RNA polymerases reshape chromatin architecture and couple transcription on individual fibers.

RNA polymerases must initiate and pause within a complex chromatin environment, surrounded by nucleosomes and other transcriptional machinery. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address this, we employed long-read chromatin fiber sequencing (Fiber-seq) in Drosophila to visualize RNA polymerase (Pol) within its native chromatin context with single-molecule precision along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of individual Pol II, nucleosome, and transcription factor footprints, revealing Pol II pausing-driven destabilization of downstream nucleosomes. Furthermore, we demonstrate pervasive direct distance-dependent transcriptional coupling between nearby Pol II genes, Pol III genes, and transcribed enhancers, modulated by local chromatin architecture. Overall, transcription initiation reshapes surrounding nucleosome architecture and couples nearby transcriptional machinery along individual chromatin fibers.

A microRNA program in the C. elegans hypodermis couples to intestinal mTORC2/PQM-1 signaling to modulate fat transport.

Animals integrate metabolic, developmental, and environmental information before committing key resources to reproduction. In Caenorhabditis elegans, adult animals transport fat from intestinal cells to the germline to promote reproduction. We identified a microRNA (miRNA)-regulated developmental timing pathway that functions in the hypodermis to nonautonomously coordinate the mobilization of intestinal fat stores to the germline upon initiation of adulthood. This developmental timing pathway, which is controlled by the lin-4 and let-7 miRNAs, engages mTOR signaling in the intestine. The intestinal signaling component is specific to mTORC2 and functions in parallel to the insulin pathway to modulate the activity of the serum/glucocorticoid-regulated kinase (SGK-1). Surprisingly, SGK-1 functions independently of DAF-16/FoxO; instead, SGK-1 promotes the cytoplasmic localization of the PQM-1 transcription factor, which antagonizes intestinal fat mobilization at the transcriptional level when localized to the nucleus. These results revealed that a non-cell-autonomous developmental input regulates intestinal fat metabolism by engaging mTORC2 signaling to promote the intertissue transport of fat reserves from the soma to the germline.

A Single-Institutional Comparative Analysis of Advanced Versus Standard Snare Removal of Inferior Vena Cava Filters.

PURPOSE: To investigate differences in procedure time, radiation exposure, and periprocedural complications associated with advanced inferior vena cava (IVC) filter retrieval compared with standard snare retrieval. MATERIALS AND METHODS: A total of 378 patients underwent standard or advanced IVC filter retrieval over a 5-year period. Technical success, retrieval techniques, fluoroscopy time, radiation dose, and complications were analyzed. All retrieval procedures with techniques other than a "snare-and-sheath" method were categorized as advanced, including failed standard attempts requiring intraprocedural conversion to advanced techniques. RESULTS: A total of 462 filter retrieval attempts were made in 378 patients (57% female). Success rates for standard and advanced retrieval attempts were 86.8% (317 of 365) and 91.8% (89 of 97), respectively. The rate of periprocedural complications was significantly higher in the advanced retrieval group (P = .006). Complication rates for standard and advanced retrievals were 0.6% (2 of 318; all minor) and 5.2% (5 of 97; 3 minor [3.1%] and 2 major [2.1%]), respectively. The 2 major complications during advanced retrievals included filter fracture and embolization. Average fluoroscopy time for advanced retrievals was significantly higher than for standard retrievals (23.1 min vs 4.3 min; P < .001). Average radiation dose for advanced retrievals was also significantly higher than for standard retrievals (557.2 mGy vs 156.9 mGy; P < .001). Use of general anesthesia was also significantly more common in advanced retrievals compared with standard retrievals (6.2% vs 0.9%; P = .002). CONCLUSIONS: Advanced filter retrieval results in a similarly high rate of technical success compared with standard snare retrieval but is associated with greater fluoroscopy time, anesthesia requirements, and radiation exposure.

Experimental maps of DNA structure at nucleotide resolution distinguish intrinsic from protein-induced DNA deformations.

Recognition of DNA by proteins depends on DNA sequence and structure. Often unanswered is whether the structure of naked DNA persists in a protein-DNA complex, or whether protein binding changes DNA shape. While X-ray structures of protein-DNA complexes are numerous, the structure of naked cognate DNA is seldom available experimentally. We present here an experimental and computational analysis pipeline that uses hydroxyl radical cleavage to map, at single-nucleotide resolution, DNA minor groove width, a recognition feature widely exploited by proteins. For 11 protein-DNA complexes, we compared experimental maps of naked DNA minor groove width with minor groove width measured from X-ray co-crystal structures. Seven sites had similar minor groove widths as naked DNA and when bound to protein. For four sites, part of the DNA in the complex had the same structure as naked DNA, and part changed structure upon protein binding. We compared the experimental map with minor groove patterns of DNA predicted by two computational approaches, DNAshape and ORChID2, and found good but not perfect concordance with both. This experimental approach will be useful in mapping structures of DNA sequences for which high-resolution structural data are unavailable. This approach allows probing of protein family-dependent readout mechanisms.

