Specific ZNF274 binding interference at SNORD116 activates the maternal transcripts in Prader-Willi syndrome neurons.

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay and hyperphagia/obesity. This disorder is caused by the absence of paternally expressed gene products from chromosome 15q11-q13. We previously demonstrated that knocking out ZNF274, a Kruppel-associated box-A-domain zinc finger protein capable of recruiting epigenetic machinery to deposit the H3K9me3 repressive histone modification, can activate expression from the normally silent maternal allele of SNORD116 in neurons derived from PWS induced pluripotent stem cells (iPSCs). However, ZNF274 has many other targets in the genome in addition to SNORD116. Depleting ZNF274 will surely affect the expression of other important genes and disrupt other pathways. Here, we used CRISPR/Cas9 to delete ZNF274 binding sites at the SNORD116 locus to determine whether activation of the maternal copy of SNORD116 could be achieved without altering ZNF274 protein levels. We obtained similar activation of gene expression from the normally silenced maternal allele in neurons derived from PWS iPSCs, compared with ZNF274 knockout, demonstrating that ZNF274 is directly involved in the repression of SNORD116. These results suggest that interfering with ZNF274 binding at the maternal SNORD116 locus is a potential therapeutic strategy for PWS.

Defining the impact of mutation accumulation on replicative lifespan in yeast using cancer-associated mutator phenotypes.

Mutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Human cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polε) or lagging strand (Polδ) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decreases cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burden.

Zinc finger protein 274 regulates imprinted expression of transcripts in Prader-Willi syndrome neurons.

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay and hyperphagia/obesity and is caused by the absence of paternal contribution to chromosome 15q11-q13. Using induced pluripotent stem cell (iPSC) models of PWS, we previously discovered an epigenetic complex that is comprised of the zinc-finger protein ZNF274 and the SET domain bifurcated 1 (SETDB1) histone H3 lysine 9 (H3K9) methyltransferase and that silences the maternal alleles at the PWS locus. Here, we have knocked out ZNF274 and rescued the expression of silent maternal alleles in neurons derived from PWS iPSC lines, without affecting DNA methylation at the PWS-Imprinting Center (PWS-IC). This suggests that the ZNF274 complex is a separate imprinting mark that represses maternal PWS gene expression in neurons and is a potential target for future therapeutic applications to rescue the PWS phenotype.

Visual scoring of aortic valve calcifications on low-dose CT in lung cancer screening.

OBJECTIVES: To evaluate risk factors for prevalence and progression of aortic valve calcification (AVC) in lung cancer screening participants and also to assess the sensitivity and reliability of visual AVCs on low-dose CT (LDCT) for predicting aortic stenosis (AS) in high-risk smokers. METHODS: We reviewed 1225 consecutive participants in annual LDCT screening for lung cancer at the Mount Sinai Hospital between 2010 and 2017. Sensitivity and specificity of moderate/severe AVC score on LDCT to identify AS on echocardiogram were calculated for 126 participants who had both within 12 months. Using regression analyses, risk factors for AVC at baseline, for progression, and for new AVC on annual rounds of screening were identified. Reliability of AVC assessment on LDCT was assessed by comparing visual AVC scores (1) with standard-dose, electrocardiography (ECG)-gated CT for 31 participants who had both within 12 months and (2) with Agatston scores of 1225 participants and by determining (3) the intra-reader agreement of 1225 participants. RESULTS: Visual AVC scores on LDCT had substantial agreement with the severity of AS on echocardiography and substantial inter-observer and excellent intra-observer agreement. Sensitivity and specificity of moderate/severe visual AVC scores for moderate/severe AS on echocardiogram were 100% and 94%, respectively. Significant predictors for baseline AVC were male sex (OR = 2.52), age (OR10 years = 2.87), and coronary artery calcification score (OR = 1.18), the significant predictor for AVC progression after baseline was pack-years of smoking (HR10 packyears = 1.14), and significant predictors for new AVC on annual LDCT were male sex (HR = 1.51), age (HR10 years = 2.17), CAC (HR = 1.09)  and BMI (HR = 1.06). CONCLUSIONS: AVC scores on LDCT should be documented, especially in lung cancer screening program. KEY POINTS: • LDCT screening for lung cancer provides an opportunity to identify lung cancer and cardiovascular disease in asymptomatic smokers. • Visual aortic valve calcification scores could be reliably evaluated on LDCT and had substantial agreement with the severity of aortic valve stenosis on echocardiography. • Sensitivity and specificity of moderate/severe visual AVC scores on LDCT for moderate/severe AS on echocardiogram were 100% and 94%, respectively.

