Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization.

Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.

Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome.

Phase transitions involving biomolecular liquids are a fundamental mechanism underlying intracellular organization. In the cell nucleus, liquid-liquid phase separation of intrinsically disordered proteins (IDPs) is implicated in assembly of the nucleolus, as well as transcriptional clusters, and other nuclear bodies. However, it remains unclear whether and how physical forces associated with nucleation, growth, and wetting of liquid condensates can directly restructure chromatin. Here, we use CasDrop, a novel CRISPR-Cas9-based optogenetic technology, to show that various IDPs phase separate into liquid condensates that mechanically exclude chromatin as they grow and preferentially form in low-density, largely euchromatic regions. A minimal physical model explains how this stiffness sensitivity arises from lower mechanical energy associated with deforming softer genomic regions. Targeted genomic loci can nonetheless be mechanically pulled together through surface tension-driven coalescence. Nuclear condensates may thus function as mechano-active chromatin filters, physically pulling in targeted genomic loci while pushing out non-targeted regions of the neighboring genome. VIDEO ABSTRACT.

Viscoelasticity and advective flow of RNA underlies nucleolar form and function.

The nucleolus is the largest biomolecular condensate and facilitates transcription, processing, and assembly of ribosomal RNA (rRNA). Although nucleolar function is thought to require multiphase liquid-like properties, nucleolar fluidity and its connection to the highly coordinated transport and biogenesis of ribosomal subunits are poorly understood. Here, we use quantitative imaging, mathematical modeling, and pulse-chase nucleotide labeling to examine nucleolar material properties and rRNA dynamics. The mobility of rRNA is several orders of magnitude slower than that of nucleolar proteins, with rRNA steadily moving away from the transcriptional sites in a slow (∼1 Å/s), radially directed fashion. This constrained but directional mobility, together with polymer physics-based calculations, suggests that nascent rRNA forms an entangled gel, whose constant production drives outward flow. We propose a model in which progressive maturation of nascent rRNA reduces its initial entanglement, fluidizing the nucleolar periphery to facilitate the release of assembled pre-ribosomal particles.

Engineered molecular sensors for quantifying cell surface crowding.

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

Condensate interfaces can accelerate protein aggregation.

Protein aggregates, formed from the assembly of aberrant, misfolded proteins, are a hallmark of neurodegenerative diseases. Disease-associated aggregates such as mutant Huntingtin polyQ inclusions, are typically enriched in p62/SQSTM1, an oligomeric protein that binds to and sequesters aberrant proteins. p62 has been suggested to sequester proteins through formation of liquid-like biomolecular condensates, but the physical mechanisms by which p62 condensates may regulate pathological protein aggregation remain unclear. Here, we use a light-inducible biomimetic condensate system to show that p62 condensates enhance coarsening of mutant polyQ aggregates through interface-mediated sequestration, which accelerates polyQ accumulation into larger aggregates. However, the resulting large aggregates accumulate polyubiquitinated proteins, which depletes free p62, ultimately suppressing further p62 condensation. This dynamic interplay between interface-mediated coarsening of solid aggregates and downstream consequences on the phase behavior of associated regulatory proteins could contribute to the onset and progression of protein aggregation diseases.

Erratum: Mechanical Frustration of Phase Separation in the Cell Nucleus by Chromatin [Phys. Rev. Lett. 126, 258102 (2021)].

This corrects the article DOI: 10.1103/PhysRevLett.126.258102.

Childhood adversity, combat experiences, and military sexual trauma: a test and extension of the stress sensitization hypothesis.

BACKGROUND: U.S. veterans report high rates of traumatic experiences and mental health symptomology [e.g. posttraumatic stress disorder (PTSD)]. The stress sensitization hypothesis posits experiences of adversity sensitize individuals to stress reactions which can lead to greater psychiatric problems. We extend this hypothesis by exploring how multiple adversities such as early childhood adversity, combat-related trauma, and military sexual trauma related to heterogeneity in stress over time and, subsequently, greater risk for PTSD. METHODS: 1230 veterans were recruited for an observational, longitudinal study. Veterans responded to questionnaires on PTSD, stress, and traumatic experiences five times over an 18-month study period. We used latent transition analysis to understand how heterogeneity in adverse experiences is related to transition into stress trajectory classes. We also explored how transition patterns related to PTSD symptomology. RESULTS: Across all models, we found support for stress sensitization. In general, combat trauma in combinations with other types of adverse experiences, namely early childhood adversity and military sexual trauma, imposed a greater probability of transitioning into higher risk stress profiles. We also showed differential effects of early childhood and military-specific adversity on PTSD symptomology. CONCLUSION: The present study rigorously integrates both military-specific and early life adversity into analysis on stress sensitivity, and is the first to examine how sensitivity might affect trajectories of stress over time. Our study provides a nuanced, and specific, look at who is risk for sensitization to stress based on previous traumatic experiences as well as what transition patterns are associated with greater PTSD symptomology.

