Cardiovascular toxicity of immune therapies for cancer.

In addition to conventional chemoradiation and targeted cancer therapy, the use of immune based therapies, specifically immune checkpoint inhibitors (ICIs) and chimeric antigen receptor T cell therapy (CAR-T), has increased exponentially across a wide spectrum of cancers. This has been paralleled by recognition of off-target immune related adverse events that can affect almost any organ system including the cardiovascular system. The use of ICIs has been associated with myocarditis, a less common but highly fatal adverse effect, pericarditis and pericardial effusions, vasculitis, thromboembolism, and potentially accelerated atherosclerosis. CAR-T resulting in a systemic cytokine release syndrome has been associated with myriad cardiovascular consequences including arrhythmias, myocardial infarction, and heart failure. This review summarizes the current state of knowledge regarding adverse cardiovascular effects associated with ICIs and CAR-T.

Molecular pathways and cellular subsets associated with adverse clinical outcomes in overlapping immune-related myocarditis and myositis.

Immune checkpoint therapies (ICTs) can induce life-threatening immune-related adverse events, including myocarditis and myositis, which are rare but often concurrent. The molecular pathways and immune subsets underlying these toxicities remain poorly understood. To address this need, we obtained heart and skeletal muscle biopsies for single-cell RNA sequencing in living patients with cancers treated with ICTs admitted to the hospital with myocarditis and/or myositis (overlapping myocarditis plus myositis, n=10; myocarditis-only, n=1) compared to ICT-exposed patients ruled out for toxicity utilized as controls (n=9) within 96 hours of clinical presentation. Analyses of 58,523 cells revealed CD8+ T cells with a cytotoxic phenotype expressing activation/exhaustion markers in both myocarditis and myositis. Furthermore, the analyses identified a population of myeloid cells expressing tissue-resident signatures and FcγRIIIa (CD16a), which is known to bind IgG and regulate complement activation. Immunohistochemistry of affected cardiac and skeletal muscle tissues revealed protein expression of pan-IgG and complement product C4d that were associated with the presence of high-titer serum autoantibodies against muscle antigens in a subset of patients. We further identified a population of inflammatory IL-1B+TNF+ myeloid cells specifically enriched in myocarditis and associated with greater toxicity severity and poorer clinical outcomes. These results are the first to recognize these myeloid subsets in human immune-related myocarditis and myositis tissues and nominate new targets for investigation into rational treatments to overcome these high-mortality toxicities.

Impact of Combinatorial Histone Modifications on Acetyllysine Recognition by the ATAD2 and ATAD2B Bromodomains.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

H3K18 & H3K23 acetylation directs establishment of MLL-mediated H3K4 methylation.

In an unmodified state, positively charged histone N-terminal tails engage nucleosomal DNA in a manner which restricts access to not only the underlying DNA, but also key tail residues subject to binding and/or modification. Charge-neutralizing modifications, such as histone acetylation, serve to disrupt this DNA-tail interaction, facilitating access to such residues. We previously showed that a polyacetylation-mediated chromatin "switch" governs the read-write capability of H3K4me3 by the MLL1 methyltransferase complex. Here, we discern the relative contributions of site-specific acetylation states along the H3 tail and extend our interrogation to other chromatin modifiers. We show that the contributions of H3 tail acetylation to H3K4 methylation by MLL1 are highly variable, with H3K18 and H3K23 acetylation exhibiting robust stimulatory effects, and that this extends to the related H3K4 methyltransferase complex, MLL4. We show that H3K4me1 and H3K4me3 are found preferentially co-enriched with H3 N-terminal tail proteoforms bearing dual H3K18 and H3K23 acetylation (H3{K18acK23ac}). We further show that this effect is specific to H3K4 methylation, while methyltransferases targeting other H3 tail residues (H3K9, H3K27, & H3K36), a methyltransferase targeting the nucleosome core (H3K79), and a kinase targeting a residue directly adjacent to H3K4 (H3T3) are insensitive to tail acetylation. Together, these findings indicate a unique and robust stimulation of H3K4 methylation by H3K18 and H3K23 acetylation and provide key insight into why H3K4 methylation is often associated with histone acetylation in the context of active gene expression.