Cardiac Applications of PET-MR.

PURPOSE OF REVIEW: The purpose of this study was to provide an overview of the clinical applications of PET-MR in the setting of cardiac imaging with emphasis on specific scenarios where both techniques together provided added information. RECENT FINDINGS: Synergy of cardiac PET and MR fusion may hold similar promise eliminating ionizing radiation and improving tissue contrast. Future development of new hybrid scanners, use of new imaging tracers, and clinical applications are significant factors which will influence its use. Both positron emission tomography (PET) and cardiac magnetic resonance imaging (CMR) provide important anatomic and physiologic information with regard to the heart. Being able to combine the data from these two examinations in a hybrid technique allows for a more complete evaluation of cardiac pathology. While hybrid PET-CT has already established the utility of a combined imaging approach, the use of CMR in lieu of CT allows for elimination of ionizing radiation and for improved tissue contrast.

Noncanonical amino acids to improve the pH response of pHLIP insertion at tumor acidity.

The pH low insertion peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membranes with a pH50 of 6.1, while most solid tumors have extracellular pH (pH(e)) of 6.5-7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH response. Replacing Asp25 with α-aminoadipic acid (Aad) adjusts the pH50 to 6.74, matching average tumor acidity, and replacing Asp14 with γ-carboxyglutamic acid (Gla) increases the sharpness of pH response (transition over 0.5 instead of 1 pH unit). These effects are additive: the Asp14Gla/Asp25Aad double variant shows a pH50 of 6.79, with sharper transition than Asp25Aad. Furthermore, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn-on fluorescence assays and anti-proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pH 6.6.

Identifying key underlying regulatory networks and predicting targets of orphan C/D box SNORD116 snoRNAs in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder characterized principally by initial symptoms of neonatal hypotonia and failure-to-thrive in infancy, followed by hyperphagia and obesity. It is well established that PWS is caused by loss of paternal expression of the imprinted region on chromosome 15q11-q13. While most PWS cases exhibit megabase-scale deletions of the paternal chromosome 15q11-q13 allele, several PWS patients have been identified harboring a much smaller deletion encompassing primarily SNORD116. This finding suggests SNORD116 is a direct driver of PWS phenotypes. The SNORD116 gene cluster is composed of 30 copies of individual SNORD116 C/D box small nucleolar RNAs (snoRNAs). Many C/D box snoRNAs have been shown to guide chemical modifications of other RNA molecules, often ribosomal RNA (rRNA). However, SNORD116 snoRNAs are termed 'orphans' because no verified targets have been identified and their sequences show no significant complementarity to rRNA. It is crucial to identify the targets and functions of SNORD116 snoRNAs because all reported PWS cases lack their expression. To address this, we engineered two different deletions modelling PWS in two distinct human embryonic stem cell (hESC) lines to control for effects of genetic background. Utilizing an inducible expression system enabled quick, reproducible differentiation of these lines into neurons. Systematic comparisons of neuronal gene expression across deletion types and genetic backgrounds revealed a novel list of 42 consistently dysregulated genes. Employing the recently described computational tool snoGloBe, we discovered these dysregulated genes are significantly enriched for predicted SNORD116 targeting versus multiple control analyses. Importantly, our results showed it is critical to use multiple isogenic cell line pairs, as this eliminated many spuriously differentially expressed genes. Our results indicate a novel gene regulatory network controlled by SNORD116 is likely perturbed in PWS patients.