Cigarette Smoking and Depression Among U.S. Veterans: Longitudinal Associations With Posttraumatic Stress Disorder.

INTRODUCTION: Cigarette smoking and depression are associated with morbidity and mortality. Among veterans, approximately 22% are current smokers and 11%-15% have been diagnosed with depression. Although prior research suggests a strong association between smoking and depression among veterans, little research has examined trajectories of smoking and depressive symptoms and their correlates over time in this population. AIMS AND METHODS: Using parallel process growth curve modeling, we examined the longitudinal relationship between smoking and depression and tested whether posttraumatic stress disorder (PTSD) symptoms predict smoking and depression trajectories over 18 months (February 2020-August 2021). Veterans were recruited for an online, longitudinal study and responded to surveys across five-time points (baseline N = 1230; retention = 79.3%-83.3% across waves). RESULTS: Associations indicated that more frequent smoking at baseline was associated with steeper increases in depression symptom severity, and greater depression severity at baseline was associated with a less steep decrease in smoking frequency over time. PTSD was associated with less smoking at time 1 but more frequent smoking at times 3-5 as well as greater depression across all time points. CONCLUSIONS: Findings provide support that the growth trajectories of smoking and depression are linked, and PTSD symptoms are associated with these trajectories among veterans. Addressing these factors simultaneously in veteran treatment centers or through tobacco cessation efforts may be beneficial. IMPLICATIONS: This study offers strong evidence that the growth trajectories of smoking and depression are linked, and PTSD symptoms affect these trajectories among veterans, who represent a largely understudied population despite high rates of substance use and mental health problems. Results of this study strengthen the case for a more integrated treatment approach in which both smoking and mental health concerns are simultaneously addressed, which may yield more beneficial physical health and clinical outcomes for post-9/11 veterans.

Maternal and adolescent depressive symptoms and family conflict: An autoregressive cross-lagged examination of competing models in multi-stressed mothers and adolescents.

Maternal and adolescent depression are challenges that often co-occur. Many studies have drawn bivariate associations between maternal depressive symptoms, adolescent depressive symptoms, and family conflict, but few have examined reciprocal effects. Even among extant studies, there is a lack of clarity related to directionality of influence. Three competing theoretical models may explain the relationship between maternal depressive symptoms, adolescent depressive symptoms, and family conflict, and these processes may differ by adolescents' sex. Using three time points of data from 187 diverse mother-adolescent dyads, we fit a taxonomy of autoregressive cross-lagged structural equation models to simultaneously evaluate the competing theoretical models and also examine differences by sex using multiple-group analyses. Results indicate a symptom-driven model whereby adolescent depressive symptoms predicted increases in family conflict. Sex differences were also found. For males, but not females, greater adolescent depressive symptoms predicted subsequent increases in maternal depressive symptoms, which then predicted lower family conflict-possibly indicating maternal disengagement/withdrawal. Our findings suggest addressing adolescent depressive symptoms in order to prevent family conflict and that distinctive targets for the prevention/intervention of family conflict should account for differences by adolescents' sex.

Molecular profiles and urinary biomarkers of upper tract urothelial carcinomas associated with aristolochic acid exposure.