Gender-Specific Factors Influencing Medical Students' Career Choice of IR.

The authors conducted an anonymous survey to assess positive and negative factors that may affect medical students' decisions to pursue a career in interventional radiology (IR). The survey was sent to registrants for the Midwest IR Student Symposium in 2016 and/or 2017, with a response rate of 13%; male and female responses were then compared. Female and male medical students shared similar rankings of factors affecting their decisions about choosing IR as a career, such as concern about lifestyle and excitement about therapeutic applications. Access to female IR mentors and diversification of the currently male-dominated workplace were important, gender-specific concerns.

GBshape: a genome browser database for DNA shape annotations.

Many regulatory mechanisms require a high degree of specificity in protein-DNA binding. Nucleotide sequence does not provide an answer to the question of why a protein binds only to a small subset of the many putative binding sites in the genome that share the same core motif. Whereas higher-order effects, such as chromatin accessibility, cooperativity and cofactors, have been described, DNA shape recently gained attention as another feature that fine-tunes the DNA binding specificities of some transcription factor families. Our Genome Browser for DNA shape annotations (GBshape; freely available at http://rohslab.cmb.usc.edu/GBshape/) provides minor groove width, propeller twist, roll, helix twist and hydroxyl radical cleavage predictions for the entire genomes of 94 organisms. Additional genomes can easily be added using the GBshape framework. GBshape can be used to visualize DNA shape annotations qualitatively in a genome browser track format, and to download quantitative values of DNA shape features as a function of genomic position at nucleotide resolution. As biological applications, we illustrate the periodicity of DNA shape features that are present in nucleosome-occupied sequences from human, fly and worm, and we demonstrate structural similarities between transcription start sites in the genomes of four Drosophila species.

Indwelling and Retrieval Complications of Denali and Celect Infrarenal Vena Cava Filters.

PURPOSE: To compare indwelling and retrieval complications of Denali and Celect filters placed in the infrarenal inferior vena cava (IVC). MATERIALS AND METHODS: A retrospective study was conducted over 2 years at a single institution in which 171 Denali and 162 Celect filters were placed in 333 patients with a mean age of 62.3 years ± 15.7 (161 men; 48.3%). Filter indications included venous thromboembolic disease (n = 320; 96.1%) and surgical prophylaxis (n = 13; 3.9%). A jugular approach was used to place 303 filters (91.0%). Computed tomography (CT) follow-up, complications, and retrieval data were obtained. RESULTS: Follow-up CT imaging was performed on 58 filters from each group with lower incidences of caval strut penetration (one vs 12) and filter tilt (one vs 15) in the Denali filter group (P = .002 and P < .001, respectively). There was no difference in incidences of breakthrough pulmonary embolism (P = .68). Retrieval attempts were performed on 43 Denali and 53 Celect filters with mean indwelling times at retrieval of 128.2 and 144.1 days, respectively (P = .40). Mean fluoroscopy time at retrieval was lower in the Denali group (3.1 min vs 6.0 min; P = .01). There were fewer cases of complex retrieval in the Denali group (n = 2 vs 10; P = .06). Tilt, fluoroscopy time, and air kerma were associated with complex retrieval (P = .04, P < .001, and P < .001, respectively). There was one Denali filter deployment complication that led to retrieval failure. CONCLUSIONS: This study suggests that Denali filters are associated with lower incidences of strut penetration and filter tilt as well as shorter fluoroscopy time at retrieval compared with Celect filters when placed in the infrarenal IVC.

Chemical probing of RNA with the hydroxyl radical at single-atom resolution.

While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5'-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5'-aldehyde. We conclude that hydroxyl radical abstracts a 5'-hydrogen atom, leading to RNA strand cleavage. We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA. Cleavage at U exhibits a large 5' deuterium kinetic isotope effect, a potential signature of a base triple. Others had noted a ribose-phosphate hydrogen bond involving the G 2'-OH and the U phosphate of the GUA triple, and suggested that this hydrogen bond contributes to backbone rigidity. Substituting deoxyguanosine for G, to eliminate this hydrogen bond, results in a substantial decrease in cleavage at G and U of the triple. We conclude that this hydrogen bond is a linchpin of backbone structure around the triple.

