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Cells fine-tune their metabolic programs according to nutrient availability in order to maintain homeostasis. This is achieved largely through integrating signaling pathways and the gene expression program, allowing cells to adapt to nutritional change. Dbp2, a member of the DEAD-box RNA helicase family in Saccharomyces cerevisiae, has been proposed to integrate gene expression with cellular metabolism. Prior work from our laboratory has reported the necessity of DBP2 in proper gene expression, particularly for genes involved in glucose-dependent regulation. Here, by comparing differentially expressed genes in dbp2Δ to those of 700 other deletion strains from other studies, we find that CYC8 and TUP1, which form a complex and inhibit transcription of numerous genes, corepress a common set of genes with DBP2. Gene ontology (GO) annotations reveal that these corepressed genes are related to cellular metabolism, including respiration, gluconeogenesis, and alternative carbon-source utilization genes. Consistent with a direct role in metabolic gene regulation, loss of either DBP2 or CYC8 results in increased cellular respiration rates. Furthermore, we find that corepressed genes have a propensity to be associated with overlapping long noncoding RNAs and that upregulation of these genes in the absence of DBP2 correlates with decreased binding of Cyc8 to these gene promoters. Taken together, this suggests that Dbp2 integrates nutrient availability with energy homeostasis by maintaining repression of glucose-repressed, Cyc8-targeted genes across the genome. [ABSTRACT FROM AUTHOR]

RNA helicases play fundamental roles in modulating RNA structures and facilitating RNA–protein (RNP) complex assembly in vivo . Previously, our laboratory demonstrated that the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae is required to promote efficient assembly of the co-transcriptionally associated mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A) + RNA. We also found that Yra1 associates directly with Dbp2 and functions as an inhibitor of Dbp2-dependent duplex unwinding, suggestive of a cycle of unwinding and inhibition by Dbp2. To test this, we undertook a series of experiments to shed light on the order of events for Dbp2 in co-transcriptional mRNP assembly. We now show that Dbp2 is recruited to chromatin via RNA and forms a large, RNA-dependent complex with Yra1 and Mex67. Moreover, single-molecule fluorescence resonance energy transfer and bulk biochemical assays show that Yra1 inhibits unwinding in a concentration-dependent manner by preventing the association of Dbp2 with single-stranded RNA. This inhibition prevents over-accumulation of Dbp2 on mRNA and stabilization of a subset of RNA polymerase II transcripts. We propose a model whereby Yra1 terminates a cycle of mRNP assembly by Dbp2. [ABSTRACT FROM AUTHOR]

Summary Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S . cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch. [ABSTRACT FROM AUTHOR]

Cellular homeostasis requires a fine balance between energy uptake, utilization, and growth. Dbp2 is a member of the DEAD-box protein family in Saccharomyces cerevisiae with characterized ATPase and helicase activity in vitro. DEAD-box RNA helicases are a class of enzymes that utilize ATP hydrolysis to remodel RNA and/or RNA-protein (RNP) composition. Dbp2 has been proposed to utilize its helicase activity in vivo to promote RNA-protein complex assembly of both messenger (m)RNAs and long noncoding (lnc)RNAs. Previous work from our laboratory demonstrated that loss of DBP2 enhances the lncRNA-dependent transcriptional induction of the GAL genes by abolishing glucose-dependent repression. Herein, we report that either a carbon source switch or glucose deprivation results in rapid export of Dbp2 to the cytoplasm. Genome-wide RNA sequencing identified a new class of antisense hexose transporter transcripts that are specifically upregulated upon loss of DBP2. Further investigation revealed that both sense and antisense hexose transporter (HXT) transcripts are aberrantly expressed in DBP2-deficient cells and that this expression pathway can be partially mimicked in wild-type cells by glucose depletion. We also find that Dbp2 promotes ribosome biogenesis and represses alternative ATP-producing pathways, as loss of DBP2 alters the transcript levels of ribosome biosynthesis (snoRNAs and associated proteins) and respiration gene products. This suggests that Dbp2 is a key integrator of nutritional status and gene expression programs required for energy homeostasis. [ABSTRACT FROM AUTHOR]

