"Invictus".

While attempting to thrombose a pseudoaneurysm, an interventional radiology resident provides important care to a patient beyond the procedure.

Characterization of the ZFX family of transcription factors that bind downstream of the start site of CpG island promoters.

Our study focuses on a family of ubiquitously expressed human C2H2 zinc finger proteins comprised of ZFX, ZFY and ZNF711. Although their protein structure suggests that ZFX, ZFY and ZNF711 are transcriptional regulators, the mechanisms by which they influence transcription have not yet been elucidated. We used CRISPR-mediated deletion to create bi-allelic knockouts of ZFX and/or ZNF711 in female HEK293T cells (which naturally lack ZFY). We found that loss of either ZFX or ZNF711 reduced cell growth and that the double knockout cells have major defects in proliferation. RNA-seq analysis revealed that thousands of genes showed altered expression in the double knockout clones, suggesting that these TFs are critical regulators of the transcriptome. To gain insight into how these TFs regulate transcription, we created mutant ZFX proteins and analyzed them for DNA binding and transactivation capability. We found that zinc fingers 11-13 are necessary and sufficient for DNA binding and, in combination with the N terminal region, constitute a functional transactivator. Our functional analyses of the ZFX family provides important new insights into transcriptional regulation in human cells by members of the large, but under-studied family of C2H2 zinc finger proteins.

Surgeon Perspectives on Benefits and Downsides of Overlapping Surgery: In-depth, Qualitative Interviews.

OBJECTIVE: The aim of the study was to characterize surgeon perspectives regarding the benefits and downsides of conducting overlapping surgery. BACKGROUND: Although surgeons are key stakeholders in current discussions surrounding overlapping surgery, little has been published regarding their opinions on the practice. Further characterization of surgeon perspectives is needed to guide future studies and policy development regarding overlapping surgery. METHODS: Study information was sent to all members of 3 professional surgical societies. Interested individuals were eligible to participate if they identified as attending surgeons in an academic setting who work with trainees. Purposive selection was used to diversify surgeons interviewed across multiple dimensions, including subspecialty and opinion regarding appropriateness of overlapping surgery. In-depth, qualitative interviews were conducted with participants regarding their opinions on overlapping surgery. RESULTS: The 51 surgeons interviewed identified a wide array of potential benefits and disadvantages of overlapping surgery, some of which have not previously been measured, including downsides to surgeon wellness and patient experience, less surgeon control over procedures, and difficulty in scheduling cases. Interviewees often disagreed as to whether overlapping surgery negatively or positively affects each dimension discussed, particularly regarding the impact on resident training. CONCLUSIONS: The utilization of the novel perspectives presented here will allow for targeted assessment of physician perspectives in future quantitative studies and increase the likelihood that variables measured encompass the range of factors that surgeons find meaningful and relevant. Priority areas of future research should include examining effects of overlapping surgery on surgical training and surgeon wellness.

ZFX acts as a transcriptional activator in multiple types of human tumors by binding downstream of transcription start sites at the majority of CpG island promoters.

High expression of the transcription factor ZFX is correlated with proliferation, tumorigenesis, and patient survival in multiple types of human cancers. However, the mechanism by which ZFX influences transcriptional regulation has not been determined. We performed ChIP-seq in four cancer cell lines (representing kidney, colon, prostate, and breast cancers) to identify ZFX binding sites throughout the human genome. We identified ~9,000 ZFX binding sites and found that the majority of the sites are in CpG island promoters. Moreover, genes with promoters bound by ZFX are expressed at higher levels than genes with promoters not bound by ZFX. To determine if ZFX contributes to regulation of the promoters to which it is bound, we performed RNA-seq analysis after knockdown of ZFX by siRNA in prostate and breast cancer cells. Many genes with promoters bound by ZFX were downregulated upon ZFX knockdown, supporting the hypothesis that ZFX acts as a transcriptional activator. Surprisingly, ZFX binds at +240 bp downstream of the TSS of the responsive promoters. Using Nucleosome Occupancy and Methylome Sequencing (NOMe-seq), we show that ZFX binds between the open chromatin region at the TSS and the first downstream nucleosome, suggesting that ZFX may play a critical role in promoter architecture. We have also shown that a closely related zinc finger protein ZNF711 has a similar binding pattern at CpG island promoters, but ZNF711 may play a subordinate role to ZFX. This functional characterization of ZFX provides important new insights into transcription, chromatin structure, and the regulation of the cancer transcriptome.

Practical strategies to reduce nosocomial transmission to healthcare professionals providing respiratory care to patients with COVID-19.