Recurrent upper tract urothelial carcinomas (UTUCs) arise in the context of nephropathy linked to exposure to the herbal carcinogen aristolochic acid (AA). Here we delineated the molecular programs underlying UTUC tumorigenesis in patients from endemic aristolochic acid nephropathy (AAN) regions in Southern Europe. We applied an integrative multiomics analysis of UTUCs, corresponding unaffected tissues and of patient urines. Quantitative microRNA (miRNA) and messenger ribonucleic acid (mRNA) expression profiling, immunohistochemical analysis by tissue microarrays and exome and transcriptome sequencing were performed in UTUC and nontumor tissues. Urinary miRNAs of cases undergoing surgery were profiled before and after tumor resection. Ribonucleic acid (RNA) and protein levels were analyzed using appropriate statistical tests and trend assessment. Dedicated bioinformatic tools were used for analysis of pathways, mutational signatures and result visualization. The results delineate UTUC-specific miRNA:mRNA networks comprising 89 miRNAs associated with 1,862 target mRNAs, involving deregulation of cell cycle, deoxyribonucleic acid (DNA) damage response, DNA repair, bladder cancer, oncogenes, tumor suppressors, chromatin structure regulators and developmental signaling pathways. Key UTUC-specific transcripts were confirmed at the protein level. Exome and transcriptome sequencing of UTUCs revealed AA-specific mutational signature SBS22, with 68% to 76% AA-specific, deleterious mutations propagated at the transcript level, a possible basis for neoantigen formation and immunotherapy targeting. We next identified a signature of UTUC-specific miRNAs consistently more abundant in the patients' urine prior to tumor resection, thereby defining biomarkers of tumor presence. The complex gene regulation programs of AAN-associated UTUC tumors involve regulatory miRNAs prospectively applicable to noninvasive urine-based screening of AAN patients for cancer presence and recurrence.

Expanding adverse child experiences to inequality and racial discrimination.

There is a well-established correlation between health and adverse childhood experiences (ACEs). Arguments have been made to expand ACE scales to include indicators of racism and structural inequalities. In this paper, we use nationally representative data to examine the relationships between latent groups of an expanded adversity scale and a broad range of child health outcomes. Data were obtained from a merger of the 2017 and 2018 National Survey of Children's Health (NSCH) and analyzed in 2021 (n = 52,129). Adversities were defined as violent victimization, violence exposure, a range of parental problems, racial discrimination, food insecurity, and unkempt housing. Latent class analysis (LCA) was used to uncover emergent groups of adversities, and logistic regression was used to assess group relationship to global and diagnosed measures of health. Four groups emerged: high all (3.6%), material and food hardship (11.9%), parental problems (10.3%), and low all (74.2%). Results showed the high all groups at greater odds of almost all outcomes. Compared to low all group, high all had particularly higher odds of any special (OR = 2.29) or complex (OR = 2.53) healthcare need, frequent severe headaches (OR = 2.07), and depression (OR = 3.4) or anxiety (OR = 2.11). Our analysis noted separation of experiences based on additional items related to structural inequalities: food insecurity, poverty, and unkempt housing. However, augmenting existing ACE scales with these indicators may be unnecessary as children most at-risk for poor health were a very small group (1 in 28) that experienced multiple forms of violence and parental problems.

Identifying factors that impact general dentists' referrals to orthodontists.

INTRODUCTION: Referrals from general dentists are a significant source of orthodontic patients, but our knowledge of factors guiding referral decisions predates recent marketplace trends of direct-to-consumer products and broad digitization. To provide orthodontists with current, nationwide data on referral values and communication preferences, a mixed-methods study was conducted. METHODS: Semistructured interviews were conducted with practicing general dentists (n = 23), and interviews were analyzed to identify factors important to communication and referrals. Qualitative data provided the foundation to develop and pretest a survey distributed nationally to American Dental Association general dentists through email (n = 373). Survey results were evaluated using bivariate and descriptive statistics. RESULTS: Although 42% of general dentists provide orthodontics, of those, 68% report insufficient orthodontic training in dental school, and 61% have increased orthodontic care in recent years. General dentists refer to orthodontists for complex cases and growing patients, with the orthodontist's perceived skill and proximity to the patient being important for choosing a specific orthodontic practice. Phone (70%) and email (61%) are the most used interoffice communication methods, with email (40%) and then phone (30%) being the most preferred. The most important aspects of a working relationship were patient satisfaction and final occlusal outcome, whereas the best way to boost referrals was through improving communication. CONCLUSIONS: Excellent care and patient satisfaction remain primary motivators for referrals from generalists to orthodontists. An improved orthodontic curriculum is needed for general dentists. Good communication with dentists is critical for growing referral streams and enhancing patient care.

Mechanical Frustration of Phase Separation in the Cell Nucleus by Chromatin.

Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that cross-linked chromatin can mechanically suppress droplets' coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.

Enantioselective Total Synthesis of (+)-Monocerin, a Dihydroisocoumarin Derivative with Potent Antimalarial Properties.

We describe here the enantioselective synthesis of (+)-monocerin and its acetate derivative. The present synthesis features an efficient optically active synthesis of the ß-hydroxy-γ-lactone derivative with high enantiomeric purity using Sharpless dihydroxylation as the key step. The synthesis also highlights a tandem Lewis acid-catalyzed, oxocarbenium ion-mediated diastereoselective syn-allylation reaction, and a methoxymethyl group promoted methylenation reaction. We investigated this reaction with a variety of Lewis acids. A selective CrO3-mediated oxidation of isochroman provided the corresponding lactone derivative. The synthesis is quite efficient and may be useful for the preparation of derivatives.

Detection of Regional Variation in Selection Intensity within Protein-Coding Genes Using DNA Sequence Polymorphism and Divergence.

Numerous approaches have been developed to infer natural selection based on the comparison of polymorphism within species and divergence between species. These methods are especially powerful for the detection of uniform selection operating across a gene. However, empirical analyses have demonstrated that regions of protein-coding genes exhibiting clusters of amino acid substitutions are subject to different levels of selection relative to other regions of the same gene. To quantify this heterogeneity of selection within coding sequences, we developed Model Averaged Site Selection via Poisson Random Field (MASS-PRF). MASS-PRF identifies an ensemble of intragenic clustering models for polymorphic and divergent sites. This ensemble of models is used within the Poisson Random Field framework to estimate selection intensity on a site-by-site basis. Using simulations, we demonstrate that MASS-PRF has high power to detect clusters of amino acid variants in small genic regions, can reliably estimate the probability of a variant occurring at each nucleotide site in sequence data and is robust to historical demographic trends and recombination. We applied MASS-PRF to human gene polymorphism derived from the 1,000 Genomes Project and divergence data from the common chimpanzee. On the basis of this analysis, we discovered striking regional variation in selection intensity, indicative of positive or negative selection, in well-defined domains of genes that have previously been associated with neurological processing, immunity, and reproduction. We suggest that amino acid-altering substitutions within these regions likely are or have been selectively advantageous in the human lineage, playing important roles in protein function.

The effects of thoracic load carriage on maximal ambulatory work tolerance and acceptable work durations.

PURPOSE: Torso loads restrict chest-wall movement and ventilation, particularly during heavy exercise. Therefore, the differential impact of load carriage and chest-wall restriction was investigated during progressive treadmill exercise. In addition, acceptable work durations were derived across a wide range of submaximal, loaded exercise intensities. METHODS: Twelve males participated in two research phases. Phase 1: Three incremental treadmill tests until exhaustion [control (clothing only), load carriage (clothing plus 22-kg vest), and clothing with chest strapping]. Phase 2: Five steady-state exercise trials (clothing plus 22-kg vest) at intensities from 30 to 80 % of peak aerobic power to determine maximal acceptable work durations. RESULTS: Maximal work tolerance [control 17.21 min (±0.93); loaded 13.44 min (±0.68); strapped 17.00 min (±0.83)] and the mass-specific peak aerobic power [control 61.61 mL kg(-1) min(-1) (±2.28); loaded 45.42 mL kg(-1) min(-1) (±1.41); strapped 59.99 mL kg(-1) min(-1) (±1.61)] were reduced only when loaded (P < 0.05). Peak minute ventilation was retained, although loading and chest strapping reduced the breathing reserve. The lower and upper acceptable work duration derivations when working at the 30 % intensity were 133.40 min (±23.77) and 220.10 min (±48.69), but at 80 % intensity, both durations were reduced to <7 min. CONCLUSION: Thoracic loading significantly reduced exercise tolerance and the breathing reserve, but did not modify peak minute ventilation or the absolute peak aerobic power. Chest strapping, as used herein, exerted minimal impact. However, the projected maximal acceptable work durations were much less than derived using previously published methods.

Mitochondrial uncoupling does not decrease reactive oxygen species production after ischemia-reperfusion.