Elevated IL-6 and Tumor Necrosis Factor-α in Immune Checkpoint Inhibitor Myocarditis.

INTRODUCTION: The impact of peripheral cytokine levels on the prognosis and treatment of immune checkpoint inhibitor (ICI) myocarditis has not been well studied. OBJECTIVES: This study aimed to identify cytokines that can prognosticate and direct the treatment of ICI myocarditis. METHODS: This was a single-center, retrospective cohort study of patients with ICI myocarditis who had available peripheral cytokine levels between January 2011 and May 2022. Major adverse cardiovascular events (MACEs) were defined as a composite of heart failure with/without cardiogenic shock, arterial thrombosis, life-threatening arrhythmias, pulmonary embolism, and sudden cardiac death. RESULTS: In total, 65 patients with ICI myocarditis had cytokine data available. Patients were mostly males (70%), with a mean age of 67.8 ± 12.7 years. Interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) were the most common cytokines to be elevated with 48/65 (74%) of patients having a peak IL-6 above normal limits (>5 pg/mL) and 44/65 (68%) of patients with peak TNF-α above normal limits (>22 pg/mL). Patients with elevated peak IL-6 had similar 90-day mortality and MACE outcomes compared to those without (10.4% vs. 11.8%, p = 0.878 and 8.8% vs. 17.7%, p = 0.366, respectively). Similarly, those with elevated peak TNF-α had similar 90-day mortality and MACEs compared to those without (29.6% vs. 14.3%, p = 0.182 and 13.6% vs. 4.8%, p = 0.413, respectively). Kaplan-Meier survival analysis also showed that there was not a significant difference between MACE-free survival when comparing elevated and normal IL-6 and TNF-α levels (p = 0.182 and p = 0.118, respectively). MACEs and overall survival outcomes were similar between those who received infliximab and those who did not among all patients and those with elevated TNF-α (p-value 0.70 and 0.83, respectively). CONCLUSION: Peripheral blood levels of IL-6 and TNF-α are the most commonly elevated cytokines in patients with ICI myocarditis. However, their role in the prognostication and guidance of immunomodulatory treatment is currently limited.

Angiographic tool to detect pulmonary arteriovenous malformations in single ventricle physiology.

OBJECTIVE: Individuals with single ventricle physiology who are palliated with superior cavopulmonary anastomosis (Glenn surgery) may develop pulmonary arteriovenous malformations. The traditional tools for pulmonary arteriovenous malformation diagnosis are often of limited diagnostic utility in this patient population. We sought to measure the pulmonary capillary transit time to determine its value as a tool to identify pulmonary arteriovenous malformations in patients with single ventricle physiology. METHODS: We defined the angiographic pulmonary capillary transit time as the number of cardiac cycles required for transit of contrast from the distal pulmonary arteries to the pulmonary veins. Patients were retrospectively recruited from a single quaternary North American paediatric centre, and angiographic and clinical data were reviewed. Pulmonary capillary transit time was calculated in 20 control patients and compared to 20 single ventricle patients at the pre-Glenn, Glenn, and Fontan surgical stages (which were compared with a linear-mixed model). Correlation (Pearson) between pulmonary capillary transit time and haemodynamic and injection parameters was assessed using angiograms from 84 Glenn patients. Five independent observers calculated pulmonary capillary transit time to measure reproducibility (intraclass correlation coefficient). RESULTS: Mean pulmonary capillary transit time was 3.3 cardiac cycles in the control population, and 3.5, 2.4, and 3.5 in the pre-Glenn, Glenn, and Fontan stages, respectively. Pulmonary capillary transit time in the Glenn population did not correlate with injection conditions. Intraclass correlation coefficient was 0.87. CONCLUSIONS: Pulmonary angiography can be used to calculate the pulmonary capillary transit time, which is reproducible between observers. Pulmonary capillary transit time accelerates in the Glenn stage, correlating with absence of direct hepatopulmonary venous flow.

