Mutual antagonism between CD44 and integrins in glioblastoma cell traction and migration.

Cell migration is the major driver of invasion and metastasis during cancer progression. For cells to migrate, they utilize the actin-myosin cytoskeleton and adhesion molecules, such as integrins and CD44, to generate traction forces in their environment. CD44 primarily binds to hyaluronic acid (HA) and integrins primarily bind to extracellular matrix (ECM) proteins such as collagen. However, the role of CD44 under integrin-mediated conditions and vice versa is not well known. Here, we performed traction force microscopy (TFM) on U251 cells seeded on collagen I-coated polyacrylamide gels to assess the functional mechanical relationship between integrins and CD44. Performing TFM on integrin-mediated adhesion conditions, i.e., collagen, we found that CD44KO U251 cells exerted more traction force than wild-type (WT) U251 cells. Furthermore, untreated WT and CD44-blocked WT exhibited comparable results. Conversely, in CD44-mediated adhesive conditions, integrin-blocked WT cells exerted a higher traction force than untreated WT cells. Our data suggest that CD44 and integrins have a mutually antagonistic relationship where one receptor represses the other's ability to generate traction force on its cognate substrate.

The contribution of chronic peer victimization in elementary school to depressive symptoms in adolescence.

Throughout his career, John Schulenberg challenged us to understand adolescent development as the confluence of distal and proximal experiences along with critical transitions. Heeding this call, we examined whether chronic childhood peer victimization predicted adolescents' depressive symptoms via early-emerging depression growth trajectories, continued victimization into adolescence, and stress-amplification at the middle school transition. Self-reported depressive symptoms and teacher-reported and self-reported peer victimization were obtained from 636 youth (338 girls; Mage = 7.96 years, 66.7% White, 21.7% Black, 11.6% other) in the 2nd-9th grades. Latent growth curve analyses revealed that, by 7th grade, chronic childhood peer victimization was associated with depressive symptoms only through an indirect association with peer victimization in adolescence, underscoring how interrelated historical and ongoing interpersonal stressors contribute to adolescent psychopathology.

"Forcing" new interpretations of molecular tension sensor studies.

Molecular tension sensors are central tools for mechanobiology studies but have limitations in interpretation. Reporting in Cell Reports Methods, Shoyer et al. discover that fluorescent protein photoswitching in concert with sensor extension may expand the use and interpretation of common force-sensing tools.

HUHgle: An Interactive Substrate Design Tool for Covalent Protein-ssDNA Labeling Using HUH-Tags.

HUH-tags have emerged as versatile fusion partners that mediate sequence specific protein-ssDNA bioconjugation through a simple and efficient reaction. Here we present HUHgle, a python-based interactive tool for the visualization, design, and optimization of substrates for HUH-tag mediated covalent labeling of proteins of interest with ssDNA substrates of interest. HUHgle streamlines design processes by integrating an intuitive plotting interface with a search function capable of predicting and displaying protein-ssDNA bioconjugate formation efficiency and specificity in proposed HUH-tag/ssDNA sequence combinations. Validation demonstrates that HUHgle accurately predicts product formation of HUH-tag mediated bioconjugation for single- and orthogonal-labeling reactions. In order to maximize the accessibility and utility of HUHgle, we have implemented it as a user-friendly Google Colab notebook which facilitates broad use of this tool, regardless of coding expertise.

Prescriptive Authority for Direct Entry Midwives in Washington State: Increasing Client Access to Contraception.