Combining Initial Radiographs and Clinical Variables Improves Deep Learning Prognostication in Patients with COVID-19 from the Emergency Department.

PURPOSE: To train a deep learning classification algorithm to predict chest radiograph severity scores and clinical outcomes in patients with coronavirus disease 2019 (COVID-19). MATERIALS AND METHODS: In this retrospective cohort study, patients aged 21-50 years who presented to the emergency department (ED) of a multicenter urban health system from March 10 to 26, 2020, with COVID-19 confirmation at real-time reverse-transcription polymerase chain reaction screening were identified. The initial chest radiographs, clinical variables, and outcomes, including admission, intubation, and survival, were collected within 30 days (n = 338; median age, 39 years; 210 men). Two fellowship-trained cardiothoracic radiologists examined chest radiographs for opacities and assigned a clinically validated severity score. A deep learning algorithm was trained to predict outcomes on a holdout test set composed of patients with confirmed COVID-19 who presented between March 27 and 29, 2020 (n = 161; median age, 60 years; 98 men) for both younger (age range, 21-50 years; n = 51) and older (age >50 years, n = 110) populations. Bootstrapping was used to compute CIs. RESULTS: The model trained on the chest radiograph severity score produced the following areas under the receiver operating characteristic curves (AUCs): 0.80 (95% CI: 0.73, 0.88) for the chest radiograph severity score, 0.76 (95% CI: 0.68, 0.84) for admission, 0.66 (95% CI: 0.56, 0.75) for intubation, and 0.59 (95% CI: 0.49, 0.69) for death. The model trained on clinical variables produced an AUC of 0.64 (95% CI: 0.55, 0.73) for intubation and an AUC of 0.59 (95% CI: 0.50, 0.68) for death. Combining chest radiography and clinical variables increased the AUC of intubation and death to 0.88 (95% CI: 0.79, 0.96) and 0.82 (95% CI: 0.72, 0.91), respectively. CONCLUSION: The combination of imaging and clinical information improves outcome predictions.Supplemental material is available for this article.© RSNA, 2020.

CT Findings and Patterns of e-Cigarette or Vaping Product Use-Associated Lung Injury: A Multicenter Cohort of 160 Cases.

BACKGROUND: e-Cigarette or vaping-induced lung injury (EVALI) causes a spectrum of CT lung injury patterns. Relative frequencies and associations with vaping behavior are unknown. RESEARCH QUESTION: What are the frequencies of imaging findings and CT patterns in EVALI and what is the relationship to vaping behavior? STUDY DESIGN AND METHODS: CT scans of 160 subjects with EVALI from 15 institutions were retrospectively reviewed. CT findings and patterns were defined and agreed on via consensus. The parenchymal organizing pneumonia (OP) pattern was defined as regional or diffuse ground-glass opacity (GGO) ± consolidation without centrilobular nodules (CNs). An airway-centered OP pattern was defined as diffuse CNs with little or no GGO, whereas a mixed OP pattern was a combination of the two. Other patterns included diffuse alveolar damage (DAD), acute eosinophilic-like pneumonia, and pulmonary hemorrhage. Cases were classified as atypical if they did not fit into a pattern. Imaging findings, pattern frequencies, and injury severity were correlated with substance vaped (marijuana derives [tetrahydrocannabinol] [THC] only, nicotine derivates only, and both), vaping frequency, regional geography, and state recreational THC legality. One-way analysis of variance, χ2 test, and multivariable analyses were used for statistical analysis. RESULTS: A total of 160 patients (79.4% men) with a mean age of 28.2 years (range, 15-68 years) with EVALI underwent CT scan. Seventy-seven (48.1%), 15 (9.4%), and 68 (42.5%) patients admitted to vaping THC, nicotine, or both, respectively. Common findings included diffuse or lower lobe GGO with subpleural (78.1%), lobular (59.4%), or peribronchovascular (PBV) sparing (40%). Septal thickening (50.6%), lymphadenopathy (63.1%), and CNs (36.3%) were common. PBV sparing was associated with younger age (P = .02). Of 160 subjects, 156 (97.5%) had one of six defined patterns. Parenchymal, airway-centered, and mixed OP patterns were seen in 89 (55.6%), 14 (8.8%), and 32 (20%) patients, respectively. Acute eosinophilic-like pneumonia (six of 160, 3.8%), DAD (nine of 160, 5.6%), pulmonary hemorrhage (six of 160, 3.8%), and atypical (four of 160, 2.5%) patterns were less common. Increased vaping frequency was associated with more severe injury (P = .008). Multivariable analysis showed a negative association between vaping for > 6 months and DAD pattern (P = .03). Two subjects (1.25%) with DAD pattern died. There was no relation between pattern and injury severity, geographic location, and state legality of recreational use of THC. INTERPRETATION: EVALI typically causes an OP pattern but exists on a spectrum of acute lung injury. Vaping habits do not correlate with CT patterns except for negative correlation between vaping > 6 months and DAD pattern. PBV sparing, not previously described in acute lung injury, is a common finding.