Two distinct regulatory systems control pulcherrimin biosynthesis in Bacillus subtilis.

Regulation of transcription is a fundamental process that allows bacteria to respond to external stimuli with appropriate timing and magnitude of response. In the soil bacterium Bacillus subtilis, transcriptional regulation is at the core of developmental processes needed for cell survival. Gene expression in cells transitioning from exponential phase to stationary phase is under the control of a group of transcription factors called transition state regulators (TSRs). TSRs influence numerous developmental processes including the decision between biofilm formation and motility, genetic competence, and sporulation, but the extent to which TSRs influence bacterial physiology remains to be fully elucidated. Here, we demonstrate two TSRs, ScoC and AbrB, along with the MarR-family transcription factor PchR negatively regulate production of the iron chelator pulcherrimin in B. subtilis. Genetic analysis of the relationship between the three transcription factors indicate that all are necessary to limit pulcherrimin production during exponential phase and influence the rate and total amount of pulcherrimin produced. Similarly, expression of the pulcherrimin biosynthesis gene yvmC was found to be under control of ScoC, AbrB, and PchR and correlated with the amount of pulcherrimin produced by each background. Lastly, our in vitro data indicate a weak direct role for ScoC in controlling pulcherrimin production along with AbrB and PchR. The layered regulation by two distinct regulatory systems underscores the important role for pulcherrimin in B. subtilis physiology.

Whole blood versus balanced resuscitation in massive hemorrhage: six of one or half dozen of the other?

INTRODUCTION: Whole blood (WB) resuscitation is increasingly used at trauma centers. Prior studies investigating outcomes in WB versus component-only (CO) resuscitation have been limited by small cohorts, low volumes of WB resuscitation, and unbalanced CO resuscitation. This study aimed to address these limitations using data from a high-volume Level I trauma center, which adopted a WB-first resuscitation paradigm in 2018. We hypothesized that the resuscitation method, WB or balanced CO, would have no impact on patient mortality. METHODS: A single-center, retrospective cohort study of adults presenting as a trauma activation from July 2016 through July 2021 was performed. Receipt of 3 or more units of WB or packed red blood cells (RBC) within the first hour of resuscitation was required for inclusion. Patients were grouped into WB versus CO resuscitation and important clinical outcomes were compared. Mortality was evaluated with Kaplan-Meier analysis, log-rank testing, and multivariable Cox proportional hazards modeling. RESULTS: There were 180 patients in the WB group and 170 patients in the CO group. Of the 180 WB patients, 110 (61%) received only WB during the first 24 hours. The WB group received a median of 5.0 units (IQR 4.0-8.0) of WB and CO group received a median of 6.0 units (IQR 4.0-11.8) of RBCs during the first 24 hours of resuscitation. In the CO group, median RBC/plasma and RBC/platelet ratios approximated 1:1:1. Groups were similar in clinicopathologic characteristics including age, injury severity score, mechanism of injury, and requirement for hemorrhage control interventions (WB 55% vs CO 59%, p = 0.60). Unadjusted survival was equivalent at 24 hours (p = 0.52) and 30 days (p = 0.70) between both groups on Kaplan-Meier analysis with log-rank testing. On multivariable Cox regression, WB resuscitation was not independently associated with improved survival after accounting for age, ISS, mechanism of injury, and receipt of hemorrhage control procedure (HR 0.85, 95% CI 0.61-1.19, p = 0.34). CONCLUSIONS: Balanced CO resuscitation is associated with similar mortality outcomes to that of WB based resuscitation. LEVEL OF EVIDENCE: Level IV; Therapeutic/Care Management.

Probing the content of affective semantic memory following caregiving-related early adversity.