Coronavirus disease (COVID-19) is an emerging viral infection that is rapidly spreading across the globe. SARS-CoV-2 belongs to the same coronavirus class that caused respiratory illnesses such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). During the SARS and MERS outbreaks, many frontline healthcare workers were infected when performing high-risk aerosol-generating medical procedures as well as when providing basic patient care. Similarly, COVID-19 disease has been reported to infect healthcare workers at a rate of ~ 3% of cases treated in the USA. In this review, we conducted an extensive literature search to develop practical strategies that can be implemented when providing respiratory treatments to COVID-19 patients, with the aim to help prevent nosocomial transmission to the frontline workers.

Association between Periprocedural Neutropenia and Early Infection-related Chest Port Removal.

Background Patients who require long-term central venous access can present for port placement with depressed immune function as a result of their treatment or disease process. At present, there is no consensus regarding whether neutropenia at the time of port placement confers a higher risk for early infection-related port removal. Purpose To compare the incidence of early infection-related chest port removal in adults when placed in neutropenic versus nonneutropenic patient groups. Materials and Methods This retrospective cohort study examined 2580 port placements in 1081 men (41.9%) and 1499 women (58.1%) at a single tertiary medical center between June 2007 and July 2017. Mean patient age ± standard deviation was 56 years ± 14 (range, 18-91 years). The electronic medical record was used to identify neutropenia (absolute neutrophil count <1500 cells/mm3) at the time of port placement and incidence of infection-related port removal. Electronic medical record follow-up was conducted for 30 days following port placement. End points were infection-related port removal or death related to port infection within 30 days. Statistical analysis compared the neutropenic (n = 159) and nonneutropenic (n = 2421) patient groups by using a χ2 test for categorical data and a Student t test for continuous variables, with a Fisher exact test to compare incidence of port removal and death related to port infection. Results Ports placed in patients with neutropenia had an infection-related removal rate of 3.8% (six of 159) versus 0.91% (22 of 2421) in patients without neutropenia (P = .003). Patients with neutropenia had a port infection-related death rate of 0.63% (one of 159) versus 0.12% (three of 2421) for patients without neutropenia (P = .22). Conclusion Neutropenia in adults at the time of implantable subcutaneous chest port placement was associated with a higher risk for early infection-related port removal. There was no difference in the incidence of death related to port infection in neutropenic or nonneutropenic populations. © RSNA, 2019 See also the editorial by Johnson in this issue.

dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression.

Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type. dCpf1 fusions were unable to repress gene expression. The most persistent gene repression required the action of several effector domains; however, KRAB-dCas9 did not contribute to persistence in contrast to previous reports. A 'direct tethering' strategy attaching the Ezh2 methyltransferase enzyme to dCas9, as well as a 'recruitment' strategy attaching the N-terminal 45 residues of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex, were both successful in targeted deposition of H3K27me3. Surprisingly, however, repression was not correlated with deposition of either H3K9me3 or H3K27me3. Our results suggest that so-called repressive histone modifications are not sufficient for gene repression. The easily programmable dCas9 toolkit allowed precise control of epigenetic information and dissection of the relationship between the epigenome and gene regulation.

Estimating tropospheric and stratospheric winds using infrasound from explosions.

The receiver-to-source backazimuth of atmospheric infrasound signals is biased when cross-winds are present along the propagation path. Infrasound from 598 surface explosions from over 30 years in northern Finland is measured with high spatial resolution on an array 178 km almost due North. The array is situated in the classical shadow-zone distance from the explosions. However, strong infrasound is almost always observed, which is most plausibly due to partial reflections from stratospheric altitudes. The most probable propagation paths are subject to both tropospheric and stratospheric cross-winds, and the wave-propagation modelling in this study yields good correspondence between the observed backazimuth deviation and cross-winds from the European Centre for Medium-Range Weather Forecasts Reanalysis (ERA)-Interim reanalysis product. This study demonstrates that atmospheric cross-winds can be estimated directly from infrasound data using propagation time and backazimuth deviation observations. This study finds these cross-wind estimates to be in good agreement with the ERA-Interim reanalysis.

MicroRNA-378-mediated suppression of Runx1 alleviates the aggressive phenotype of triple-negative MDA-MB-231 human breast cancer cells.

