Diurnal Oscillations in Liver Mass and Cell Size Accompany Ribosome Assembly Cycles.

The liver plays a pivotal role in metabolism and xenobiotic detoxification, processes that must be particularly efficient when animals are active and feed. A major question is how the liver adapts to these diurnal changes in physiology. Here, we show that, in mice, liver mass, hepatocyte size, and protein levels follow a daily rhythm, whose amplitude depends on both feeding-fasting and light-dark cycles. Correlative evidence suggests that the daily oscillation in global protein accumulation depends on a similar fluctuation in ribosome number. Whereas rRNA genes are transcribed at similar rates throughout the day, some newly synthesized rRNAs are polyadenylated and degraded in the nucleus in a robustly diurnal fashion with a phase opposite to that of ribosomal protein synthesis. Based on studies with cultured fibroblasts, we propose that rRNAs not packaged into complete ribosomal subunits are polyadenylated by the poly(A) polymerase PAPD5 and degraded by the nuclear exosome.

ALL-tRNAseq enables robust tRNA profiling in tissue samples.

Transfer RNAs (tRNAs) are small adaptor RNAs essential for mRNA translation. Alterations in the cellular tRNA population can directly affect mRNA decoding rates and translational efficiency during cancer development and progression. To evaluate changes in the composition of the tRNA pool, multiple sequencing approaches have been developed to overcome reverse transcription blocks caused by the stable structures of these molecules and their numerous base modifications. However, it remains unclear whether current sequencing protocols faithfully capture tRNAs existing in cells or tissues. This is specifically challenging for clinical tissue samples that often present variable RNA qualities. For this reason, we developed ALL-tRNAseq, which combines the highly processive MarathonRT and RNA demethylation for the robust assessment of tRNA expression, together with a randomized adapter ligation strategy prior to reverse transcription to assess tRNA fragmentation levels in both cell lines and tissues. Incorporation of tRNA fragments not only informed on sample integrity but also significantly improved tRNA profiling of tissue samples. Our data showed that our profiling strategy effectively improves classification of oncogenic signatures in glioblastoma and diffuse large B-cell lymphoma tissues, particularly for samples presenting higher levels of RNA fragmentation, further highlighting the utility of ALL-tRNAseq for translational research.

Critical roles of transcriptional coactivator MED1 in the formation and function of mouse adipose tissues.

The MED1 subunit has been shown to mediate ligand-dependent binding of the Mediator coactivator complex to multiple nuclear receptors, including the adipogenic PPARγ, and to play an essential role in ectopic PPARγ-induced adipogenesis of mouse embryonic fibroblasts. However, the precise roles of MED1, and its various domains, at various stages of adipogenesis and in adipose tissue have been unclear. Here, after establishing requirements for MED1, including specific domains, for differentiation of 3T3L1 cells and both primary white and brown preadipocytes, we used multiple genetic approaches to assess requirements for MED1 in adipocyte formation, maintenance, and function in mice. We show that MED1 is indeed essential for the differentiation and/or function of both brown and white adipocytes, as its absence in these cells leads to, respectively, defective brown fat function and lipodystrophy. This work establishes MED1 as an essential transcriptional coactivator that ensures homeostatic functions of adipocytes.

Blood-borne circadian signal stimulates daily oscillations in actin dynamics and SRF activity.

In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.

Gene-Specific Control of tRNA Expression by RNA Polymerase II.

Increasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.

NORMSEQ: a tool for evaluation, selection and visualization of RNA-Seq normalization methods.

RNA-sequencing has become one of the most used high-throughput approaches to gain knowledge about the expression of all different RNA subpopulations. However, technical artifacts, either introduced during library preparation and/or data analysis, can influence the detected RNA expression levels. A critical step, especially in large and low input datasets or studies, is data normalization, which aims at eliminating the variability in data that is not related to biology. Many normalization methods have been developed, each of them relying on different assumptions, making the selection of the appropriate normalization strategy key to preserve biological information. To address this, we developed NormSeq, a free web-server tool to systematically assess the performance of normalization methods in a given dataset. A key feature of NormSeq is the implementation of information gain to guide the selection of the best normalization method, which is crucial to eliminate or at least reduce non-biological variability. Altogether, NormSeq provides an easy-to-use platform to explore different aspects of gene expression data with a special focus on data normalization to help researchers, even without bioinformatics expertise, to obtain reliable biological inference from their data. NormSeq is freely available at: https://arn.ugr.es/normSeq.