Cognitive science has demonstrated that we construct knowledge about the world by abstracting patterns from routinely encountered experiences and storing them as semantic memories. This preregistered study tested the hypothesis that caregiving-related early adversities (crEAs) shape affective semantic memories to reflect the content of those adverse interpersonal-affective experiences. We also tested the hypothesis that because affective semantic memories may continue to evolve in response to later-occurring positive experiences, child-perceived attachment security will inform their content. The sample comprised 160 children (ages 6-12 at Visit 1; 87F/73 M), 66% of whom experienced crEAs (n = 105). At Visit 1, crEA exposure prior to study enrollment was operationalized as parental-reports endorsing a history of crEAs (abuse/neglect, permanent/significant parent-child separation); while child-reports assessed concurrent attachment security. A false memory task was administered online ∼2.5 years later (Visit 2) to probe the content of affective semantic memories-specifically attachment schemas. Results showed that crEA exposure (vs. no exposure) was associated with a higher likelihood of falsely endorsing insecure (vs. secure) schema scenes. Attachment security moderated the association between crEA exposure and insecure schema-based false recognition. Findings suggest that interpersonal-affective semantic schemas include representations of parent-child interactions that may capture the quality of one's own attachment experiences and that these representations shape how children remember attachment-relevant narrative events. Findings are also consistent with the hypothesis that these affective semantic memories can be modified by later experiences. Moving forward, the approach taken in this study provides a means of operationalizing Bowlby's notion of internal working models within a cognitive neuroscience framework. RESEARCH HIGHLIGHTS: Affective semantic memories representing insecure schema knowledge (child needs + needs-not-met) may be more salient, elaborated, and persistent among youths exposed to early caregiving adversity. All youths, irrespective of early caregiving adversity exposure, may possess affective semantic memories that represent knowledge of secure schemas (child needs + needs-met). Establishing secure relationships with parents following early-occurring caregiving adversity may attenuate the expression of insecure semantic memories, suggesting potential malleability. Affective semantic memories include schema representations of parent-child interactions that may capture the quality of one's own attachment experiences and shape how youths remember attachment-relevant events.

Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress.

BACKGROUND: Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Our preliminary analyses of RNA-Seq data uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. RESULTS: Motivated to understand how BAT function is regulated epigenetically, we developed a novel method for the first-ever unbiased top-down proteomic quantitation of histone modifications in BAT and validated our results with a multi-omic approach. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C) and BAT was analyzed for DNA methylation and histone modifications. Methylation of promoters and intragenic regions in genomic DNA decrease in response to chronic cold exposure. Integration of DNA methylation and RNA expression datasets suggest a role for epigenetic modification of DNA in regulation of gene expression in response to cold. In response to cold housing, we observe increased bulk acetylation of histones H3.2 and H4, increased histone H3.2 proteoforms with di- and trimethylation of lysine 9 (K9me2 and K9me3), and increased histone H4 proteoforms with acetylation of lysine 16 (K16ac) in BAT. CONCLUSIONS: Our results reveal global epigenetically-regulated transcriptional "on" and "off" signals in murine BAT in response to varying degrees of chronic cold stimuli and establish a novel methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Additionally, we make histone PTM and proteoform quantitation, RNA splicing, RRBS, and transcriptional footprint datasets available as a resource for future research.

Determinants of non-vaccination against seasonal influenza during pregnancy.