DEAD-box proteins are a class of RNA-dependent ATP hydrolysis enzymes that rearrangeRNAand RNA-protein (ribonucleoprotein) complexes. In an effort to characterize the cellular function of individual DEAD-box proteins, our laboratory has uncovered a previously unrecognized link between the DEAD-box protein Dbp2 and the regulation of transcription in Saccharomyces cerevisiae. Here, we report that Dbp2 is a double- stranded RNA-specific ATPase that associates directly with chromatin and is required for transcriptional fidelity. In fact, loss of DBP2 results in multiple gene expression defects, including accumulation of noncoding transcripts, inefficient 3_ end formation, and appearance of aberrant transcriptional initiation products.Wealso show that loss of DBP2 is synthetic lethal with deletion of the nuclearRNAdecay factor, RRP6, pointing to a global role for Dbp2 in prevention of aberrant transcriptional products. Taken together, we present a model whereby Dbp2 functions to cotranscriptionally modulate RNA structure, a process that facilitates ribonucleoprotein assembly and clearance of transcripts from genomic loci. These studies suggest that Dbp2 is a missing link in RNA quality control that functions to maintain the fidelity of transcriptional processes. [ABSTRACT FROM AUTHOR]

Proteins bound to the poly(A) tail of mRNA transcripts, called poly(A)-binding proteins (Pabs), play critical roles in regulating RNA stability, translation, and nuclear export. Like many mRNA-binding proteins that modulate post-transcriptional processing events, assigning specific functions to Pabs is challenging because these processing events are tightly coupled to one another. To investigate the role that a novel class of zinc finger-containing Pabs plays in these coupled processes, we defined the mode of polyadenosine RNA recognition for the conserved Saccharomyces cerevisiae Nab2 protein and assessed in vivo consequences caused by disruption of RNA binding. The polyadenosine RNA recognition domain of Nab2 consists of three tandem Cys-Cys-Cys-His (CCCH) zinc fingers. Cells expressing mutant Nab2 proteins with decreased binding to polyadenosine RNA show growth defects as well as defects in poly(A) tail length but do not accumulate poly(A) RNA in the nucleus. We also demonstrate genetic interactions between mutant nab2 alleles and mutant alleles of the mRNA 3′-end processing machinery. Together, these data provide strong evidence that Nab2 binding to RNA is critical for proper control of poly(A) tail length. [ABSTRACT FROM AUTHOR]

Prostaglandin D[sub2] activation of the seven-transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases, such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2.Wetested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, the presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced Gα[sub1] and ERK1/2 activation despite enhanced constitutive and agonist-mediated internalization. Enhanced activation of Gα[sub1] proteins and downstream signaling cascades is probably due to the inability of the tail-truncated receptor to recruit β-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits Gα[sub1] signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation. [ABSTRACT FROM AUTHOR]

Introduction: Advanced prostate cancer treatment has improved with androgen receptor signaling inhibitors (ARPI), yet many patients develop metastatic castration-resistant prostate cancer (mCRPC), characterized by sustained androgen receptor (AR) signaling. Bipolar androgen therapy (BAT) introduces supraphysiologic testosterone levels to inhibit tumor growth, offering novel treatment for mCRPC by exploiting AR-dependent mechanisms., Case Presentations: Case 1: A 53-year-old man with mCRPC, post multiple systemic therapies, initiated BAT and pembrolizumab, achieving PSA reduction and improved quality of life before progression. The patient exhibited AR amplification, which may have contributed to favorable response to BAT. Case 2: A 73-year-old man with recurrent prostate cancer, stable on ADT and abiraterone, experienced PSA decline with BAT to an undetectable level, maintaining stability post-therapy discontinuation. Case 3: A 73-year-old man with metastatic prostate cancer, initially resistant to enzalutamide, achieved clinical benefit and disease control with BAT, although he did not meet PSA response criteria, patient had remarkable response upon enzalutamide rechallenge. Case 4: A 90-year-old man with localized prostate cancer, refractory to multiple treatments, experienced symptom relief and PSA reduction with BAT before progression., Conclusion: BAT represents a promising treatment strategy for mCRPC. This case series underscores BAT's potential to induce significant clinical and biochemical responses, resensitize tumors to ARPIs, and improve patients' quality of life. Despite eventual progression in some cases, BAT offers a period of disease control. Further research is needed to optimize patient selection and understand the molecular determinants of BAT responsiveness., (© 2024 Wiley Periodicals LLC.)