Improving health and achieving health equity includes access to sexual and reproductive health care for all populations, especially those most in need. However, access to life-saving and life-affirming contraception with an individual's chosen perinatal provider can be impeded by restrictive regulations that limit scope and practice authority. This is especially true for the majority of community and direct entry midwives in the United States who have historically been unable to legally provide effective contraceptive methods. Recently, licensed midwives in Washington state were the first in the nation to achieve prescriptive authority, enabling their clients to directly obtain contraception and access to medications for common prenatal and postpartum conditions. Sustained advocacy efforts in the state's capitol enabled the Midwives' Association of Washington State to build relationships over time with legislators and government agencies to achieve this long-term goal. We present a successful midwifery-led innovation that achieved scope expansion for licensed midwives whose practice authority was limited by restrictive laws. Lessons learned are described and strategies offered to aid midwives and their advocates in other locales who want to improve health equity and access to contraception. Midwives are well positioned to provide this essential care to individuals living in underserved rural and urban areas and those from historically marginalized communities, but their ability to do so is limited by restrictive legislation.

The crystal structure of the human smacovirus 1 Rep domain.

Replication initiator proteins (Reps) from the HUH endonuclease family process specific single-stranded DNA sequences to initiate rolling-circle replication in viruses. Here, the first crystal structure of the apo state of a Rep domain from the smacovirus family is reported. The structure of the human smacovirus 1 Rep domain was obtained at 1.33 Šresolution and represents an expansion of the HUH endonuclease superfamily, allowing greater diversity in bioconjugation-tag applications.

Multiparametric domain insertional profiling of adeno-associated virus VP1.

Several evolved properties of adeno-associated virus (AAV), such as broad tropism and immunogenicity in humans, are barriers to AAV-based gene therapy. Most efforts to re-engineer these properties have focused on variable regions near AAV's 3-fold protrusions and capsid protein termini. To comprehensively survey AAV capsids for engineerable hotspots, we determined multiple AAV fitness phenotypes upon insertion of six structured protein domains into the entire AAV-DJ capsid protein VP1. This is the largest and most comprehensive AAV domain insertion dataset to date. Our data revealed a surprising robustness of AAV capsids to accommodate large domain insertions. Insertion permissibility depended strongly on insertion position, domain type, and measured fitness phenotype, which clustered into contiguous structural units that we could link to distinct roles in AAV assembly, stability, and infectivity. We also identified engineerable hotspots of AAV that facilitate the covalent attachment of binding scaffolds, which may represent an alternative approach to re-direct AAV tropism.

Reconnecting to Internal Sensations and Experiences (RISE): An online, multi-session intervention improves interoceptive sensibility for military personnel.

OBJECTIVE: This preregistered randomized controlled trial tested the effects of a four-session, online interoceptive awareness intervention relative to an active comparator, matched for time and attention on interoception and suicidal ideation. METHOD: Participants (N = 195; 69% male; mean age = 37) were active duty service members (62%) and veterans (38%) who completed measures of interoceptive sensibility, interoceptive accuracy, and suicidal ideation at baseline. They were randomized to either the interoceptive awareness intervention, Reconnecting to Internal Sensations and Experiences (RISE), or the comparator, Healthy Habits. Participants completed the assessment battery again at posttest as well as a 1 and 3-month follow-up. RESULTS: RISE was rated as acceptable and demonstrated excellent feasibility per completion rates (85% completed all four modules). RISE improved the majority of interoceptive sensibility domains assessed (noticing body sensations, not worrying about sensations of pain or discomfort, emotional awareness, self-regulation, body listening, and body trust), and most of these gains remained at 1 and 3-month follow-ups. There were no differences between conditions on suicidal ideation, perhaps due to the low levels of ideation reported, or interoceptive accuracy. CONCLUSIONS: RISE is a disseminable, cost-effective, and transdiagnostic intervention that improves interoceptive sensibility up to 3 months.

Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase.

Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

Multiparametric domain insertional profiling of Adeno-Associated Virus VP1.