OBJECTIVE: The objective of this study was to identify the determinants of influenza non-vaccination during pregnancy in Canada. METHODS: Biological mothers of children born between December 2018 and March 2019 were surveyed about vaccinations they had received during pregnancy, reasons for non-vaccination, obstetrical history, and demographics. Simple and multiple logistic regression models were used to measure associations between various sociodemographic factors as well as obstetrical history, and non-vaccination against influenza. We analyzed data from 2361 mothers. RESULTS: Factors associated with non-vaccination included being followed during pregnancy by a midwife compared to by an obstetrician-gynecologist (OR 2.02; 95% CI, 1.17‒3.50); having two or more past live births compared to none (OR 1.58; 95% CI, 1.01‒2.49); having an education level below high school diploma compared to a bachelor's degree or above (OR 2.50; 95% CI, 1.06‒5.90); and having a household income below $60,000 (OR 2.46; 95% CI, 1.42‒4.24) or between $60,000 and $99,999 (OR 2.77; 95% CI, 1.70‒4.52) compared to a household income of $140,000 or more. The province or territory of prenatal care proved to be an important factor in non-vaccination, with statistically significant odds ratios for certain provinces: OR 7.50 (95% CI, 1.40‒40.26) for Ontario, 8.23 (95% CI, 1.53‒44.23) for Newfoundland and Labrador, and 11.39 (95% CI, 2.14‒60.60) for Quebec, as compared to the territories. CONCLUSION: Despite universal access to influenza vaccines in Canada during pregnancy, regional variations and socioeconomic disparities in non-vaccination are still observable.

RéSUMé: OBJECTIF: Identifier les déterminants de la non-vaccination contre la grippe pendant la grossesse au Canada. MéTHODES: Notre étude porte sur 2 361 mères biologiques d'enfants nés entre décembre 2018 et mars 2019 qui ont été interrogées sur les vaccins reçus pendant leur grossesse, les raisons de non-vaccination, leurs antécédents obstétricaux, et leurs caractéristiques démographiques. Des modèles de régression logistique simple et multiple ont été utilisés pour mesurer les associations entre divers facteurs sociodémographiques, les antécédents obstétricaux, et la non-vaccination contre l'influenza. RéSULTATS: Les facteurs associés à la non-vaccination comprennent le suivi de grossesse par une sage-femme par rapport à un obstétricien-gynécologue (RC 2,02; IC 95% : 1,17‒3,50); avoir eu deux naissances vivantes ou plus par rapport à aucune (RC 1,58; IC 95% : 1,01‒2,49); avoir une scolarité inférieure au diplôme d'études secondaires par rapport à un baccalauréat ou plus (RC 2,50; IC 95% : 1,06‒5,90); et avoir un revenu du ménage inférieur à 60 000 $ (RC 2,46; IC 95% : 1,42‒4,24) ou entre 60 000 $ et 99 999 $ (RC 2,77; IC 95% : 1,70‒4,52) par rapport à un revenu ménager de 140 000 $ ou plus. La province ou le territoire de soins prénataux s'est avéré un facteur important de la non-vaccination avec des rapports de cote statistiquement significatifs pour certaines provinces : RC 7,50 (IC 95% : 1,40‒40,26) pour l'Ontario, 8,23 (IC 95% : 1,53‒44,23) pour Terre-Neuve-et-Labrador, et 11,39 (IC 95% : 2,14‒60,60) pour le Québec, comparativement aux territoires. CONCLUSION: Malgré l'accès universel aux vaccins antigrippaux au Canada durant la grossesse, des variations régionales et des disparités socioéconomiques en non-vaccination persistent.

Development of the hyolaryngeal architecture in horseshoe bats: insights into the evolution of the pulse generation for laryngeal echolocation.