Purpose: To study geographic patterns in ophthalmologist supply and patient demand for services in the United States., Methods: Google Trends data for the keywords "ophthalmology" and "ophthalmologist" between 2004 and 2019 were queried and normalized to determine relative search volumes (RSV) for each United States state. Ophthalmologist density was calculated by dividing the number of practicing ophthalmologists by the State Census Bureau population estimates. RSV values were divided by ophthalmologist density and normalized to calculate the relative demand index (RDI) for each state. The number of accredited ophthalmology programs per state was acquired through the Accreditation Council for Graduate Medical Education., Results: Ophthalmologist concentration was highly heterogeneous across the country. The states with the highest concentration of ophthalmologist per 10,000 people were Washington, DC (1.42), Maryland (0.94), Massachusetts (0.87), and New York (0.86), while the lowest were Wyoming (0.19), Idaho (0.36), New Mexico (0.38), and Nevada (0.39). RSVs ranged from 36 (Alaska and North Dakota) to 100 (Michigan). The highest RDI was found in South Dakota (100), Delaware (84), Michigan (66), and Arizona (56). The lowest RDI was in Washington, DC (0), Hawaii (7), Oregon (8), and Montana (14). The highest number of ophthalmology residency programs were in New York (18), Texas (9), and California (9), whereas 12 states lacked residency programs altogether., Conclusions: In this study, we found a wide range in the geographic distribution of ophthalmologists and residency programs in the United States. States with the highest relative demand index may represent areas most at risk of unmet medical needs.

A considerable proportion of the eukaryotic genome undergoes transcription, leading to the generation of noncoding RNA molecules that lack protein-coding information and are not subjected to translation. These noncoding RNAs (ncRNAs) are well recognized to have essential roles in several biological processes. Long noncoding RNAs (lncRNAs) represent the most extensive category of ncRNAs found in the human genome. Much research has focused on investigating the roles of cis-acting lncRNAs in the regulation of specific target gene expression. In the majority of instances, the regulation of sense gene expression by its corresponding antisense pair occurs in a negative (discordant) manner, resulting in the suppression of the target genes. The notion that a negative correlation exists between sense and antisense pairings is, however, not universally valid. In fact, several recent studies have reported a positive relationship between corresponding cis antisense pairs within plants, budding yeast, and mammalian cancer cells. The positive (concordant) correlation between anti-sense and sense transcripts leads to an increase in the level of the sense transcript within the same genomic loci. In addition, mechanisms such as altering chromatin structure, the formation of R loops, and the recruitment of transcription factors can either enhance transcription or stabilize sense transcripts through their antisense pairs. The primary objective of this work is to provide a comprehensive understanding of both aspects of antisense regulation, specifically focusing on the positive correlation between sense and antisense transcripts in the context of eukaryotic gene expression, including its implications towards cancer progression. This article is categorized under: RNA Processing > 3' End Processing Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs., (© 2024 The Author(s). WIREs RNA published by Wiley Periodicals LLC.)