Use of three-dimensional power Doppler sonography in the diagnosis of uterine arteriovenous malformation and follow-up after uterine artery embolization: Case report and brief review of literature.

Arteriovenous malformations (AVM) of the uterus can cause life-threatening hemorrhage. Unexplained, heavy vaginal bleeding in a reproductive age woman should raise suspicion for an AVM. Here a 37-year-old woman had increasingly severe vaginal bleeding for 15 days. Serum ß-hCG was elevated. Two-dimensional transvaginal ultrasound suggested retained products of conception. Before dilation and curettage (D&C), color Doppler and three-dimensional (3D) power Doppler demonstrated findings indicative of uterine AVM. A bilateral uterine artery embolization was performed without complications. Three months after uterine artery embolization, 3D power Doppler ultrasonography found complete resolution of the AVM. This case illustrates the importance of assessing both gray-scale and 3D power Doppler, and the ability of postprocedure Doppler to assess resolution.

Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA.

Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution. We found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction, with most nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are co-occupied by transcription and replication components and selectively form a triple-stranded displacement loop structure. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings reveal the primary architecture of mtDNA packaging and regulation in human cells.

RNA polymerases reshape chromatin and coordinate transcription on individual fibers.

During eukaryotic transcription, RNA polymerases must initiate and pause within a crowded, complex environment, surrounded by nucleosomes and other transcriptional activity. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address these limitations, we employed long-read chromatin fiber sequencing (Fiber-seq) to visualize RNA polymerases within their native chromatin context at single-molecule and near single-nucleotide resolution along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of single-molecule RNA Polymerase (Pol) II and III transcription associated footprints, which, in aggregate, mirror bulk short-read sequencing-based measurements of transcription. We show that Pol II pausing destabilizes downstream nucleosomes, with frequently paused genes maintaining a short-term memory of these destabilized nucleosomes. Furthermore, we demonstrate pervasive direct coordination and anti-coordination between nearby Pol II genes, Pol III genes, transcribed enhancers, and insulator elements. This coordination is largely limited to spatially organized elements within 5 kb of each other, implicating short-range chromatin environments as a predominant determinant of coordinated polymerase initiation. Overall, transcription initiation reshapes surrounding nucleosome architecture and coordinates nearby transcriptional machinery along individual chromatin fibers.

Comparing outcomes of percutaneous cholecystostomy drain placement between patients within and outside of Tokyo guidelines diagnostic criteria for acute cholecystitis.

OBJECTIVE: To compare outcomes following percutaneous cholecystostomy drain placement based on presence or absence of Tokyo Guidelines diagnostic criteria for acute cholecystitis. METHODS: Chart review was performed to identify the presence or absence of Tokyo Guidelines diagnostic criteria for acute cholecystitis in 146 patients who underwent percutaneous cholecystostomy between 2012 and 2015. Those who met criteria were compared to those who did not in terms of demographics, laboratory values, drain indwelling time, treatment response, eventual surgical management, and 30-day mortality. RESULTS: 94 patients (64%) met Tokyo Guidelines diagnostic criteria, while 52 did not (36%). Patients within criteria had a shorter mean length of stay (13.5 days vs 18.9 days), were more likely to have a positive gallbladder fluid culture (64.5% vs 28.6%), demonstrated greater response to treatment (87.2% vs 32.7%), and had lower 30-day mortality (6.4% vs 37.8%). There was no significant difference in terms of ICU requirement (38.3% vs 38.9%), mean drain indwelling time (58.8 days vs 65.3 days), eventual laparoscopic cholecystectomy (40.4% vs 25.0%), or open cholecystectomy performed (9.5% vs 9.6%). CONCLUSION: Patients outside of Tokyo Guidelines diagnostic criteria for acute cholecystitis were less likely to respond to treatment with percutaneous cholecystostomy and had worse outcomes. Further research may be indicated to better define the indications for percutaneous cholecystostomy placement in this group.

Update on Preoperative Embolization of Bone Metastases.

Management of patients with bone metastasis is complex and should include different specialties. Goals of therapy should be identified for each individual patient prior to the start of treatment. Preoperative embolization has generally been considered a safe and effective means of reducing intraoperative blood loss with recent studies and advances in technique reported. Update on indications, contraindications, technique, and efficacy, as well as prognostic factors and complications of preoperative embolization of bone metastases will be reviewed. New trends such as transradial arterial access and usage of liquid embolic agents will be discussed. Large tumor size, increased preprocedural tumor vascularity, longer embolization-to-surgery interval, and radical surgical procedures are associated with greater intraoperative blood loss and prolonged operative time. An accurate, noninvasive method to evaluate tumor vascularity prior to angiography is needed to identify patients who are most likely to benefit from preoperative embolization. Particular attention will be paid to skeletal metastases and spinal metastases as each has its own set of complexity.

