Spindle Cell Carcinoma Presenting as a Massive Pleural Effusion.

The lung's sarcomatoid carcinomas (SC) are a heterogeneous sporadic group of non-small cell lung carcinomas (NSCLCs) and are very challenging to diagnose and treat. Spindle cell carcinoma (SpCC) is a very rare subset of this group. Hence, the prognosis and treatments are unclear due to the limited literature available. The presentation of this cancer varies based on the site of the neoplasm and the complications and metastases observed at the time of diagnosis. Here, we report a 73-year-old man who presented to the emergency room after two months of worsening dyspnea and fatigue. Chest X-ray showed an extensive left-sided pleural effusion. A computed tomography (CT) scan of the chest showed a pleural-based mass that came back as SpCC, for which he was referred to a university hospital.

Splenic Surprise: Investigating a Case of Splenic Infarct as an Isolated COVID-19 Manifestation.

Coronavirus disease 2019 (COVID-19) infection has been associated with a multitude of complications, one established complication being thromboembolism, a result of the proinflammatory state induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This prothrombotic state is a cumulation of many inflammatory pathways at work. Here, we present an interesting case of a 43-year-old female who did not present with the typical COVID-19 clinical picture. Instead, she presented with periumbilical pain, nausea, and vomiting. Upon further investigation, she was found to have a splenic infarct on a computed tomography (CT) scan. An extensive workup was performed to explore possible etiologies; however, it was concluded that her splenic infarct was secondary to her COVID-19 infection. With this case, we aim to add to the literature regarding the manifestations of the prothrombotic state of SARS-CoV-2.

Cranial Conundrum: An Unusual Case of an Epidermoid Cyst in the Prepontine Cistern.

We present a fascinating case of a patient who suffered from persistent headaches for three months due to an epidermoid cyst located in the prepontine cistern. Epidermoid cysts are a very uncommon type of intracranial tumor, known for their slow growth and gradual onset of neurological symptoms. In this particular case, our patient, a 35-year-old, experienced a headache that was accompanied by dizziness, photophobia, and pain when moving their eyes. Further imaging revealed a cystic lesion in the prepontine cistern, which had a mass effect on the pons. After confirming the lesion was likely an epidermoid cyst through an MRI, the patient underwent surgery to have it removed. We hope to highlight the rarity of this type of tumor and its unique features when viewed through imaging.

Leveraging multisource feedback to address diversity, equity, and inclusivity.

Multisource feedback has long been a recommended tool to assess clinical competencies within graduate medical education. Additionally, incorporating feedback supplied by patients and other members of the healthcare team can provide the framework to bridge perspectives and viewpoints that may be different from their own. This, in effect, can aid in fortifying values in diversity, equity, and inclusivity by developing more knowledgeable, empathetic, and respectful future healthcare providers.

Protocol for the generation of Symbiodiniaceae mutants using UV mutagenesis.

Genetic approaches are limited in the dinoflagellate family, Symbiodiniaceae, causing a bottleneck in the discovery of useful mutants toward the goal of preventing future coral bleaching events. In this protocol, we demonstrate the application of UV exposure, coupled with downstream phenotypic screening and mutant isolation, to form a UV mutagenesis pipeline. This pipeline provides an avenue to generate Symbiodiniaceae mutants to help link genotype to phenotype, as well as address previously unanswered questions surrounding relationships with host organisms, like coral. For complete details on the use and execution of this protocol, please refer to Jinkerson et al. (2022).1.

Yeast Puf3p-mediated mRNA decay is regulated by carbon source-specific differential interaction of Puf3p with Pop2p and Yak1p.