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Background: Little is known whether electronic cigarettes (ECIG) increase vulnerability to future atherosclerotic cardiovascular disease. We determined, using an ex vivo mechanistic atherogenesis assay, whether proatherogenic changes including monocyte transendothelial migration and monocyte-derived foam cell formation are increased in people who use ECIGs., Methods: In a cross-sectional single-center study using plasma and peripheral blood mononuclear cells from healthy participants who are nonsmokers or with exclusive use of ECIGs or tobacco cigarettes (TCIGs), autologous peripheral blood mononuclear cells with patient plasma and pooled peripheral blood mononuclear cells from healthy nonsmokers with patient plasma were utilized to dissect patient-specific ex vivo proatherogenic circulating factors present in plasma and cellular factors present in monocytes. Our main outcomes were monocyte transendothelial migration (% of blood monocyte cells that undergo transendothelial migration through a collagen gel) and monocyte-derived foam cell formation as determined by flow cytometry and the median fluorescence intensity of the lipid-staining fluorochrome BODIPY in monocytes of participants in the setting of an ex vivo model of atherogenesis., Results: Study participants (N=60) had median age of 24.0 years (interquartile range [IQR], 22.0-25.0 years), and 31 were females. Monocyte transendothelial migration was increased in people who exclusively used TCIGs (n=18; median [IQR], 2.30 [ 1.29-2.82]; P <0.001) and in people who exclusively used ECIGs (n=21; median [IQR], 1.42 [ 0.96-1.91]; P <0.01) compared with nonsmoking controls (n=21; median [IQR], 1.05 [0.66-1.24]). Monocyte-derived foam cell formation was increased in people who exclusively used TCIGs (median [IQR], 2.01 [ 1.59-2.49]; P <0.001) and in people who exclusively used ECIGs (median [IQR], 1.54 [ 1.10-1.86]; P <0.001) compared with nonsmoker controls (median [IQR], 0.97 [0.86-1.22]). Both monocyte transendothelial migration and monocyte-derived foam cell formation were higher in TCIG smokers compared with ECIG users and in ECIG users who were former smokers versus ECIG users who were never smokers ( P <0.05 for all comparisons)., Conclusions: The finding of alterations in proatherogenic properties of blood monocytes and plasma in TCIG smokers compared with nonsmokers validates this assay as a strong ex vivo mechanistic tool with which to measure proatherogenic changes in people who use ECIGs. Similar yet significantly less severe alterations in proatherogenic properties of monocytes and plasma were detected in the blood from ECIG users. Future studies are necessary to determine whether these findings are attributable to a residual effect of prior smoking or are a direct effect of current ECIG use., Competing Interests: Disclosures None.

PHO84 is a budding yeast gene reported to be negatively regulated by its cognate antisense transcripts both in cis and in trans. In this study, we performed Transient-transcriptome sequencing (TT-seq) to investigate the correlation of sense/antisense pairs in a dbp2Δ strain and found over 700 sense/antisense pairs, including PHO84, to be positively correlated, contrasting the prevailing model. To define what mechanism regulates the PHO84 gene and how this regulation could have been originally attributed to repression by the antisense transcript, we conducted a series of molecular biology and genetics experiments. We now report that the 3' untranslated region (3'UTR) of PHO84 plays a repressive role in sense expression, an activity not linked to the antisense transcripts. Moreover, we provide results of a genetic screen for 3'UTR-dependent repression of PHO84 and show that the vast majority of identified factors are linked to negative regulation. Finally, we show that the PHO84 promoter and terminator form gene loops which correlate with transcriptional repression, and that the RNA-binding protein, Tho1, increases this looping and the 3'UTR-dependent repression. Our results negate the current model for antisense non-coding transcripts of PHO84 and suggest that many of these transcripts are byproducts of open chromatin., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Revised diagnostic criteria for myeloid neoplasms (MN) issued by the International Consensus Classification (ICC) and the World Health Organization (WHO) recommended major change pertaining to TP53-mutated (TP53 mut ) MN. However, these assertions have not been specifically examined in therapy-related myeloid neoplasm (t-MN), a subset enriched with TP53 mut . We analyzed 488 t-MN patients for TP53 mut . At least one TP53 mut with variant allele frequency (VAF) ≥ 2% with or without loss of TP53 locus was noted in 182 (37.3%) patients and 88.2% of TP53 mut t-MN had a VAF ≥10%. TP53 mut t-MN with VAF ≥ 10% had a distinct clinical and biological profile compared to both TP53 mut VAF < 10% and wild-type TP53 (TP53 wt ) cases. Notably, TP53 mut VAF ≥ 10% had a significantly shorter survival compared to TP53 wt (8.3 vs. 21.6 months; P < 0.001), while the survival of TP53 mut VAF < 10% was comparable to TP53 wt . Within TP53 mut VAF ≥ 10% cohort, the inferior outcomes persisted irrespective of the single- or multi-hit status, co-mutation pattern, or treatments received. Finally, survival of TP53 mut patients was poor across all the blast categories and MDS patients with >10% blasts had inferior survival compared to <5%. In summary, TP53 mut VAF ≥10% signified a clinically and molecularly homogenous cohort regardless of the allelic status., (© 2023. Crown.)