Cardiac ischemia-reperfusion (IR) leads to myocardial dysfunction by increasing production of reactive oxygen species (ROS). Mitochondrial H(+) leak decreases ROS formation; it has been postulated that increasing H(+) leak may be a mechanism of decreasing ROS production after IR. Ischemic preconditioning (IPC) decreases ROS formation after IR, but the mechanism is unknown. We hypothesize that pharmacologically increasing mitochondrial H(+) leak would decrease ROS production after IR. We further hypothesize that IPC would be associated with an increase in the rate of H(+) leak. Isolated male Sprague-Dawley rat hearts were subjected to either control or IPC. Mitochondria were isolated at end equilibration, end ischemia, and end reperfusion. Mitochondrial membrane potential (mΔΨ) was measured using a tetraphenylphosphonium electrode. Mitochondrial uncoupling was achieved by adding increasing concentrations of FCCP. Mitochondrial ROS production was measured by fluorometry using Amplex-Red. Pyridine dinucleotide levels were measured using HPLC. Before IR, increasing H(+) leak decreased mitochondrial ROS production. After IR, ROS production was not affected by increasing H(+) leak. H(+) leak increased at end ischemia in control mitochondria. IPC mitochondria showed no change in the rate of H(+) leak throughout IR. NADPH levels decreased after IR in both IPC and control mitochondria while NADH increased. Pharmacologically, increasing H(+) leak is not a method of decreasing ROS production after IR. Replenishing the NADPH pool may be a means of scavenging the excess ROS thereby attenuating oxidative damage after IR.

Transcutaneous electrical nerve stimulation for the treatment of acute postoperative pain following spine surgery: a scoping review.

OBJECTIVE: Given the ubiquity and severity of postoperative pain following spine surgery, developing adequate pain management modalities is critical. Transcutaneous electrical nerve stimulation (TENS) is a promising noninvasive modality that is well studied for managing postoperative pain following a variety of surgeries, but data on using TENS for pain management in the acute postoperative period of spine surgery are limited. Therefore, this review aimed to recapitulate the existing evidence for the use of TENS in postoperative pain management for spine surgery and explore the potential of this modality moving forward. METHODS: A scoping review was conducted according to 2020 PRISMA guidelines. Two independently operating reviewers then conducted a systematic search of PubMed, Embase, and Scopus databases to identify studies that reported the use of TENS for the treatment of acute postoperative pain following spine surgery. The following data were abstracted from included studies: study type, sample size, demographics, surgery details, comparison group, assessment parameters, timing of postoperative assessment, TENS technical characteristics, relevant findings, length of hospital stay, complications with TENS, and notable limitations. RESULTS: Nine hundred thirty-two publications were screened, resulting in 6 studies included in this review, all of which were prospective clinical trials. The publication dates ranged from 1980 to 2011. Spine surgery types varied; the most common was posterior lumbar interbody fusion. No studies evaluated pain control in cervical- or thoracic-only surgeries. All 6 studies evaluated the level of postoperative pain directly. Five of the 6 studies that directly examined postoperative pain reported lower levels of pharmacological analgesia usage in the TENS groups compared with controls, with 4 of these studies reporting this difference as statistically significant. Length of hospital stay was evaluated in 2 studies, both of which reported decreases in mean length of stay, but these differences were not significant. Notably, every study reported distinct TENS administration parameters while also reporting similar results. CONCLUSIONS: This review concludes that TENS is effective at reducing postoperative pain in spine surgery. Further investigation is needed regarding the optimal settings for TENS administration, as well as efficacy in the thoracic and cervical spine.

American Veterans in the Era of COVID-19: Reactions to the Pandemic, Posttraumatic Stress Disorder, and Substance Use Behaviors.

The COVID-19 pandemic may have a compounding effect on the substance use of American veterans with posttraumatic stress disorder (PTSD). This study investigated the relationship between PTSD and current reactions to COVID-19 on alcohol and cannabis use among veterans who completed a survey 1 month prior to the pandemic in the USA and a 6-month follow-up survey. We hypothesized that veterans with PTSD would experience more negative reactions to COVID-19 and increased alcohol and cannabis use behaviors over those without PTSD. Veterans with PTSD prior to the pandemic, relative to those without, endorsed poorer reactions, greater frequency of alcohol use, and greater cannabis initiation and use during the pandemic. Veterans with PTSD may use substances to manage COVID-related stress. Clinicians may see an increase in substance use among this group during and after the pandemic and may need to implement specific behavioral interventions to mitigate the negative effects of COVID-19.