The choreography of chromatin in RNA polymerase III regulation.

Regulation of eukaryotic gene expression involves a dynamic interplay between the core transcriptional machinery, transcription factors, and chromatin organization and modification. While this applies to transcription by all RNA polymerase complexes, RNA polymerase III (RNAPIII) seems to be atypical with respect to its mechanisms of regulation. One distinctive feature of most RNAPIII transcribed genes is that they are devoid of nucleosomes, which relates to the high levels of transcription. Moreover, most of the regulatory sequences are not outside but within the transcribed open chromatin regions. Yet, several lines of evidence suggest that chromatin factors affect RNAPIII dynamics and activity and that gene sequence alone does not explain the observed regulation of RNAPIII. Here we discuss the role of chromatin modification and organization of RNAPIII transcribed genes and how they interact with the core transcriptional RNAPIII machinery and regulatory DNA elements in and around the transcribed genes.

Persuading US White evangelicals to vaccinate for COVID-19: Testing message effectiveness in fall 2020 and spring 2021.

The development of COVID-19 vaccines was an important breakthrough for ending the pandemic. However, people refusing to get vaccinated diminish the level of community protection afforded to others. In the United States, White evangelicals have proven to be a particularly difficult group to convince to get vaccinated. Here we investigate whether this group can be persuaded to get vaccinated. To do this, we leverage data from two survey experiments, one fielded prior to approval of COVID-19 vaccines (study 1) and one fielded after approval (study 2). In both experiments, respondents were randomly assigned to treatment messages to promote COVID-19 vaccination. In study 1, we find that a message that emphasizes community interest and reciprocity with an invocation of embarrassment for choosing not to vaccinate is the most effective at increasing uptake intentions, while values-consistent messaging appears to be ineffective. In contrast, in study 2 we observe that this message is no longer effective and that most messages produce little change in vaccine intent. This inconsistency may be explained by the characteristics of White evangelicals who remain unvaccinated vis à vis those who got vaccinated. These results demonstrate the importance of retesting messages over time, the apparent limitations of values-targeted messaging, and document the need to consider heterogeneity even within well-defined populations. This work also cautions against drawing broad conclusions from studies carried out at a single point in time during the COVID-19 pandemic.

Theory of Mind and Social Informant Discrepancy in Autism.

When autistic youth are asked to assess their own social skills, they frequently rate themselves more favorably than their parents rate them. The magnitude of this informant discrepancy has been shown to relate to key clinical outcomes such as treatment response. It has been proposed that this discrepancy arises from difficulties with Theory of Mind. Participants were 167 youth 11 to 17 years old; 72% male, and their parents. Youth completed self-report measures of social skills and social cognitive tasks, while their parents completed questionnaires regarding social skills. A repeated-measures ANOVA indicated both non-autistic and autistic youth rated themselves more favorably than their parents rated them across all measures. Zero-order correlations revealed that raw differences between parent- and participant-report were negatively correlated with scores on parent-reported Theory of Mind measures. However, polynomial analysis did not indicate interaction effects between parent- and participant-report on any of the measures used. Polynomial regression revealed that increases in parent-reported social skill predicted larger increases in parent-report Theory of Mind at low levels of parent-reported social skill compared to high levels of parent-reported social skill. Participant-report social skills predicted performance on a behavioral Theory of Mind test in a curvilinear fashion, such that the relationship was positive at low levels of participant-reported social skills, but negative at high levels. This study replicates the finding that raw difference score analyses may result in illusory effects that are not supported when using more contemporary analysis methods, and that more complex and subtle relationships between social insight and perspective-taking exist within autistic youth.

Structure-Based Design of Bicyclic Helical Peptides That Target the Oncogene ß-Catenin.

The inhibition of intracellular protein-protein interactions is challenging, in particular, when involved interfaces lack pronounced cavities. The transcriptional co-activator protein and oncogene ß­catenin is a prime example of such a challenging target. Despite extensive targeting efforts, available high-affinity binders comprise only large molecular weight Inhibitors. This hampers the further development of therapeutically useful compounds. Herein, we report the design of a considerably smaller peptidomimetic scaffold derived from the α-helical ß­catenin-binding motif of Axin. Sequence maturation and bicyclization provided a stitched peptide with an unprecedented crosslink architecture. The binding mode and site were confirmed by a crystal structure. Further derivatization yielded a ß-catenin inhibitor with single-digit micromolar activity in a cell-based assay. This study sheds a light on how to design helix mimetics with reduced molecular weight thereby improving their biological activity.

Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions.

Enzymes are of central importance to many biotechnological and biomedical applications. However, for many potential applications, the required conditions impede enzyme folding and therefore function. The enzyme Sortase A is a transpeptidase that is widely used to perform bioconjugation reactions with peptides and proteins. Thermal and chemical stress impairs Sortase A activity and prevents its application under harsh conditions, thereby limiting the scope for bioconjugation reactions. Here, we report the stabilization of a previously reported, activity-enhanced Sortase A, which suffered from particularly low thermal stability, using the in situ cyclization of proteins (INCYPRO) approach. After introduction of three spatially aligned solvent-exposed cysteines, a triselectrophilic cross-linker was attached. The resulting bicyclic INCYPRO Sortase A demonstrated activity both at elevated temperature and in the presence of chemical denaturants, conditions under which both wild-type Sortase A and the activity-enhanced version are inactive.

Quantifying social skill deficits and strengths profiles in autistic youth.

While social difficulties in autism are well-established, questions remain regarding whether these represent challenges in acquiring or performing such skills, reduced social strengths, or a unique distribution across these domains (i.e., social profile). This study empirically derived social profiles of 211 autistic and non-autistic youth (Mage  = 13.50; Autistic N = 150; Male N = 151; 85.3% White). Assessments occurred between 2016 and 2020. Results showed that autistic youth exhibit significantly more social acquisition and performance deficits and fewer strengths than nonautistic youth (ds = -.44 to .65). Performance deficits were most-and acquisition deficits least-prominent within autistic profiles, potentially implicating longstanding theoretical models of social difficulties in autism, and supporting new, idiographic approaches for conceptualizing, assessing, and treating social challenges.

Longitudinal Impact of the Pandemic on Social Disruption and Loneliness in Autistic and Non-Autistic Youth.

OBJECTIVE: The coronavirus pandemic drastically increased social isolation. Autistic youth already experience elevated social isolation and loneliness, making them highly vulnerable to the impact of the pandemic. We examined trajectories of social disruption and loneliness in autistic and non-autistic youth during a six-month period of the pandemic (June 2020 until November 2020). METHOD: Participants were 76 youth, ages 8 through 17, (Mage = 12.82, Nautistic = 51) with an IQ ≥ 70. Youth completed a biweekly measure of loneliness (Revised UCLA Loneliness Scale) and their parent completed a measure of pandemic-related family social disruption (Epidemic Pandemic Impacts Inventory). RESULTS: There were no time trends in loneliness across all youth, however, social disruption displayed linear, quadratic, and cubic trends. Non-autistic youth reported relatively greater declines in social disruption compared to autistic youth. Additionally, autistic youth reported relatively greater declines in loneliness relative to non-autistic youth. Greater social disruption was associated with higher loneliness, however, autistic youth demonstrated a relatively stronger relationship between social disruption and loneliness compared to non-autistic youth. CONCLUSIONS: The current study was one of the first to investigate social disruption and loneliness in autistic youth during the COVID-19 pandemic. Results indicated that autistic youth experienced relative decreases in loneliness during this time, perhaps due to reductions in social demands. Nonetheless, when autistic youth did experience social disruption, they reported relatively higher levels of loneliness. This work contributes to our understanding of risk factors for loneliness and highlights the need to understand the benefits, as well as the challenges, to remote schooling and social interactions.

Functions of paralogous RNA polymerase III subunits POLR3G and POLR3GL in mouse development.

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIß). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.

Polarization, the Pandemic, and Public Trust in Health System Actors.