Evolved properties of Adeno-Associated Virus (AAV), such as broad tropism and immunogenicity in humans, are barriers to AAV-based gene therapy. Previous efforts to re-engineer these properties have focused on variable regions near AAV’s 3-fold protrusions and capsid protein termini. To comprehensively survey AAV capsids for engineerable hotspots, we determined multiple AAV fitness phenotypes upon insertion of large, structured protein domains into the entire AAV-DJ capsid protein VP1. This is the largest and most comprehensive AAV domain insertion dataset to date. Our data revealed a surprising robustness of AAV capsids to accommodate large domain insertions. There was strong positional, domain-type, and fitness phenotype dependence of insertion permissibility, which clustered into correlated structural units that we could link to distinct roles in AAV assembly, stability, and infectivity. We also identified new engineerable hotspots of AAV that facilitate the covalent attachment of binding scaffolds, which may represent an alternative approach to re-direct AAV tropism.

RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes.

Mechanical forces drive critical cellular processes that are reflected in mechanical phenotypes, or mechanotypes, of cells and their microenvironment. We present here "Rupture And Deliver" Tension Gauge Tethers (RAD-TGTs) in which flow cytometry is used to record the mechanical history of thousands of cells exerting forces on their surroundings via their propensity to rupture immobilized DNA duplex tension probes. We demonstrate that RAD-TGTs recapitulate prior DNA tension probe studies while also yielding a gain of fluorescence in the force-generating cell that is detectable by flow cytometry. Furthermore, the rupture propensity is altered following disruption of the cytoskeleton using drugs or CRISPR-knockout of mechanosensing proteins. Importantly, RAD-TGTs can differentiate distinct mechanotypes among mixed populations of cells. We also establish oligo rupture and delivery can be measured via DNA sequencing. RAD-TGTs provide a facile and powerful assay to enable high-throughput mechanotype profiling, which could find various applications, for example, in combination with CRISPR screens and -omics analysis.

Patterns of natural health product use at an outpatient heart function clinic.

Background: Natural health product (NHP) use is common among Canadians, but the NHPs used by outpatients with cardiovascular conditions such as atrial fibrillation and heart failure have not been identified. Objectives: Describe NHP use among outpatient cardiac patients, assess drug interactions and their potential implications and determine NHP documentation by health care providers. Methods: Telephone interviews were conducted by the main researcher with patients who attended the Cardiac Clinics at the Royal Columbian Hospital. Medication reconciliation was performed to elicit information regarding NHP use and clinic charts were used to supplement demographic information. Results: There were 119 successful interviews. Most patients were approximately 65 years old and male, were diagnosed with atrial fibrillation, had 2 to 3 queried comorbidities and took 2 cardiovascular medications. It was found that 62% of patients use NHPs, and 239 individual NHPs were identified. The most common NHPs used were vitamins and minerals (63%), especially vitamin D (13%), multivitamins (8%) and omega-3s (8%). Interactions between cardiac medications and NHPs occurred in 86% of patients. NHP use was completely documented by health care providers in 24% of patients. Conclusion: NHP use is common among patients who attend outpatient cardiac clinics. Interactions between NHPs and cardiovascular medications are prevalent and may carry specific individual patient risks. NHP documentation by health care providers is often incomplete.

Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin.

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies.

Need for approval and antisocial behavior moderate the effect of socioemotional cues on adolescent girls' cognitive control.

To examine whether need for approval (NFA) and antisocial behavior (ASB) moderate the effects of socioemotional stimuli on cognitive control, 88 girls (Mage  = 16.31 years; SD = 0.84; 65.9% White) completed a socioemotional Go/No-go and questionnaires. At high approach NFA, girls responded more slowly during appetitive than control (b = -8.80, p < .01) and aversive (b = -5.58, p = .01) trials. At high ASB, girls responded more slowly (b = -6.12, p = .02) and less accurately (OR = 1.11, p = .03) during appetitive than aversive trials; at low ASB, girls responded more slowly during aversive than control trials (b = -4.42, p = .04). Thus, both context and individual differences influence adolescents' cognitive control.

Watson-Crick Base-Pairing Requirements for ssDNA Recognition and Processing in Replication-Initiating HUH Endonucleases.

