Shining light on DHX9: UV-induced stress granules illuminate protective mechanisms for daughter cell resilience.

In a recent article in Cell, Zhou et al. investigate the origins, composition, and biological consequences of UV-induced stress granules. They find that UV-induced stress granules are triggered by the formation of RNA-protein crosslinks, uniquely contain DHX9 as a marker, form during mitosis independently of translation repression, and are enriched in intron-containing RNAs and splicing factors. Moreover, UV-induced granules contain double-stranded RNA (dsRNA) and trigger a dsRNA response. This work identifies a mechanism for resolving UV-damaged RNA and broadens the types of cytosolic "stress granules" that form.

First demonstration of a novel nerve-targeting fluorophore in a cohort of ex vivo human tissues.

Iatrogenic nerve injury is a common complication across all surgical specialties. Better nerve visualization and identification during surgery will improve outcomes and reduce nerve injuries. The Gibbs Laboratory at Oregon Health and Science University has developed a library of near-infrared, nerve-specific fluorophores to highlight nerves intraoperatively and aid surgeons in nerve identification and visualization; the current lead agent is LGW16-03. Prior to this study, testing of LGW16-03 was restricted to animal models; therefore, it was unknown how LGW16-03 performs in human tissue. To advance LGW16-03 to clinic, we sought to test this current lead agent in ex vivo human tissues from a cohort of patients and determine if the route of administration affects LGW16-03 fluorescence contrast between nerves and adjacent background tissues (muscle and adipose). LGW16-03 was applied to ex vivo human tissue from lower limb amputations via two strategies: (1) systemic administration of the fluorophore using our first-in-kind model for fluorophore testing, and (2) topical application of the fluorophore. Results showed no statistical difference between topical and systemic administration. However, in vivo human validation of these findings is required.

Acceptability and Feasibility of Delivering Decision Aids to Veterans for Management of Knee Osteoarthritis - A Pilot Study.

Introduction: Decision aids are effective tools in facilitating patient-centered care and patient involvement in the decision-making process. Given unique barriers to providing patient-centered care for Veterans, implementation of decision aids may improve overall quality of care. We aimed to assess the acceptability and feasibility of video-based and pamphlet-based decision aid use in Veterans with knee osteoarthritis. Materials and Methods: Veterans considering treatment for knee osteoarthritis received either an online video-based aid, pamphlet-based aid, or both before their surgical consult. At their visit, patients completed written pre-visit and post-visit questionnaires. The pre-visit questionnaire included questions about the patient's demographics, decision-making preferences, experiences using the assigned decision aids, and the Hip-Knee Decision Quality Instrument. The post-visit questionnaire assessed the patient's overall experience with the decision-making process and how use of the decision aid influenced their discussion with the physician. Results: All 16 patients who received the pamphlet-based aid reviewed the decision aid before their visit, compared to only five of the 12 patients who received the video-based aid. Thirteen of 20 patients indicated that they preferred to share treatment decision-making with their physician. Seventeen of 20 patients believed they would feel comfortable questioning the treatment recommendation of their surgeon after decision aid use. Most patients reported a positive experience using their decision aid, regardless of modality, and found it easily comprehensible and useful in visit preparation. A preference for a pamphlet-based aid was expressed by the majority of patients. Conclusion: Veterans considering treatment for knee osteoarthritis are well prepared to engage in a patient-centered care experience. Most patients preferred sharing the decision-making process with their physician and felt comfortable questioning them about treatment recommendations. Decision aids helped Veterans feel more informed about their treatment options and improved engagement and discussion with their physician. Pamphlet-based aids were utilized more reliably than video-based aids.

Surgical Opioid Stewardship for Orthopedic Surgery: A Quality Improvement Initiative.