Prader-Willi syndrome: reflections on seminal studies and future therapies.

Prader-Willi syndrome (PWS) is caused by the loss of function of the paternally inherited 15q11-q13 locus. This region is governed by genomic imprinting, a phenomenon in which genes are expressed exclusively from one parental allele. The genomic imprinting of the 15q11-q13 locus is established in the germline and is largely controlled by a bipartite imprinting centre. One part, termed the Prader-Willi syndrome imprinting center (PWS-IC), comprises a CpG island that is unmethylated on the paternal allele and methylated on the maternal allele. The second part, termed the Angelman syndrome imprinting centre, is required to silence the PWS_IC in the maternal germline. The loss of the paternal contribution of the imprinted 15q11-q13 locus most frequently occurs owing to a large deletion of the entire imprinted region but can also occur through maternal uniparental disomy or an imprinting defect. While PWS is considered a contiguous gene syndrome based on large-deletion and uniparental disomy patients, the lack of expression of only non-coding RNA transcripts from the SNURF-SNRPN/SNHG14 may be the primary cause of PWS. Patients with small atypical deletions of the paternal SNORD116 cluster alone appear to have most of the PWS related clinical phenotypes. The loss of the maternal contribution of the 15q11-q13 locus causes a separate and distinct condition called Angelman syndrome. Importantly, while much has been learned about the regulation and expression of genes and transcripts deriving from the 15q11-q13 locus, there remains much to be learned about how these genes and transcripts contribute at the molecular level to the clinical traits and developmental aspects of PWS that have been observed.

Residue-specific structures and membrane locations of pH-low insertion peptide by solid-state nuclear magnetic resonance.

The pH-low insertion peptide (pHLIP) binds to a membrane at pH 7.4 unstructured but folds across the bilayer as a transmembrane helix at pH∼6. Despite their promising applications as imaging probes and drug carriers that target cancer cells for cytoplasmic cargo delivery, the mechanism of pH modulation on pHLIP-membrane interactions has not been completely understood. Here, we show the first study on membrane-associated pHLIP using solid-state NMR spectroscopy. Data on residue-specific conformation and membrane location describe pHLIP in various surface-bound and membrane-inserted states at pH 7.4, 6.4 and 5.3. The critical membrane-adsorbed state is more complex than previously envisioned. At pH 6.4, for the major unstructured population, the peptide sinks deeper into the membrane in a state II' that is distinct from the adsorbed state II observed at pH 7.4, which may enable pHLIP to sense slight change in acidity even before insertion.

Phase I study of oxaliplatin in combination with gemcitabine, irinotecan, and 5-fluorouracil/leucovorin (G-FLIE) in patients with metastatic solid tumors including adenocarcinoma of the pancreas.