CONTEXT: Public opinion on the performance of health system actors is polarized today, but it remains unclear which actors enjoy the most (least) trust among Democrats and Republicans, whether the Covid-19 pandemic has influenced how people view their own physicians, and whether doctors have retained the ability to influence public beliefs about policy issues. METHODS: We conducted two national surveys in 2022 and 2023 to examine these questions. FINDINGS: Democrats rate the performance of medical research scientists and public health experts during the pandemic more highly than do Republicans and independents. About three in ten Republicans say that the pandemic decreased their trust in their personal doctors. Nonetheless, most Americans report confidence in physicians. We replicate the findings of Gerber et al. (2014) to demonstrate that respondents continue to have more positive views of doctors than other professionals, and that public opinion is responsive to cues from a doctors' group. CONCLUSIONS: What polarizes Democrats and Republicans today is not whether medical scientists and public health experts are competent, but whether the advice offered by these actors is in the public interest and should guide policymakers' decisions. Democrats strongly believe the answer to these questions is yes, while Republicans exhibit skepticism.

Bicyclic ß-Sheet Mimetics that Target the Transcriptional Coactivator ß-Catenin and Inhibit Wnt Signaling.

Protein complexes are defined by the three-dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied for example, as inhibitors of protein-protein interactions (PPIs). Even though ß-sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure-based design of ß-sheet mimetics targeting the intracellular protein ß-catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of ß-catenin, a macrocyclic peptide was designed and its crystal structure in complex with ß-catenin obtained. Using this structure, we designed a library of bicyclic ß-sheet mimetics employing a late-stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to ß-catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other ß-sheet-mediated PPIs.

A field experiment shows that subtle linguistic cues might not affect voter behavior.

One of the most important recent developments in social psychology is the discovery of minor interventions that have large and enduring effects on behavior. A leading example of this class of results is in the work by Bryan et al. [Bryan CJ, Walton GM, Rogers T, Dweck CS (2011) Proc Natl Acad Sci USA 108(31):12653-12656], which shows that administering a set of survey items worded so that subjects think of themselves as voters (noun treatment) rather than as voting (verb treatment) substantially increases political participation (voter turnout) among subjects. We revisit these experiments by replicating and extending their research design in a large-scale field experiment. In contrast to the 11 to 14% point greater turnout among those exposed to the noun rather than the verb treatment reported in the work by Bryan et al., we find no statistically significant difference in turnout between the noun and verb treatments (the point estimate of the difference is approximately zero). Furthermore, when we benchmark these treatments against a standard get out the vote message, we estimate that both are less effective at increasing turnout than a much shorter basic mobilization message. In our conclusion, we detail how our study differs from the work by Bryan et al. and discuss how our results might be interpreted.

Informant discrepancy defines discrete, clinically useful autism spectrum disorder subgroups.

BACKGROUND: Discrepancy between informants (parents and teachers) in severity ratings of core symptoms commonly arise when assessing autism spectrum disorder (ASD). Whether such discrepancy yields unique information about the ASD phenotype and its clinical correlates has not been examined. We examined whether degree of discrepancy between parent and teacher ASD symptom ratings defines discrete, clinically meaningful subgroups of youth with ASD using an efficient, cost-effective procedure. METHODS: Children with ASD (N = 283; 82% boys; Mage  = 10.5 years) were drawn from a specialty ASD clinic. Parents and teachers provided ratings of the three core DSM-IV-TR domains of ASD symptoms (communication, social, and perseverative behavior) with the Child and Adolescent Symptom Inventory-4R (CASI-4R). External validators included child psychotropic medication status, frequency of ASD-relevant school-based services, and the Autism Diagnostic Observation Schedule (ADOS-2). RESULTS: Four distinct subgroups emerged that ranged from large between-informant discrepancy (informant-specific) to relative lack of discrepancy (i.e. informant agreement; cross-situational): Moderate Parent/Low Teacher or Low Parent/Moderate Teacher Severity (Discrepancy), and Moderate or High Symptom Severity (Agreement). Subgroups were highly distinct (mean probability of group assignment = 94%). Relative to Discrepancy subgroups, Agreement subgroups were more likely to receive psychotropic medication, school-based special education services, and an ADOS-2 diagnosis. These differential associations would not have been identified based solely on CASI-4R scores from one informant. CONCLUSIONS: The degree of parent-teacher discrepancy about ASD symptom severity appears to provide more clinically useful information than reliance on a specific symptom domain or informant, and thus yields an innovative, cost-effective approach to assessing functional impairment. This conclusion stands in contrast to existing symptom clustering approaches in ASD, which treat within-informant patterns of symptom severity as generalizable across settings. Within-child variability in symptom expression across settings may yield uniquely useful information for characterizing the ASD phenotype.