The aim of this quality improvement initiative was to reduce unnecessary opioid prescribing by sharing data with prescribers on opioid use by patients. In our study, transition of care clinicians performed follow-up phone calls to select postoperative orthopedic patients to determine opioid use. We implemented a standardized postoperative 7-day opioid wean and designed a dashboard to track the information gathered. We calculated descriptive statistics for continuous and categorical variables. In the initial assessment of opioid use by orthopedic patients, the study consisted of 296 patients with a mean age of 64.8±11.4 years, 147 females (49.7%) and 149 males (50.3%), 59.1% joint replacements (hip, knee, shoulder), and 40.9% spine surgeries (lumbar decompression, cervical fusion, hemilaminectomy). Among those prescribed an opioid, 50% received a prescription for 30 pills or less and 52.4% reported taking more than 80% of the opioid pills, while 35.1% reported taking less than 60%. In the prescribing quality improvement assessment, there were a total of 1547 hospitalizations for joint replacement surgeries from June 2018 to June 2020: 774 (50.0%) hips and 773 (50.0%) knees. There was a significant difference in morphine milligram equivalents per day and quantity prescribed when comparing the preintervention period with the postintervention period without significant increases in opioid refill requests or return visit rates. In our study, sharing data around patient opioid use and provider-facing prescribing metrics reduced postoperative opioid prescribing without significantly increasing opioid refill or emergency department return visit rates. [Orthopedics. 2023;46(4):e230-e236.].

The Effect of Intra-articular Hyaluronic Acid Injections and Payer Coverage on Total Knee Arthroplasty Procedures: Evidence From Large US Claims Database.

Background: There is debate regarding the efficacy of intra-articular (IA) hyaluronic acid (HA) injections for the management of knee osteoarthritis (OA). This study aimed to determine if IA HA utilization and payer coverage of viscosupplementation affected the prevalence of total knee arthroplasty (TKA) procedures and the age of TKA patients. Methods: We performed a retrospective analysis from 2014 to 2020 using a large national commercial claims data set. We analyzed the number of TKA procedures and the age of the patients in states that covered IA HA vs those with limited coverage. Mixed random effects and slopes models were used to identify the impact of the IA HA injections. Results: Of 7,335,301 patients with knee OA, 440,606 (6.0%) received a TKA procedure at an average age of 59 years. The rate of TKA procedures increased by 0.56% per year (95% confidence interval [CI] 0.46-0.66; P < .001). Payer coverage of IA HA injections had no effect on TKA prevalence (P = .926). The age of surgical patients increased yearly by 0.15 years (95% CI 0.12-0.18; P < .001), regardless of IA HA injections (P = .990). After controlling for demographics and comorbidities, patients that received an IA HA injection had a higher probability of receiving a subsequent TKA (odds ratio = 2.83; 95% CI 2.80-2.87; P < .001); this finding was conditional of patients' age at the first diagnosis of knee OA. Conclusions: Additional clinical trials should be employed to identify the role of HA injections in the treatment armamentarium for knee OA.

The ERM-1 membrane-binding domain directs erm-1 mRNA localization to the plasma membrane in the C. elegans embryo.

mRNA localization and transport are integral in regulating gene expression. In Caenorhabditis elegans embryos, the maternally inherited mRNA erm-1 (Ezrin/Radixin/Moesin) becomes concentrated in anterior blastomeres. erm-1 mRNA localizes within those blastomeres to the plasma membrane where the essential ERM-1 protein, a membrane-actin linker, is also found. We demonstrate that the localization of erm-1 mRNA to the plasma membrane is translation dependent and requires its encoded N-terminal, membrane-binding (FERM) domain. By perturbing translation through multiple methods, we found that erm-1 mRNA localization at the plasma membrane persisted only if the nascent peptide remained in complex with the translating mRNA. Indeed, re-coding the erm-1 mRNA coding sequence while preserving the encoded amino acid sequence did not disrupt erm-1 mRNA localization, corroborating that the information directing mRNA localization resides within its membrane-binding protein domain. A single-molecule inexpensive fluorescence in situ hybridization screen of 17 genes encoding similar membrane-binding domains identified three plasma membrane-localized mRNAs in the early embryo. Ten additional transcripts showed potential membrane localization later in development. These findings point to a translation-dependent pathway for localization of mRNAs encoding membrane-associated proteins.

It's Just a Phase: Exploring the Relationship Between mRNA, Biomolecular Condensates, and Translational Control.

Cells spatially organize their molecular components to carry out fundamental biological processes and guide proper development. The spatial organization of RNA within the cell can both promote and result from gene expression regulatory control. Recent studies have demonstrated diverse associations between RNA spatial patterning and translation regulatory control. One form of patterning, compartmentalization in biomolecular condensates, has been of particular interest. Generally, transcripts associated with cytoplasmic biomolecular condensates-such as germ granules, stress granules, and P-bodies-are linked with low translational status. However, recent studies have identified new biomolecular condensates with diverse roles associated with active translation. This review outlines RNA compartmentalization in various condensates that occur in association with repressed or active translational states, highlights recent findings in well-studied condensates, and explores novel condensate behaviors.