PURPOSE: The aims of this study were to establish the maximum tolerated dose (MTD) of oxaliplatin in combination with fixed doses of gemcitabine, irinotecan, and 5-fluorouracil/leucovorin (G-FLIE) in solid tumors, including advanced pancreatic cancer, and to evaluate the toxicity of the regimen. METHODS: Patients with metastatic solid tumors were treated with a regimen consisting of gemcitabine (500 mg/m(2) by fixed-dose-rate infusion), irinotecan (120 mg/m(2)), leucovorin 300 mg, bolus/infusion 5-fluorouracil (400 and 1,500 mg/m2, respectively), and oxaliplatin at doses from 50 to 85 mg/m(2) according to the escalation schema. Treatment was repeated every 14 days. RESULTS: The study enrolled 25 patients with a median age of 64 years and median Karnofsky performance score of 80. Patients had metastatic adenocarcinomas of pancreas (n = 9), as well as gastroesointestinal, hepatobiliary, or unknown primary tumors. With only one dose limiting toxicity (neutropenia and constipation), the MTD of oxaliplatin was not reached up to the pre-specified maximum level of 85 mg/m(2). Other toxicities predictably included cytopenias, fatigue, sensory neuropathy, nausea/vomiting, diarrhea, and constipation. Four partial responses and ten disease stabilizations were observed. The overall median time to disease progression was 17 weeks (2-110 weeks) with median overall survival of 31.5 weeks (7-139 weeks). CONCLUSIONS: G-FLIE is a tolerable multi-agent chemotherapy regimen with the oxaliplatin dose up to 85 mg/m(2). The combination of full-dose oxaliplatin with gemcitabine, irinotecan, and 5-fluorouracil is feasible with attenuated doses of the drugs, but further optimization is necessary before assessment of efficacy.

Rational design of DNA sequence-based strategies for subtyping Listeria monocytogenes.

The ability to differentiate bacteria beyond the species level is essential for identifying and tracking infectious disease outbreaks and to improve our knowledge of the population genetics, epidemiology, and ecology of bacterial pathogens. Commonly used subtyping methods, such as serotyping, phage typing, ribotyping, and pulsed-field gel electrophoresis, can yield ambiguous results that are difficult to standardize and share among laboratories. DNA sequence-based subtyping strategies can reduce interpretation ambiguity. We report the development of a rational approach for designing sequence-based subtyping methods. Listeria monocytogenes was selected as the model organism for testing the efficacy of this approach. Two housekeeping genes (recA and prs), one stress response gene (sigB), two virulence genes (actA and inlA), and two intergenic regions (hly-mpl and plcA-hly) were sequenced for 15 L. monocytogenes isolates. Isolates were chosen from a representative collection of more than 1,000 L. monocytogenes isolates to reflect the genetic diversity of this species. DNA sequences were aligned, and sliding window analyses were performed for each gene to define 600-bp-long regions that were (i) most polymorphic (using ProSeq) or (ii) most discriminatory (using a new algorithm implemented in WINDOWMIN). Complete gene sequences for actA (1,929 bp) and inlA (2,235 bp) provided the highest discrimination (identifying 15 and 14 allelic types, respectively). WINDOWMIN allowed identification of 600-bp regions within these genes that provided similar discriminatory power (yielding 15 and 13 allelic types, respectively). The most discriminatory 600-bp fragments identified in the housekeeping and stress response genes differentiated the isolates into 8 to 10 subtypes; intergenic region sequences yielded 8 and 12 allelic types based on 335- and 242-bp sequences for hly-mpl and plcA-hly, respectively. Regions identified as most polymorphic were not necessarily most discriminatory; therefore, application of the WINDOWMIN algorithm provided a powerful tool for determining the best target regions for DNA sequence-based subtyping. Our specific results also show that inclusion of virulence gene target sequences in a DNA sequence-based subtyping scheme for L. monocytogenes is necessary to achieve maximum subtype differentiation.