The Royal College of Anaesthetists was commissioned by the United Kingdom Health Quality Partnership to conduct the National Emergency Laparotomy Audit of England and Wales (NELA), to compare outcomes of patients undergoing emergency laparotomy in order to promote quality improvement. Prior to 2016 there were minimal data for emergency laparotomy patients in Australia. The aim of this cohort study was to assess the utility and applicability of the NELA model in a tertiary centre in Western Australia. NELA-related data of patients who underwent emergency laparotomy, between June 2018 and May 2020, were merged with other administrative databases and clinical records. The discriminative ability and calibration of the model were assessed by the area under the receiver operating characteristic (AUROC) curve and calibration plot, respectively. Cox proportional hazards regression was used to assess whether the NELA-predicted risks were an independent predictor of hospital mortality. Of the 502 patients included, 168 (33.5%) patients had a NELA-predicted risk >10%, and of these, 93 (55.4%) were admitted to a critical care unit in a planned fashion immediately after surgery. The NELA model had a good ability to discriminate between survivors and non-survivors (AUROC 0.892, 95% confidence intervals 0.854 to 0.93, P <0.001). However, the model was not perfectly calibrated, with the predicted risks tending to overestimate the observed risks of mortality, especially when the predicted risks were >50%. A high NELA-predicted risk remained significantly associated with mortality after adjusting for other covariates, including sepsis and plasma lactate concentration, suggesting that it is a reliable screening tool for identifying high-risk patients requiring emergency laparotomy.