Puf3p regulates the stability of nuclear-encoded mRNAs acting in mitochondrial biogenesis and function in Saccharomyces cerevisiae. This work identifies the phosphorylation of Pop2p, a component of the deadenylase complex, as being critical for adapting Puf3p-mediated mRNA decay upon carbon source alterations. We demonstrate that the Puf3p-Pop2p association diminishes in mitochondria-reliant conditions and establish Yak1p, a kinase that phosphorylates Pop2p at threonine 97, as a new player in Puf3p-mediated regulation of mRNA decay. Yak1p deletion alters the half-life of Puf3p target mRNAs. Our findings outline a metabolism-driven regulatory switch, whereby, in mitochondria-independent conditions, Puf3p recruits Pop2p and the decay machinery to bound mRNAs for rapid decay. Conversely, in mitochondria-reliant conditions, the association of Puf3p with Yak1p increases, placing Yak1p proximal to neighboring Pop2p. Subsequent Pop2p phosphorylation reduces the Puf3p-Pop2p interaction and stabilizes Puf3p target mRNAs.

Cnidarian-Symbiodiniaceae symbiosis establishment is independent of photosynthesis.

Photosynthesis shapes the symbiotic relationships between cnidarians and Symbiodiniaceae algae-with many cnidarian hosts requiring symbiont photosynthate for survival-but little is known about how photosynthesis impacts symbiosis establishment. Here, we show that during symbiosis establishment, infection, proliferation, and maintenance can proceed without photosynthesis, but the ability to do so is dependent on specific cnidarian-Symbiodiniaceae relationships. The evaluation of 31 pairs of symbiotic relationships (five species of Symbiodiniaceae in sea anemone, coral, and jellyfish hosts) revealed that infection can occur without photosynthesis. A UV mutagenesis method for Symbiodiniaceae was established and used to generate six photosynthetic mutants that can infect these hosts. Without photosynthesis, Symbiodiniaceae cannot proliferate in the sea anemone Aiptasia or jellyfish Cassiopea but can proliferate in the juvenile polyps of the coral Acropora. After 6 months of darkness, Breviolum minutum is maintained within Aiptasia, indicating that Symbiodiniaceae maintenance can be independent of photosynthesis. Manipulating photosynthesis provides insights into cnidarian-Symbiodiniaceae symbiosis.

Development of CVN424: A Selective and Novel GPR6 Inverse Agonist Effective in Models of Parkinson Disease.

GPR6 is an orphan G-protein-coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT: CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.

YTHDF2 destabilizes m6A-modified neural-specific RNAs to restrain differentiation in induced pluripotent stem cells.

N6-methyladenosine (m6A) is an abundant post-transcriptional modification that can impact RNA fate via interactions with m6A-specific RNA binding proteins. Despite accumulating evidence that m6A plays an important role in modulating pluripotency, the influence of m6A reader proteins in pluripotency is less clear. Here, we report that YTHDF2, an m6A reader associated with mRNA degradation, is highly expressed in induced pluripotent stem cells (iPSCs) and down-regulated during neural differentiation. Through RNA sequencing, we identified a group of m6A-modified transcripts associated with neural development that are directly regulated by YTDHF2. Depletion of YTHDF2 in iPSCs leads to stabilization of these transcripts, loss of pluripotency, and induction of neural-specific gene expression. Collectively, our results suggest YTHDF2 functions to restrain expression of neural-specific mRNAs in iPSCs and facilitate their rapid and coordinated up-regulation during neural induction. These effects are both achieved by destabilization of the targeted transcripts.

Zika virus noncoding sfRNAs sequester multiple host-derived RNA-binding proteins and modulate mRNA decay and splicing during infection.

Insect-borne flaviviruses produce a 300-500-base long noncoding RNA, termed subgenomic flavivirus RNA (sfRNA), by stalling the cellular 5'-3'-exoribonuclease 1 (XRN1) via structures located in their 3' UTRs. In this study, we demonstrate that sfRNA production by Zika virus represses XRN1 analogous to what we have previously shown for other flaviviruses. Using protein-RNA reconstitution and a stringent RNA pulldown assay with human choriocarcinoma (JAR) cells, we demonstrate that the sfRNAs from both dengue type 2 and Zika viruses interact with a common set of 21 RNA-binding proteins that contribute to the regulation of post-transcriptional processes in the cell, including splicing, RNA stability, and translation. We found that four of these sfRNA-interacting host proteins, DEAD-box helicase 6 (DDX6) and enhancer of mRNA decapping 3 (EDC3) (two RNA decay factors), phosphorylated adaptor for RNA export (a regulator of the biogenesis of the splicing machinery), and apolipoprotein B mRNA-editing enzyme catalytic subunit 3C (APOBEC3C, a nucleic acid-editing deaminase), inherently restrict Zika virus infection. Furthermore, we demonstrate that the regulations of cellular mRNA decay and RNA splicing are compromised by Zika virus infection as well as by sfRNA alone. Collectively, these results reveal the large extent to which Zika virus-derived sfRNAs interact with cellular RNA-binding proteins and highlight the potential for widespread dysregulation of post-transcriptional control that likely limits the effective response of these cells to viral infection.