BACKGROUND: The hyolaryngeal apparatus generates biosonar pulses in the laryngeally echolocating bats. The cartilage and muscles comprising the hyolarynx of laryngeally echolocating bats are morphologically modified compared to those of non-bat mammals, as represented by the hypertrophied intrinsic laryngeal muscle. Despite its crucial contribution to laryngeal echolocation, how the development of the hyolarynx in bats differs from that of other mammals is poorly documented. The genus Rhinolophus is one of the most sophisticated laryngeal echolocators, with the highest pulse frequency in bats. The present study provides the first detailed description of the three-dimensional anatomy and development of the skeleton, cartilage, muscle, and innervation patterns of the hyolaryngeal apparatus in two species of rhinolophid bats using micro-computed tomography images and serial tissue sections and compares them with those of laboratory mice. Furthermore, we measured the peak frequency of the echolocation pulse in active juvenile and adult individuals to correspond to echolocation pulses with hyolaryngeal morphology at each postnatal stage. RESULTS: We found that the sagittal crests of the cricoid cartilage separated the dorsal cricoarytenoid muscle in horseshoe bats, indicating that this unique morphology may be required to reinforce the repeated closure movement of the glottis during biosonar pulse emission. We also found that the cricothyroid muscle is ventrally hypertrophied throughout ontogeny, and that the cranial laryngeal nerve has a novel branch supplying the hypertrophied region of this muscle. Our bioacoustic analyses revealed that the peak frequency shows negative allometry against skull growth, and that the volumetric growth of all laryngeal cartilages is correlated with the pulse peak frequency. CONCLUSIONS: The unique patterns of muscle and innervation revealed in this study appear to have been obtained concomitantly with the acquisition of tracheal chambers in rhinolophids and hipposiderids, improving sound intensity during laryngeal echolocation. In addition, significant protrusion of the sagittal crest of the cricoid cartilage and the separated dorsal cricoarytenoid muscle may contribute to the sophisticated biosonar in this laryngeally echolocating lineage. Furthermore, our bioacoustic data suggested that the mineralization of these cartilages underpins the ontogeny of echolocation pulse generation. The results of the present study provide crucial insights into how the anatomy and development of the hyolaryngeal apparatus shape the acoustic diversity in bats.

Structure and interactions of prion-like domains in transcription factor Efg1 phase separation.

Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Efg1's prion-like domain (PrLD) and the protein's ability to undergo phase separation for the white-to-opaque transition of C. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered but have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase-separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding of C. albicans transcription factor interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch.

Variation in leaf dark respiration among C3 and C4 grasses is associated with use of different substrates.

Measurements of respiratory properties have often been made at a single time point either during daytime using dark-adapted leaves or during nighttime. The influence of the day-night cycle on respiratory metabolism has received less attention but is crucial to understand photosynthesis and photorespiration. Here, we examined how CO2- and O2-based rates of leaf dark respiration (Rdark) differed between midday (after 30-min dark adaptation) and midnight in 8 C3 and C4 grasses. We used these data to calculate the respiratory quotient (RQ; ratio of CO2 release to O2 uptake), and assessed relationships between Rdark and leaf metabolome. Rdark was higher at midday than midnight, especially in C4 species. The day-night difference in Rdark was more evident when expressed on a CO2 than O2 basis, with the RQ being higher at midday than midnight in all species, except in rice (Oryza sativa). Metabolomic analyses showed little correlation of Rdark or RQ with leaf carbohydrates (sucrose, glucose, fructose, or starch) but strong multivariate relationships with other metabolites. The results suggest that rates of Rdark and differences in RQ were determined by several concurrent CO2-producing and O2-consuming metabolic pathways, not only the tricarboxylic acid cycle (organic acids utilization) but also the pentose phosphate pathway, galactose metabolism, and secondary metabolism. As such, Rdark was time-, type- (C3/C4) and species-dependent, due to the use of different substrates.

Expanding the molecular grammar of polar residues and arginine in FUS prion-like domain phase separation and aggregation.

A molecular grammar governing low-complexity prion-like domains phase separation (PS) has been proposed based on mutagenesis experiments that identified tyrosine and arginine as primary drivers of phase separation via aromatic-aromatic and aromatic-arginine interactions. Here we show that additional residues make direct favorable contacts that contribute to phase separation, highlighting the need to account for these contributions in PS theories and models. We find that tyrosine and arginine make important contacts beyond only tyrosine-tyrosine and tyrosine-arginine, including arginine-arginine contacts. Among polar residues, glutamine in particular contributes to phase separation with sequence/position-specificity, making contacts with both tyrosine and arginine as well as other residues, both before phase separation and in condensed phases. For glycine, its flexibility, not its small solvation volume, favors phase separation by allowing favorable contacts between other residues and inhibits the liquid-to-solid (LST) transition. Polar residue types also make sequence-specific contributions to aggregation that go beyond simple rules, which for serine positions is linked to formation of an amyloid-core structure by the FUS low-complexity domain. Hence, here we propose a revised molecular grammar expanding the role of arginine and polar residues in prion-like domain protein phase separation and aggregation.