Enterobacterales have developed a specialized outer membrane polysaccharide (enterobacterial common antigen [ECA]). ECA biosynthesis begins on the cytoplasmic side of the inner membrane (IM) where glycosyltransferases sequentially add sugar moieties to form a complete repeat unit which is then translocated across the IM by WzxE before being polymerized into short linear chains by WzyE/WzzE. Research into WecG, the enzyme responsible for generating ECA lipid-II, has not progressed beyond Barr et al. (1988) who described WecG as a membrane protein. Here we revise our understanding of WecG and re-characterize it as a peripherally associated membrane protein. Through the use of Western immunoblotting we show that WecG in Shigella flexneri is maintained to the IM via its three C-terminal helices and further identify key residues in helix II which are critical for this interaction which has allowed us to identify WecG as a GT-E glycosyltransferase. We investigate the possibility of protein complexes and ultimately show that ECA lipid-I maintains WecG to the membrane which is crucial for its function. This research is the first since Barr et al. (1988) to investigate the biochemistry of WecG and reveals possible novel drug targets to inhibit WecG and thus ECA function and cell viability., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Enterobacteriales have evolved a specialized outer membrane polysaccharide [Enterobacterial Common Antigen (ECA)] which allows them to persist in various environmental niches. Biosynthesis of ECA initiates on the cytoplasmic leaflet of the inner membrane (IM) where glycosyltransferases assemble ECA repeat units (RUs). Complete RUs are then translocated across the IM and assembled into polymers by ECA-specific homologues of the Wzy-dependent pathway. Consisting of the membrane proteins Wzx, Wzy and Wzz, the Wzy-dependent pathway is the most common polysaccharide biosynthetic pathway in Gram-negative bacteria where it is most notably involved in LPS O antigen (Oag) biosynthesis. As such, the majority of research directed towards these proteins has been orientated towards Oag biosynthetic homologues with little directed towards ECA homologues. Belonging to the Shape, Elongation, Division and Sporulation (SEDS) protein family, Wzy proteins are polymerases, and are characterized as possessing little or no peptide homology among homologues as well as being polytopic membrane proteins with functionally relevant residues within periplasmic loops, as defined by C-terminal reporter fusion topology mapping. Here, we present the first the first major study into the ECA polymerase WzyE. Multiple sequence alignments and topology mapping showed that WzyE is unlike WzyB proteins involved with Oag biosynthesis WzyE displays high peptide conservation across Enterobacteriales . In silico structures and reporter mapping allowed us to identify possible functionally conserved residues with WzyE SF 's periplasmic loops, which we showed were crucial for its function. This work provides novel insight into Wzy proteins and suggests that WzyE is an optimal model to investigate Wzy proteins and the Wzy-dependent pathway.

Purpose: The underlying premise of prostate cancer active surveillance (AS) is that cancers likely to metastasize will be recognized and eliminated before cancer-related disease can ensue. Our study was designed to determine the prostate cancer upgrading rate when biopsy guided by magnetic resonance imaging (MRGBx) is used before entry and during AS., Materials and Methods: The cohort included 519 men with low- or intermediate-risk prostate cancer who enrolled in prospective studies (NCT00949819 and NCT00102544) between February 2008 and February 2020. Subjects were preliminarily diagnosed with Gleason Grade Group (GG) 1 cancer; AS began when subsequent MRGBx confirmed GG1 or GG2. Participants underwent confirmatory MRGBx (targeted and systematic) followed by surveillance MRGBx approximately every 12 to 24 months. The primary outcome was tumor upgrading to ≥GG3., Results: Upgrading to ≥GG3 was found in 92 men after a median followup of 4.8 years (IQR 3.1-6.5) after confirmatory MRGBx. Upgrade-free probability after 5 years was 0.85 (95% CI 0.81-0.88). Cancer detected in a magnetic resonance imaging lesion at confirmatory MRGBx increased risk of subsequent upgrading during AS (HR 2.8; 95% CI 1.3-6.0), as did presence of GG2 (HR 2.9; 95% CI 1.1-8.2) In men who upgraded ≥GG3 during AS, upgrading was detected by targeted cores only in 27%, systematic cores only in 25% and both in 47%. In 63 men undergoing prostatectomy, upgrading from MRGBx was found in only 5 (8%)., Conclusions: When AS begins and follows with MRGBx (targeted and systematic), upgrading rate (≥GG3) is greater when tumor is initially present within a magnetic resonance imaging lesion or when pathology is GG2 than when these features are absent.

[Figure: see text].

RNA helicases function in all aspects of RNA biology mainly through remodeling structures of RNA and RNA-protein (RNP) complexes. Among them, DEAD-box proteins form the largest family in eukaryotes and have been shown to remodel RNA/RNP structures and clamping of RNA-binding proteins, both in vitro and in vivo. Nevertheless, for the majority of these enzymes, it is largely unclear what RNAs are targeted and where they modulate RNA/RNP structures to promote RNA metabolism. Several methods have been developed to probe secondary and tertiary structures of specific transcripts or whole transcriptomes in vivo. In this chapter, we describe a protocol for identification of RNA structural changes that are dependent on a Saccharomyces cerevisiae DEAD-box helicase Dbp2. Experiments detailed here can be adapted to the study of other RNA helicases and identification of putative remodeling targets in vivo.

