUSSION: EEAs are associated with less blood loss, are less likely to hinder normal growth of the skull and midface, and allow for the resection of even malignant lesions. Despite the limitations of the frontal, ethmoid, and sphenoid sinuses before age 3, reports have not documented insurmountable difficulty with EEAs even in infants. 2.7-mm diameter endoscopes are favored unilaterally or bilaterally to treat both benign and malignant lesions and preserve the young patient's facial anatomy better than older methods. Ever improving technology has facilitated the use of this approach in patients it would otherwise be infeasible for in the past, but it still cannot overcome the anatomical constraints of certain young patients in which this approach remains unindicated. Patient selection is therefore of utmost importance and the risks and benefits of more extensive approaches in these cases must be considered.

Optimizing Reconstruction in Craniosynostosis: Review of Nonsyndromic Patients Treated With a Novel Technique.

PURPOSE: Open cranial vault remodeling (CVR) with autologous split calvarial bone grafts redistributes and recontours an abnormal calvarium to create an expanded cranial vault in patients with craniosynostosis. We report a 12-year retrospective review of 162 nonsyndromic patients who underwent operative repair using our previously-described technique which portends excellent surgical outcomes and can be applied to patients of any age group and with any variety of suture fusion. METHODS: Data was gathered on patients who underwent CVR from 2005 to 2016. Surgical records for each patient were analyzed and included operative time, estimated blood loss, and intraoperative transfusion volumes. Intraoperative and postoperative complications, the need for revision surgery, postoperative length of stay, and follow-up records were also reviewed. Syndromic patients were excluded, as well as patients with incomplete data sets. Patients who underwent either anterior or posterior vault remodeling were compared. RESULTS: A total of 162 patients were included in this case series. Patients undergoing anterior CVR were significantly older than those undergoing posterior CVR (13.3 versus 11.0 months, P < 0.015) and also had significantly greater intraoperative red blood transfusion volumes (20.3 versus 15.3cc/kg, P < 0.0207) and longer operative time than posterior CVR patients (274.9 versus 216.7 minutes, P < 0.0001). No patients required reoperation for resorption or recurrence or persistent contour irregularities. There were no visual or neurological complications. Calvarial bone was successfully split in 100% of cases. CONCLUSIONS: This surgical approach to CVR results in good surgical outcomes with a low recurrence rate, while also maximizing operative efficiency, and minimizing total blood loss and transfusion volume. This technique can be applied to any affected suture in a patient with craniosynostosis and in patients of any age group.

Reaction products of hexamethylene diisocyanate vapors with "self" molecules in the airways of rabbits exposed via tracheostomy.

1. Hexamethylenediisocyanate (HDI) is a widely used aliphatic diisocyanate and a well-recognized cause of occupational asthma. 2. "Self" molecules (peptides/proteins) in the lower airways, susceptible to chemical reactivity with HDI, have been hypothesized to play a role in asthma pathogenesis and/or chemical metabolism, but remain poorly characterized. 3. This study employed unique approaches to identify and characterize "self" targets of HDI reactivity in the lower airways. Anesthetized rabbits free breathed through a tracheostomy tube connected to chambers containing either, O2, or O2 plus ∼200 ppb HDI vapors. Following 60 minutes of exposure, the airways were lavaged and the fluid was analyzed by LC-MS and LC-MS/MS. 4. The low-molecular weight (<3 kDa) fraction of HDI exposed, but not control rabbit bronchoalveolar lavage (BAL) fluid identified 783.26 and 476.18 m/z [M+H]+ ions with high energy collision-induced dissociation (HCD) fragmentation patterns consistent with bis glutathione (GSH)-HDI and mono(GSH)-HDI. Proteomic analyses of the high molecular weight (>3 kDa) fraction of exposed rabbit BAL fluid identified HDI modification of specific lysines in uteroglobin (aka clara cell protein) and albumin. 5. In summary, this study utilized a unique approach to chemical vapor exposure in rabbits, to identify HDI reaction products with "self" molecules in the lower airways.

Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity.

Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity.

The history of intracranial infections.

The 19th and 20th centuries heralded the advancement of our understanding of surgical infections, reducing the risk of morbidity and mortality to patients by overturning long-held dogmas surrounding infections and perioperative care. These advancements impacted the development and establishment of the field of neurological surgery by minimizing surgical risk through aseptic techniques and promoting surgical benefit via improved neurological localization and surgical technique. Infections were significant contributors to morbidity and mortality for all surgical patients, and historically almost half of patients lost their lives as a consequence of perioperative wound contamination. With advancing understanding of germ theory, contagion, antisepsis, and subsequently asepsis, the surgeon began embracing the knowledge and techniques which would hone their craft and allow for a renaissance in the management of neurological disorders in an unprecedented manner.

Virtual modeling, stereolithography, and intraoperative CT guidance for the optimization of sagittal synostosis reconstruction: a technical note.

INTRODUCTION: Sagittal synostosis affects 1 in 1000 live births and may result in increased intracranial pressure, hindrance of normal neural development, and cosmetic deformity due to scaphocephaly. Historically, several approaches have been utilized for surgical correction and recently, computed tomography (CT)-guided reconstruction procedures are increasingly used. In this report, the authors describe the use of a CT-derived virtual and stereolithographic (3D printed) craniofacial models, which are used to guide intraoperative bone placement, and intraoperative CT guidance for confirmation of bone placement, to ensure the accuracy of surgical correction of scaphocephaly, as demonstrated to parents. METHODS: Preoperative high-resolution CT imaging was used to construct 3D image sets of the skulls of two infants (a 14-month-old female and a 6-month-old male) with scaphocephaly. These 3D image sets were then used to create a virtual model of the proposed surgical correction for each of the infants' deformities, which was then printed and made available for use intraoperatively to plan the bone flap, fashion the bone cuts, and optimize graft placement. After the remodeling, adherence to the preoperative plan was assessed by overlaying a CT scan of the remodeled skull with the virtual model. Deviations from the preoperative model were noted. RESULTS: Both patients had excellent postoperative cosmetic correction of head shape and contouring. The mean operative time was 5 h, blood loss was 100 ml, and one child required modification of the subocciput after intraoperative imaging showed a deviation of the reconstruction from the surgical goal as depicted by the preoperative model. CONCLUSION: The addition of neuro-navigation to stereolithographic modeling ensured the accuracy of the reconstruction for our patients and provided greater confidence to both surgeons and parents. While unisutural cases are presented for clarity, correction was still required for one patient. The cost of the models and the additional CT required must be weighed against the complexity of the procedure and possibly reserved for patients with potentially complicated corrections.

Understanding the Impact of Mesh on Tank Overflow System Capacity.

A 2016 incident that resulted in damage to a water storage tank's roof motivated pilot-scale experiments to be conducted to determine the impact of mesh on tank overflow capacity. A clean mesh installed near the outlet of an overflow system did not reduce the capacity during the weir dominated flow regime. The impact of a mesh was found to be a reduction in the area available to flow, which was found to lower the achievable capacity through the system. Considering only the head loss or pressure drop associated with the mesh and not area reduction resulted in an overestimation of achievable capacity, which could lead to an undersized overflow system. The results and formulas presented will help water utilities ensure overflow systems with mesh are appropriately sized.

RNA polymerase II acts as an RNA-dependent RNA polymerase to extend and destabilize a non-coding RNA.

RNA polymerase II (Pol II) is a well-characterized DNA-dependent RNA polymerase, which has also been reported to have RNA-dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non-coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3'-end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α-amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3'-end.

Ixmyelocel-T for patients with ischaemic heart failure: a prospective randomised double-blind trial.