The Runx1 transcription factor, known for its essential role in normal hematopoiesis, was reported in limited studies to be mutated or associated with human breast tumor tissues. Runx1 increases concomitantly with disease progression in the MMTV-PyMT transgenic mouse model of breast cancer. Compelling questions relate to mechanisms that regulate Runx1 expression in breast cancer. Here, we tested the hypothesis that dysregulation of Runx1-targeting microRNAs (miRNAs) allows for pathologic increase of Runx1 during breast cancer progression. Microarray profiling of the MMTV-PyMT model revealed significant downregulation of numerous miRNAs predicted to target Runx1. One of these, miR-378, was inversely correlated with Runx1 expression during breast cancer progression in mice and in human breast cancer cell lines MCF7 and triple-negative MDA-MB-231 that represent early- and late-stage diseases, respectively. MiR-378 is nearly absent in MDA-MB-231 cells. Luciferase reporter assays revealed that miR-378 binds the Runx1 3' untranslated region (3'UTR) and inhibits Runx1 expression. Functionally, we demonstrated that ectopic expression of miR-378 in MDA-MB-231 cells inhibited Runx1 and suppressed migration and invasion, while inhibition of miR-378 in MCF7 cells increased Runx1 levels and cell migration. Depletion of Runx1 in late-stage breast cancer cells resulted in increased expression of both the miR-378 host gene PPARGC1B and pre-miR-378, suggesting a feedback loop. Taken together, our study identifies a novel and clinically relevant mechanism for regulation of Runx1 in breast cancer that is mediated by a PPARGC1B-miR-378-Runx1 regulatory pathway. Our results highlight the translational potential of miRNA replacement therapy for inhibiting Runx1 in breast cancer.

ChIP-DIP: A multiplexed method for mapping hundreds of proteins to DNA uncovers diverse regulatory elements controlling gene expression.

Gene expression is controlled by the dynamic localization of thousands of distinct regulatory proteins to precise regions of DNA. Understanding this cell-type specific process has been a goal of molecular biology for decades yet remains challenging because most current DNA-protein mapping methods study one protein at a time. To overcome this, we developed ChIP-DIP (ChIP Done In Parallel), a split-pool based method that enables simultaneous, genome-wide mapping of hundreds of diverse regulatory proteins in a single experiment. We demonstrate that ChIP-DIP generates highly accurate maps for all classes of DNA-associated proteins, including histone modifications, chromatin regulators, transcription factors, and RNA Polymerases. Using these data, we explore quantitative combinations of protein localization on genomic DNA to define distinct classes of regulatory elements and their functional activity. Our data demonstrate that ChIP-DIP enables the generation of 'consortium level', context-specific protein localization maps within any molecular biology lab.

SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a potential mechanism for selective translational suppression upon cellular stress.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5'-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

Prolonged succinylcholine action during electroconvulsive therapy (ECT) after cytarabine, vincristine, and rituximab chemotherapy.

Succinylcholine is a depolarizing neuromuscular blocker frequently used during electroconvulsive therapy. In most patients, the duration of paralysis is brief, allowing for spontaneous respiration shortly after the therapy. We report a case of delayed return of neuromuscular function after succinylcholine administered during electroconvulsive therapy in a 72-year-old man receiving cytarabine, vincristine, and rituximab chemotherapy for chronic lymphocytic leukemia. We hypothesize that an interaction between succinylcholine and one of the chemotherapeutic agents caused the prolongation of paralysis and believe that this is the first reported case of prolonged duration of succinylcholine following this regimen of chemotherapy. Despite this unexpected prolonged neuromuscular blockade, the patient could be treated uneventfully, with attention paid to his respiratory support and with subsequent succinylcholine dose titration to effect.

The Association Between Upper Airway Patency and Speaking Valve Trial Tolerance for Patients With Tracheostomy: A Clinical Retrospective Study and an In Vitro Study.

Purpose Upper airway patency is crucial in a patient's ability to tolerate a one-way speaking valve (SV). Traditional assessment of airway patency is mainly subjective. We developed four noninvasive methods to assess patency (leak volume, transtracheal pressure [TTP], end-tidal CO2, and Mallampati score) in our institution. This study was aimed to evaluate the effectiveness of the four methods and explore the relationship between the patient's upper airway patency and SV trial tolerance. Method A retrospective cohort study was conducted to enroll adult patients with tracheostomies eligible for an SV trial from April 2019 through January 2020. An in vitro study was also implemented to explore the relationship between upper airway patency and noninvasive measurements. Results Forty patients (22 men and 18 women) were included; 16 used SV in-line with mechanical ventilation. Twenty-four patients tolerated an SV trial of > 10 min; they had lower TTP (3.0 [2.0-9.0] vs. 15.0 [9.3-21.3] cm H2O, p < .001), higher leak volume (268.5 ± 177.2 vs. 88.6 ± 99.6 ml, p = .038), and lower percentage of patients with Mallampati Classification IV (16.7 vs. 50.0%, p = .035), compared to the 16 patients who did not tolerate an SV trial. Twenty-two patients with a TTP of ≤ 9 cm H2O had higher percentage tolerating an SV trial than those with a TTP of > 9 cm H2O (86.4 vs. 35.3%, p = .002). The in vitro study demonstrated a strong correlation between upper airway patency and TTP, peak inspiratory flow, and tidal volume inhaled from the upper airway. Conclusions TTP, Mallampati classification, and leak volume can be used to assess upper airway patency for adult patients with tracheostomies undergoing an SV trial. A TTP of ≤ 9 cm H2O might indicate adequate upper airway patency to tolerate the SV trial.