Acute infection, if not rapidly and accurately detected, can lead to sepsis, organ failure and even death. Current detection of acute infection as well as assessment of a patient's severity of illness are imperfect. Characterization of a patient's immune response by quantifying expression levels of specific genes from blood represents a potentially more timely and precise means of accomplishing both tasks. Machine learning methods provide a platform to leverage this host response for development of deployment-ready classification models. Prioritization of promising classifiers is dependent, in part, on hyperparameter optimization for which a number of approaches including grid search, random sampling and Bayesian optimization have been shown to be effective. We compare HO approaches for the development of diagnostic classifiers of acute infection and in-hospital mortality from gene expression of 29 diagnostic markers. We take a deployment-centered approach to our comprehensive analysis, accounting for heterogeneity in our multi-study patient cohort with our choices of dataset partitioning and hyperparameter optimization objective as well as assessing selected classifiers in external (as well as internal) validation. We find that classifiers selected by Bayesian optimization for in-hospital mortality can outperform those selected by grid search or random sampling. However, in contrast to previous research: 1) Bayesian optimization is not more efficient in selecting classifiers in all instances compared to grid search or random sampling-based methods and 2) we note marginal gains in classifier performance in only specific circumstances when using a common variant of Bayesian optimization (i.e. automatic relevance determination). Our analysis highlights the need for further practical, deployment-centered benchmarking of HO approaches in the healthcare context.

Cisplatin is a potent drug used in about 40% of cancer treatment but also leads to severe deafness in 60-80% of the cases. Although the mechanism is known to be related to the accumulation of reactive oxygen species (ROS), no drug or FDA approved treatment is currently available to prevent cisplatin ototoxicity. With this study, we show for the first time that honokiol (HNK), a pleiotropic poly-phenol prevents cisplatin-induced hearing loss. HNK also improves the wellbeing of the mice during the treatment, determined by the increase in the number of surviving animals. In a transgenic tumor mouse model, HNK does not hinder cisplatin's antitumor effect. The mechanism is related to the activation of sirtuin 3, a deacetylase in mitochondria essential for ROS detoxification. We expect a paradigm shift in cisplatin chemotherapy based on the current study and future clinical trials, where honokiol is applied to reduce side effects including hearing loss., Competing Interests: None., (AJCR Copyright © 2020.)

Background Tobacco cigarettes (TCs) increase oxidative stress and inflammation, both instigators of atherosclerotic cardiac disease. It is unknown if electronic cigarettes (ECs) also increase immune cell oxidative stress. We hypothesized an ordered, "dose-response" relationship, with tobacco-product type as "dose" (lowest in nonsmokers, intermediate in EC vapers, and highest in TC smokers), and the "response" being cellular oxidative stress (COS) in immune cell subtypes, in otherwise, healthy young people. Methods and Results Using flow cytometry and fluorescent probes, COS was determined in immune cell subtypes in 33 otherwise healthy young people: nonsmokers (n=12), EC vapers (n=12), and TC smokers (n=9). Study groups had similar baseline characteristics, including age, sex, race, and education level. A dose-response increase in proinflammatory monocytes and lymphocytes, and their COS content among the 3 study groups was found: lowest in nonsmokers, intermediate in EC vapers, and highest in TC smokers. These findings were most striking in CD14 dim CD16 + and CD14 ++ CD16 + proinflammatory monocytes and were reproduced with 2 independent fluorescent probes of COS. Conclusions These findings portend the development of premature cardiovascular disease in otherwise healthy young people who chronically vape ECs. On the other hand, that the COS is lower in EC vapers compared with TC smokers warrants additional investigation to determine if switching to ECs may form part of a harm-reduction strategy. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03823885.