Engineered viral RNA decay intermediates to assess XRN1-mediated decay.

Both RNA synthesis and decay must be balanced within a cell to achieve proper gene expression. Additionally, modulation of RNA decay specifically offers the cell an opportunity to rapidly reshape the transcriptome in response to specific stimuli or cues. Therefore, it is critical to understand the underlying mechanisms through which RNA decay contribute to gene expression homeostasis. Cell-free reconstitution approaches have been used successfully to reveal mechanisms associated with numerous post-transcriptional RNA processes. Historically, it has been difficult to examine all aspects of RNA decay in such an in vitro setting due, in part, to limitations on the ability to resolve larger RNAs through denaturing polyacrylamide gels. Thus, in vitro systems to study RNA decay rely on smaller, less biologically relevant RNA fragments. Herein, we present an approach to more confidently examine RNA decay parameters of large mRNA size transcripts through the inclusion of an engineered XRN1-resistant reporter RNA (xrRNA). By placing a 67 nucleotide xrRNA near the 3' end of any in vitro transcribed RNA with variable size or sequence context, investigators can observe the accumulation of the xrRNA as a readout of exoribonuclease-mediated 5'-3' decay. This approach may allow in vitro RNA decay assays to include full biologically relevant mRNA/mRNPs, extending their utility and allow improved experimental design considerations to promote biologically relevant outcomes.

Sequences encoding C2H2 zinc fingers inhibit polyadenylation and mRNA export in human cells.

The large C2H2-Zinc Finger (C2H2-ZNF) gene family has rapidly expanded in primates through gene duplication. There is consequently considerable sequence homology between family members at both the nucleotide and amino acid level, allowing for coordinated regulation and shared functions. Here we show that multiple C2H2-ZNF mRNAs experience differential polyadenylation resulting in populations with short and long poly(A) tails. Furthermore, a significant proportion of C2H2-ZNF mRNAs are retained in the nucleus. Intriguingly, both short poly(A) tails and nuclear retention can be specified by the repeated elements that encode zinc finger motifs. These Zinc finger Coding Regions (ZCRs) appear to restrict polyadenylation of nascent RNAs and at the same time impede their export. However, the polyadenylation process is not necessary for nuclear retention of ZNF mRNAs. We propose that inefficient polyadenylation and export may allow C2H2-ZNF mRNAs to moonlight as non-coding RNAs or to be stored for later use.

Weather-Based Models for Assessing the Risk of Sclerotinia sclerotiorum Apothecial Presence in Soybean (Glycine max) Fields.