BACKGROUND: Ixmyelocel-T is an expanded, multicellular therapy produced from a patient's own bone marrow by selectively expanding two key types of bone marrow mononuclear cells: CD90+ mesenchymal stem cells and CD45+ CD14+ auto-fluorescent+ activated macrophages. Early phase clinical trials suggest that intramyocardial delivery of ixmyelocel-T might improve clinical, functional, symptomatic, and quality-of-life outcomes in patients with heart failure due to ischaemic dilated cardiomyopathy. We aimed to assess the safety and efficacy of catheter-based transendocardial injection of ixmyelocel-T cell therapy in patients with heart failure and reduced ejection fractions. METHODS: In this randomised, double-blind, placebo-controlled phase 2B trial (ixCELL-DCM), patients from 31 sites in North America with New York Heart Association class III or IV symptomatic heart failure due to ischaemic dilated cardiomyopathy, who had left ventricular ejection fraction 35% or less, an automatic implantable cardioverter defibrillator, and who were ineligible for revascularisation procedures were randomly assigned (1:1) to receive ixmyelocel-T or placebo at the time of bone marrow aspiration and followed for 12 months. Randomisation was done through an interactive (voice/web) response system. The pharmacist, treating physician, and coordinator at each site were unblinded, but the the follow-up team was completely blinded. The primary endpoint was a composite of all-cause death, cardiovascular admission to hospital, and unplanned clinic visits to treat acute decompensated heart failure based on the blinded adjudication of an independent clinical endpoint committee. Primary efficacy endpoint analyses and safety analyses were done by modified intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01670981. FINDINGS: Between April 2, 2013, and Jan 28, 2015, 126 participants were randomly assigned to receive either ixmyelocel-T (n=66) or placebo (n=60). 114 (90%) patients comprised the modified intention-to-treat population and 109 (87%) patients were included in the per-protocol primary efficacy analysis (58 in the ixmyelocel-T group and 51 in the placebo group). The primary efficacy endpoint was observed in 47 patients: 50 events in 25 (49%) of 51 patients in the placebo group and 38 events in 22 (38%) of 58 patients in the ixmyelocel-T group, which represents a 37% reduction in cardiac events compared with placebo (risk ratio 0·63 [95% CI 0·42-0·97]; p=0·0344). 41 (75%) of 51 participants in the placebo group had serious adverse events versus 31 (53%) of 58 in the ixmyelocel-T group (p=0·0197). INTERPRETATION: To the best of our knowledge, ixCELL-DCM is the largest cell therapy study done in patients with heart failure so far. The transendocardial delivery of ixmyelocel-T in patients with heart failure and reduced ejection fraction due to ischaemic dilated cardiomyopathy resulted in a significant reduction in adjudicated clinical cardiac events compared with placebo leading to improved patient outcomes. FUNDING: Vericel Corporation.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration.

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

DETENTION OUTLET RETROFIT IMPROVES THE FUNCTIONALITY OF EXISTING DETENTION BASINS BY REDUCING EROSIVE FLOWS IN RECEIVING CHANNELS.

By discharging excess stormwater at rates that more frequently exceed the critical flow for stream erosion, conventional detention basins often contribute to increased channel instability in urban and suburban systems that can be detrimental to aquatic habitat and water quality, as well as adjacent property and infrastructure. However, these ubiquitous assets, valued at approximately $600,000 per km2 in a representative suburban watershed, are ideal candidates to aid in reversing such cycles of channel degradation because improving their functionality would not necessarily require property acquisition or heavy construction. The objective of this research was to develop a simple, cost-effective device that could be installed in detention basin outlets to reduce the erosive power of the relatively frequent storm events (~ < two-year recurrence) and provide a passive bypass to maintain flood control performance during infrequent storms (such as the 100-year recurrence). Results from a pilot installation show that the Detain H2O device reduced the cumulative sediment transport capacity of the preretrofit condition by greater than 40%, and contributed to reduced flashiness and prolonged baseflows in receiving streams. When scaling the strategy across a watershed, these results suggest that potential gains in water quality and stream channel stability could be achieved at costs that are orders of magnitude less than comparable benefits from newly constructed stormwater control measures.