Epigenetic regulation is important for organismal development and response to the environment. Alteration in epigenetic status has been known mostly from the perspective of enzymatic actions of DNA methylation and/or histone modifications. In a genetic screen for cellular factors involved in preventing epigenetic silencing, we isolated an Arabidopsis mutant defective in SAC3B, a component of the conserved TREX-2 complex that couples mRNA transcription with nuleo-cytoplasmic export. Arabidopsis SAC3B dysfunction causes gene silencing at transgenic and endogenous loci, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy. SAC3B dysfunction does not alter promoter DNA methylation level of the transgene d35S::LUC, although the DNA demethylase ROS1 is also required for d35S::LUC anti-silencing. THP1 and NUA were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing. RNA-DNA hybrid exists at the repressed loci but is unrelated to gene suppression by the sac3b mutation. Genome-wide analyses demonstrated minor but clear involvement of SAC3B in regulating siRNAs and DNA methylation, particularly at a group of TAS and TAS-like loci. Together our results revealed not only a critical role of mRNA-export factors in transcriptional anti-silencing but also the contribution of SAC3B in shaping plant epigenetic landscapes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We sought to report incidence, risk factors, and survival related to bowel obstruction in 311 ovarian cancer patients with recurrent disease. A total of 68 (22%) had a documented bowel obstruction during their cancer course, and 49 (16%) developed it after cancer recurrence. Surprisingly, 142 (45%) fit into an "unknown" category (3+ months of data lacking from last contact/death). No risk factors were identified; management included surgery (n = 21), conservative measures (n = 21), and other (n = 7). Documented bowel obstruction was not associated with a statistically significant reduction in survival after cancer recurrence. In conclusion, although bowel obstruction occurs in only a subgroup of patients with ovarian cancer and does not appear to detract from survival after cancer recurrence, limited end-of-life information may be resulting in an underestimation of incidence., (© The Author(s) 2014.)

Autotransporters are a superfamily of virulence factors secreted by Gram negative bacteria. They are comprised of an N-terminal passenger domain that is translocated across the outer membrane and a C-terminal domain that inserts into the outer membrane forming a β-barrel anchor. It is still poorly understood how the passenger is efficiently translocated in the absence of external energy inputs. Several mechanisms have been proposed in solution of this problem, yet due to the vast diversity of size, sequence and function of the passenger, it is not clear how widely these mechanisms are employed. In this study we functionally characterize a conserved repeat found in many passengers that we designate the Passenger-associated Transport Repeat (PATR). Using the autotransporter IcsA from the enteropathogen Shigella flexneri, we identified conserved PATR residues that are required for efficient export of the passenger during growth and infection. Furthermore, PATR-containing autotransporters are significantly larger than non-PATR autotransporters, with PATR copy number correlating with passenger size. We also show that PATR-containing autotransporters delineate a subgroup that associates with specific virulence traits and architectures. These results advance our understanding of autotransporter composition and indicate that an additional transport mechanism is important for thousands of these proteins., (© 2015 John Wiley & Sons Ltd.)

Despite the highly conserved helicase core, individual DEAD-box proteins are specialized in diverse RNA metabolic processes. One mechanism that determines DEAD-box protein specificity is enzymatic regulation by other protein cofactors. In this chapter, we describe a protocol for purifying the Saccharomyces cerevisiae DEAD-box RNA helicase Dbp2 and RNA-binding protein Yra1 and subsequent analysis of helicase regulation. The experiments described here can be adapted to other RNA helicases and their purified cofactor(s).

Current drug discovery campaigns for G protein-coupled receptors (GPCRs) heavily rely on assay technologies that use artificial cell systems tailored to a point-of-contact readout and as a consequence are mostly pathway biased. Recently, we have developed label-free optical biosensor cellular assays that are capable of examining systems cell biology of endogenous receptors and systems cell pharmacology of GPCR ligands in both physiologically and disease relevant environments. We have shown that these biosensor assays enable high-throughput screening of pathway-biased ligands acting on endogenous beta(2)-adrenergic receptor in cells. These biosensor cellular assays hold the potential to reduce attrition rates in drug discovery and development process.