A phylogenomic perspective on interspecific competition.

Evolutionary processes may have substantial impacts on community assembly, but evidence for phylogenetic relatedness as a determinant of interspecific interaction strength remains mixed. In this perspective, we consider a possible role for discordance between gene trees and species trees in the interpretation of phylogenetic signal in studies of community ecology. Modern genomic data show that the evolutionary histories of many taxa are better described by a patchwork of histories that vary along the genome rather than a single species tree. If a subset of genomic loci harbour trait-related genetic variation, then the phylogeny at these loci may be more informative of interspecific trait differences than the genome background. We develop a simple method to detect loci harbouring phylogenetic signal and demonstrate its application through a proof-of-principle analysis of Penicillium genomes and pairwise interaction strength. Our results show that phylogenetic signal that may be masked genome-wide could be detectable using phylogenomic techniques and may provide a window into the genetic basis for interspecific interactions.

The levels of p53 govern the hierarchy of DNA damage tolerance pathway usage.

It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the consequences of DNA damage. Recent data retrieved in tumor and stem cells demonstrated that p53 also carries out non-canonical functions when interacting with the translesion synthesis (TLS) polymerase iota (POLι) at DNA replication forks. This protein complex triggers a DNA damage tolerance (DDT) mechanism controlling the DNA replication rate. Given that the levels of p53 trigger non-binary rheostat-like functions in response to stress or during differentiation, we explore the relevance of the p53 levels for its DDT functions at the fork. We show that subtle changes in p53 levels modulate the contribution of some DDT factors including POLι, POLη, POLζ, REV1, PCNA, PRIMPOL, HLTF and ZRANB3 to the DNA replication rate. Our results suggest that the levels of p53 are central to coordinate the balance between DDT pathways including (i) fork-deceleration by the ZRANB3-mediated fork reversal factor, (ii) POLι-p53-mediated fork-slowing, (iii) POLι- and POLη-mediated TLS and (iv) PRIMPOL-mediated fork-acceleration. Collectively, our study reveals the relevance of p53 protein levels for the DDT pathway choice in replicating cells.

Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress.

Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Our preliminary analyses uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. Motivated to understand how BAT function is regulated epigenetically, we developed a novel method for the first-ever unbiased top-down proteomic quantitation of histone modifications in BAT and validated our results with a multi-omic approach. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28.8°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C) and BAT was analyzed for DNA methylation and histone modifications. Methylation of promoters and intragenic regions in genomic DNA decrease in response to chronic cold exposure. Integration of DNA methylation and RNA expression data suggest a role for epigenetic modification of DNA in gene regulation in response to cold. In response to cold housing, we observe increased bulk acetylation of histones H3.2 and H4, increased histone H3.2 proteoforms with di- and trimethylation of lysine 9 (K9me2 and K9me3), and increased histone H4 proteoforms with acetylation of lysine 16 (K16ac) in BAT. Taken together, our results reveal global epigenetically-regulated transcriptional "on" and "off" signals in murine BAT in response to varying degrees of chronic cold stimuli and establish a novel methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Additionally, we make histone PTM and proteoform quantitation, RNA splicing, RRBS, and transcriptional footprint datasets available as a resource for future research.

Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates.

Cytosolic aggregation of the nuclear protein TDP-43 is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43 enriched phase within stress granules, which subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We conclude that up-concentration inside condensates and simultaneous exposure to environmental stress could be a general pathway for protein aggregation, with intra-condensate demixing constituting a key intermediate step.