Differential item functioning to validate setting of delivery compatibility in PROMIS-global health.

PURPOSE: Patient-reported outcomes measures (PROMs) such as PROMIS are increasingly utilized in healthcare to assess patient perception and functional status, but the effect of delivery setting remains to be fully investigated. To our knowledge, no current study establishes the absence of differential item functioning (DIF) across delivery setting for these PROMIS- Global Health (PROMIS-GH) measures among orthopedic patients. We sought to investigate the correlation of PROMIS-GH scores across in-clinic versus remote delivery by evaluating DIF within the Global Physical Health (GPH) and Global Mental Health (GMH) items. We hypothesize that the setting of delivery of the GPH and GMH domains of PROMIS-GH will not impact the results of the measure, allowing direct comparison between the two delivery settings. METHODS: Five thousand and seven hundred and eighty-five complete PROMIS-Global Health measures were analyzed retrospectively using the 'Lordif' package on the R platform. DIF was measured for GPH and GMH domains across setting of response (in-clinic vs remote) during the pre-operative period, immediate post-operative period, and 1-year post-operative period using Monte Carlo estimation. McFadden pseudo-R2 thresholds (> 0.02) were used to assess the magnitude of DIF for individual PROMIS items. RESULTS: No GPH or GMH items contained in the PROMIS-GH instrument yielded DIF across in-clinic vs remote delivery setting during the pre-operative, immediate post-operative, or 1-year post-operative window. CONCLUSION: The GPH and GMH domains within the PROMIS-GH instrument may be delivered in the clinic or remotely with comparable accuracy. This cross-delivery setting validation analysis may aid to improve the quality of patient care by allowing mixed platform PROMIS-GH data tailored to individual patient circumstance.

Improved Methods for Single-Molecule Fluorescence In Situ Hybridization and Immunofluorescence in Caenorhabditis elegans Embryos.

Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule fluorescence in situ hybridization (smFISH) and immunofluorescence (IF) enable the visualization of the abundance and localization of mRNAs and proteins, respectively, allowing researchers to ultimately elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol poses challenges for use in the C. elegans embryo. smFISH protocols suffer from high initial costs and can photobleach rapidly, and immunofluorescence requires technically challenging permeabilization steps and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH in C. elegans embryos either in sequence or simultaneously. We also outline the steps to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols feature improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, a streamlined, in-tube approach for antibody staining that negates freeze-cracking, a validated method to perform the cost-reducing single molecule inexpensive FISH (smiFISH) adaptation, slide preparation using empirically determined optimal antifade products, and straightforward quantification and data analysis methods. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols simplify existing workflows for single-molecule RNA and protein detection. Moreover, simultaneous, high-resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sequential immunofluorescence and single-molecule fluorescence in situ hybridization Alternate Protocol: Abbreviated protocol for simultaneous immunofluorescence and single-molecule fluorescence in situ hybridization Basic Protocol 2: Simplified immunofluorescence in C. elegans embryos Basic Protocol 3: Single-molecule fluorescence in situ hybridization or single-molecule inexpensive fluorescence in situ hybridization.

Mobility-Guided Estimation of COVID-19 Transmission Rates.

It is of critical importance to estimate changing disease-transmission rates and their dependence on population mobility. A common approach to this problem involves fitting daily transmission rates using a susceptible-exposed-infected-recovered-(SEIR) model (regularizing to avoid overfitting) and then computing the relationship between the estimated transmission rate and mobility. Unfortunately, there are often several very different transmission-rate trajectories that can fit the reported cases well, meaning that the choice of regularization determines the final solution (and thus the mobility-transmission rate relationship) selected by the SEIR model. Moreover, the classical approaches to regularization-penalizing the derivative of the transmission rate trajectory-do not correspond to realistic properties of pandemic spread. Consequently, models fitted using derivative-based regularization are often biased toward underestimating the current transmission rate and future deaths. In this work, we propose mobility-driven regularization of the SEIR transmission rate trajectory. This method rectifies the artificial regularization problem, produces more accurate and unbiased forecasts of future deaths, and estimates a highly interpretable relationship between mobility and the transmission rate. For this analysis, mobility data related to the coronavirus disease 2019 pandemic was collected by Safegraph (San Francisco, California) from major US cities between March and August 2020.

mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage.

Caenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription, thereby presenting an opportunity to study mechanisms of post-transcriptional regulatory control. We observed that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage) and P-bodies (associated with RNA processing). The subcellular distribution of transcripts differed in their dependence on 3'UTRs and RNA binding proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling translation from mRNA localization, we untangled a long-standing question: Are mRNAs directed to P granules to be translationally repressed, or do they accumulate there as a consequence of this repression? We found that translational repression preceded P granule localization and could occur independently of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. These results implicate transcriptional repression as a means to deliver essential maternal transcripts to the progenitor germ lineage for later translation.

Widespread roles for piRNAs and WAGO-class siRNAs in shaping the germline transcriptome of Caenorhabditis elegans.

Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) are distinct classes of small RNAs required for proper germline development. To identify the roles of piRNAs and siRNAs in regulating gene expression in Caenorhabditis elegans, we subjected small RNAs and mRNAs from the gonads of piRNA and siRNA defective mutants to high-throughput sequencing. We show that piRNAs and an abundant class of siRNAs known as WAGO-class 22G-RNAs are required for proper expression of spermatogenic and oogenic genes. WAGO-class 22G-RNAs are also broadly required for transposon silencing, whereas piRNAs are largely dispensable. piRNAs, however, have a critical role in controlling histone gene expression. In the absence of piRNAs, histone mRNAs are misrouted into the nuclear RNAi pathway involving the Argonaute HRDE-1, concurrent with a reduction in the expression of many histone mRNAs. We also show that high-level gene expression in the germline is correlated with high level 22G-RNA production. However, most highly expressed genes produce 22G-RNAs through a distinct pathway that presumably involves the Argonaute CSR-1. In contrast, genes targeted by the WAGO branch of the 22G-RNA pathway are typically poorly expressed and respond unpredictably to loss of 22G-RNAs. Our results point to broad roles for piRNAs and siRNAs in controlling gene expression in the C. elegans germline.

A Strategy To Isolate Modifiers of Caenorhabditis elegans Lethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2.

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen's design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10-4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations.

Clinical doses of radiation reduce collagen matrix stiffness.

Cells receive mechanical cues from their extracellular matrix (ECM), which direct migration, differentiation, apoptosis, and in some cases, the transition to a cancerous phenotype. As a result, there has been significant research to develop methods to tune the mechanical properties of the ECM and understand cell-ECM dynamics more deeply. Here, we show that ionizing radiation can reduce the stiffness of an ex vivo tumor and an in vitro collagen matrix. When non-irradiated cancer cells were seeded in the irradiated matrix, adhesion, spreading, and migration were reduced. These data have ramifications for both in vitro and in vivo systems. In vitro, these data suggest that irradiation may be a method that could be used to create matrices with tailored mechanical properties. In vivo, these suggest that therapeutic doses of radiation may alter tissue mechanics directly.

Quantitative imaging of magnesium distribution at single-cell resolution in brain tumors and infiltrating tumor cells with secondary ion mass spectrometry (SIMS).

Glioblastoma multiforme (GBM) is one of the deadliest forms of human brain tumors. The infiltrative pattern of growth of these tumors includes the spread of individual and/or clusters of tumor cells at some distance from the main tumor mass in parts of the brain protected by an intact blood-brain-barrier. Pathophysiological studies of GBM could be greatly enhanced by analytical techniques capable of in situ single-cell resolution measurements of infiltrating tumor cells. Magnesium homeostasis is an area of active investigation in high grade gliomas. In the present study, we have used the F98 rat glioma as a model of human GBM and an elemental/isotopic imaging technique of secondary ion mass spectrometry, a CAMECA IMS-3f ion microscope, for studying Mg distribution with single-cell resolution in freeze-dried brain tissue cryosections. Quantitative observations were made on tumor cells in the main tumor mass, contiguous brain tissue, and infiltrating tumor cells in adjacent normal brain. The brain tissue contained a significantly lower total Mg concentration of 4.70 ± 0.93 mmol/kg wet weight (mean ± SD) in comparison to 11.64 ± 1.96 mmol/kg wet weight in tumor cells of the main tumor mass and 10.72 ± 1.76 mmol/kg wet weight in infiltrating tumor cells (p < 0.05). The nucleus of individual tumor cells contained elevated levels of bound Mg. These observations have established that there was enhanced influx and increased binding of Mg in tumor cells. They provide strong support for further investigation of altered Mg homeostasis and activation of Mg-transporting channels in GBMs as possible therapeutic targets.