Sclerotinia stem rot (SSR) epidemics in soybean, caused by Sclerotinia sclerotiorum, are currently responsible for annual yield reductions in the United States of up to 1 million metric tons. In-season disease management is largely dependent on chemical control but its efficiency and cost-effectiveness depends on both the chemistry used and the risk of apothecia formation, germination, and further dispersal of ascospores during susceptible soybean growth stages. Hence, accurate prediction of the S. sclerotiorum apothecial risk during the soybean flowering period could enable farmers to improve in-season SSR management. From 2014 to 2016, apothecial presence or absence was monitored in three irrigated (n = 1,505 plot-level observations) and six nonirrigated (n = 2,361 plot-level observations) field trials located in Iowa (n = 156), Michigan (n = 1,400), and Wisconsin (n = 2,310), for a total of 3,866 plot-level observations. Hourly air temperature, relative humidity, dew point, wind speed, leaf wetness, and rainfall were also monitored continuously, throughout the season, at each location using high-resolution gridded weather data. Logistic regression models were developed for irrigated and nonirrigated conditions using apothecial presence as a binary response variable. Agronomic variables (row width) and weather-related variables (defined as 30-day moving averages, prior to apothecial presence) were tested for their predictive ability. In irrigated soybean fields, apothecial presence was best explained by row width (r = -0.41, P < 0.0001), 30-day moving averages of daily maximum air temperature (r = 0.27, P < 0.0001), and daily maximum relative humidity (r = 0.16, P < 0.05). In nonirrigated fields, apothecial presence was best explained by using moving averages of daily maximum air temperature (r = -0.30, P < 0.0001) and wind speed (r = -0.27, P < 0.0001). These models correctly predicted (overall accuracy of 67 to 70%) apothecial presence during the soybean flowering period for four independent datasets (n = 1,102 plot-level observations or 30 daily mean observations).

Trick or TREAT: A Scary-Good New Approach for Single-Molecule mRNA Decay Analysis.

In this issue of Molecular Cell, Horvathova et al. (2017) have developed a powerful approach to single-molecule assessment of RNA decay in living cells by exploiting the ability of flavivirus RNA structural elements to trap XRN1 decay intermediates in dual-labeled reporter constructs.

Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability.

Changes in the rate of mRNA decay are closely coordinated with transcriptional changes and together these events have profound effects on gene expression during development and disease. Traditional approaches to assess mRNA decay have relied on inhibition of transcription, which can alter mRNA decay rates and confound interpretation. More recently, metabolic labeling combined with chemical modification and fractionation of labeled RNAs has allowed the isolation of nascent transcripts and the subsequent calculation of mRNA decay rates. This approach has been widely adopted for measuring mRNA half-lives on a global scale, but has proven challenging to use for analysis of single genes. We present a series of normalization and quality assurance steps to be used in combination with 4-thiouridine pulse labeling of cultured eukaryotic cells. Importantly, we demonstrate how the relative amount of 4sU-labeled nascent RNA influences accurate quantification. The approach described facilitates reproducible measurement of individual mRNA half-lives using 4-thiouridine and could be adapted for use with other nucleoside analogs.

The CELF1 RNA-Binding Protein Regulates Decay of Signal Recognition Particle mRNAs and Limits Secretion in Mouse Myoblasts.

We previously identified several mRNAs encoding components of the secretory pathway, including signal recognition particle (SRP) subunit mRNAs, among transcripts associated with the RNA-binding protein CELF1. Through immunoprecipitation of RNAs crosslinked to CELF1 in myoblasts and in vitro binding assays using recombinant CELF1, we now provide evidence that CELF1 directly binds the mRNAs encoding each of the subunits of the SRP. Furthermore, we determined the half-lives of the Srp transcripts in control and CELF1 knockdown myoblasts. Our results indicate CELF1 is a destabilizer of at least five of the six Srp transcripts and that the relative abundance of the SRP proteins is out of balance when CELF1 is depleted. CELF1 knockdown myoblasts exhibit altered secretion of a luciferase reporter protein and are impaired in their ability to migrate and close a wound, consistent with a defect in the secreted extracellular matrix. Importantly, similar defects in wound healing are observed when SRP subunit imbalance is induced by over-expression of SRP68. Our studies support the existence of an RNA regulon containing Srp mRNAs that is controlled by CELF1. One implication is that altered function of CELF1 in myotonic dystrophy may contribute to changes in the extracellular matrix of affected muscle through defects in secretion.

Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors.

Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active.

Fragment-based drug discovery of potent and selective MKK3/6 inhibitors.

The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38.

Do You Believe in ReincaRNAtion? Herpesviruses Reveal Connection between RNA Decay and Synthesis.

Many viruses degrade host mRNAs to reduce competition for proteins/ribosomes and promote viral gene expression. In this issue of Cell Host & Microbe, Abernathy et al. (2015) demonstrate that a herpesviral RNA endonuclease induces host transcriptional repression that is mediated through the decay factor Xrn1 and evaded by viral genes.