Spontaneous Formation of Melanin from Dopamine in the Presence of Iron.

Parkinson's disease is associated with degeneration of neuromelanin (NM)-containing substantia nigra dopamine (DA) neurons and subsequent decreases in striatal DA transmission. Dopamine spontaneously forms a melanin through a process called melanogenesis. The present study examines conditions that promote/prevent DA melanogenesis. The kinetics, intermediates, and products of DA conversion to melanin in vitro, and DA melanogenesis under varying levels of Fe3+, pro-oxidants, and antioxidants were examined. The rate of melanogenesis for DA was substantially greater than related catecholamines norepinephrine and epinephrine and their precursor amino acids tyrosine and l-Dopa as measured by UV-IR spectrophotometry. Dopamine melanogenesis was concentration dependent on the pro-oxidant species and Fe3+. Melanogenesis was enhanced by the pro-oxidant hydrogen peroxide (EC50 = 500 µM) and decreased by the antioxidants ascorbate (IC50 = 10 µM) and glutathione (GSH; IC50 = 5 µM). Spectrophotometric results were corroborated by tuning a fast-scan cyclic voltammetry system to monitor DA melanogenesis. Evoked DA release in striatal brain slices resulted in NM formation that was prevented by GSH. These findings suggest that DA melanogenesis occurs spontaneously under physiologically-relevant conditions of oxidative stress and that NM may act as a marker of past exposure to oxidative stress.

A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome.

To better understand the impact of chromatin structure on regulation of the prostate cancer transcriptome, we develop high-resolution chromatin interaction maps in normal and prostate cancer cells using in situ Hi-C. By combining the in situ Hi-C data with active and repressive histone marks, CTCF binding sites, nucleosome-depleted regions, and transcriptome profiling, we identify topologically associating domains (TADs) that change in size and epigenetic states between normal and prostate cancer cells. Moreover, we identify normal and prostate cancer-specific enhancer-promoter loops and involved transcription factors. For example, we show that FOXA1 is enriched in prostate cancer-specific enhancer-promoter loop anchors. We also find that the chromatin structure surrounding the androgen receptor (AR) locus is altered in the prostate cancer cells with many cancer-specific enhancer-promoter loops. This creation of 3D epigenomic maps enables a better understanding of prostate cancer biology and mechanisms of gene regulation.

Semiautomated Small-Scale Purification Method for High-Throughput Expression Analysis of Recombinant Proteins.

The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalian transient expression systems. This method assists in the selection of the most promising expression construct(s) or the most favorable expression condition(s) to move forward into large-scale protein production.

CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops.

BACKGROUND: Recent genome-wide association studies (GWAS) have identified more than 100 loci associated with increased risk of prostate cancer, most of which are in non-coding regions of the genome. Understanding the function of these non-coding risk loci is critical to elucidate the genetic susceptibility to prostate cancer. RESULTS: We generate genome-wide regulatory element maps and performed genome-wide chromosome confirmation capture assays (in situ Hi-C) in normal and tumorigenic prostate cells. Using this information, we annotate the regulatory potential of 2,181 fine-mapped prostate cancer risk-associated SNPs and predict a set of target genes that are regulated by prostate cancer risk-related H3K27Ac-mediated loops. We next identify prostate cancer risk-associated CTCF sites involved in long-range chromatin loops. We use CRISPR-mediated deletion to remove prostate cancer risk-associated CTCF anchor regions and the CTCF anchor regions looped to the prostate cancer risk-associated CTCF sites, and we observe up to 100-fold increases in expression of genes within the loops when the prostate cancer risk-associated CTCF anchor regions are deleted. CONCLUSIONS: We identify GWAS risk loci involved in long-range loops that function to repress gene expression within chromatin loops. Our studies provide new insights into the genetic susceptibility to prostate cancer.