Percutaneous treatment of IVC obstruction due to post-resection hepatic torsion associated with IVC thrombosis.

BACKGROUND: Migration of the left hepatic lobe into the potential space following right lobe resection can result in torsion and hepatic venous outflow obstruction with compromised venous return from the IVC. If untreated, significant morbidity and mortality can develop. CASE PRESENTATION: We report a case of a 29-year-old female with Lynch syndrome who underwent right lobe resection for a metastatic hepatic tumor. There was subsequent migration of the liver remnant, torsion of the IVC, and impaired hepatic outflow, successfully treated with thrombectomy and stenting. CONCLUSION: Following right hepatectomy, hepatic venous outflow obstruction should be consdered in the setting of hepatorenal failure and hemodynamic instability. Endovascular stenting is a viable treatment option.

The relationship between fine scale DNA structure, GC content, and functional elements in 1% of the human genome.

GC content has been shown to be an important aspect of human genomic function. Extending beyond the scope of GC content alone, there is a class of regions in the genome that have especially high GC content and are enriched for the CG dinucleotide--called CpG islands. CpG islands have been linked to biologically functional genomic elements. DNA structure also contributes to biological function. Recent studies found that some DNA structural properties are correlated with CpG island functionality. Here, we use hydroxyl radical cleavage patterns as a measure of DNA structure, to explore the relationship between GC content and fine-scale DNA structure. We show that there is a positive correlation between GC content and the solvent-accessible structural properties of a DNA sequence, and that the strength of this correlation decreases as genomic resolution increases. We demonstrate that regions of the genome that have highly solvent-accessible DNA structure tend to overlap functional genomic elements. Our results suggest that fine-scale DNA structural properties that are encoded in the genome are important for biological function, and that the highly solvent-accessible nature of high GC content regions and some CpG islands may account for some of their functional properties.

Complications and Retrieval Data of Vena Cava Filters Based on Specific Infrarenal Location.

PURPOSE: Although recommended placement of IVC filters is with their tips positioned at the level of the renal vein inflow, in practice, adherence is limited due to clinical situation or IVC anatomy. We seek to evaluate the indwelling and retrieval complications of IVC filters based on their specific position within the infrarenal IVC. MATERIALS AND METHODS: Retrospective, single institution study of 333 consecutive infrarenal vena cava filters placed by interventional radiologists in patients with an average age of 62.2 ± 15.7 years was performed between 2013 and 2015. Primary indication was venous thromboembolic disease (n = 320, 96.1%). Filters were classified based on location of the apex below the lowest renal vein inflow on the procedural venogram: less than 1 cm (n = 180, 54.1%), 1-2 cm (n = 96, 28.8%), and greater than 2 cm (n = 57, 17.1%). Denali (n = 171, 51.4%) and Celect (n = 162, 48.6%) filters were evaluated. CT follow-up, indwelling complications, and retrieval data were obtained. RESULTS: Follow-up CT imaging performed for symptomatic indications occurred for 38.3% of filters placed < 1 cm below the lowest renal vein, 27.1% of filters placed 1-2 cm, and 36.8% placed > 2 cm (p = .16). There was no difference in caval strut penetration, penetration of adjacent viscera, time to penetration, filter migration, or tilt (p = .15, .27, .41, .57, .93). No filter fractures occurred. There was no difference in the incidence of breakthrough PE or complex filter retrieval (p = .83, .59). Only one retrieval failure occurred. CONCLUSIONS: This study suggests filter apex location within the infrarenal IVC, including placement > 2 cm below the level of the renal vein inflow, is not associated with differences in indwelling or retrieval complications. LEVEL OF EVIDENCE: Level 3 non-randomized controlled follow-up study.

Mapping nucleic acid structure by hydroxyl radical cleavage.

Hydroxyl radical footprinting is a widely used method for following the folding of RNA molecules in solution. This method has the unique ability to provide experimental information on the solvent accessibility of each nucleotide in an RNA molecule, so that the folding of all domains of the RNA species can be followed simultaneously at single-nucleotide resolution. In recent work, hydroxyl radical footprinting has been used, often in combination with other global measures of structure, to work out detailed folding pathways and three-dimensional structures for increasingly large and complicated RNA molecules. These include synthetic ribozymes, and group I and group II ribozymes, from yeast, the Azoarcus cyanobacterium and Tetrahymena thermophila. Advances have been made in methods for analysis of hydroxyl radical data, so that the large datasets that result from kinetic folding experiments can be analyzed in a semi-automated and quantitative manner.

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm.

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.