Replication-initiating HUH endonucleases (Reps) are sequence-specific nucleases that cleave and rejoin single-stranded DNA (ssDNA) during rolling-circle replication. These functions are mediated by covalent linkage of the Rep to its substrate post cleavage. Here, we describe the structures of the endonuclease domain from the Muscovy duck circovirus Rep in complex with its cognate ssDNA 10-mer with and without manganese in the active site. Structural and functional analyses demonstrate that divalent cations play both catalytic and structural roles in Reps by polarizing and positioning their substrate. Further structural comparisons highlight the importance of an intramolecular substrate Watson-Crick (WC) base pairing between the -4 and +1 positions. Subsequent kinetic and functional analyses demonstrate a functional dependency on WC base pairing between these positions regardless of the pair's identity (i.e., A·T, T·A, G·C, or C·G), highlighting a structural specificity for substrate interaction. Finally, considering how well WC swaps were tolerated in vitro, we sought to determine to what extent the canonical -4T·+1A pairing is conserved in circular Rep-encoding single-stranded DNA viruses and found evidence of noncanonical pairings in a minority of these genomes. Altogether, our data suggest that substrate intramolecular WC base pairing is a universal requirement for separation and reunion of ssDNA in Reps. IMPORTANCE Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses are a ubiquitous group of viruses that infect organisms across all domains of life. These viruses negatively impact both agriculture and human health. All members of this viral family employ a multifunctional nuclease (Rep) to initiate replication. Reps are structurally similar throughout this family, making them targets of interest for viral inhibition strategies. Here, we investigate the functional dependencies of the Rep protein from Muscovy duck circovirus for ssDNA interaction. We demonstrate that this Rep requires an intramolecular Watson-Crick base pairing for origin of replication (Ori) recognition and interaction. We show that noncognate base pair swaps are well tolerated, highlighting a local structural specificity over sequence specificity. Bioinformatic analysis found that the vast majority of CRESS-DNA Oris form base pairs in conserved positions, suggesting this pairing is a universal requirement for replication initiation in the CRESS-DNA virus family.

Relationships between eating disorder symptoms, muscle dysmorphia symptoms, and suicidal ideation: A random intercepts cross-lagged panel approach.

OBJECTIVE: Eating disorder (ED) symptoms correlate with suicidality; yet the strength of these relationships in men is unclear. Muscle dysmorphia (MD) symptoms may reflect a more accurate index of body-related concerns for men, as they better target muscularity concerns typical of men. However, no studies have tested a model in which ED/MD symptoms and suicidality are simultaneously examined. We longitudinally tested whether ED/MD symptoms were related to suicidal ideation among a community sample of men. METHODS: Men with MD symptoms (N = 272) were recruited to complete three surveys over 6 weeks. A random intercepts cross-lagged panel model tested predictive associations between ED/MD symptoms and suicidal ideation, while disaggregating between/within-person variance. RESULTS: ED/MD symptoms were significantly associated with suicidal ideation at the between-subjects level (ED: b = .04; MD: b = .09) and showed significant within-wave covariances with suicidal ideation (ED: b = .02-.04; MD: b = .02-.05). Those who experienced increases in ED symptoms showed increased suicidal ideation at the next wave (b = .32). Those who experienced increases in suicidal ideation showed increases in MD symptoms at the next wave (b = .85). DISCUSSION: Results highlight ED symptoms as a potential risk factor for suicidal ideation among men. Further, suicidal ideation predicted MD symptoms. ED symptoms may create intra- and interpersonal distress predicting suicidal ideation. Suicidal ideation may lead to muscle-building behaviors to cope with suicidal thoughts. Clinicians should assess for suicidal ideation among men at risk for MD/EDs, and for MD symptoms among those reporting suicidal ideation. PUBLIC SIGNIFICANCE: Eating disorder (ED) symptoms are related to suicidality, but these relationships are understudied among men. Since men report concerns surrounding muscularity, muscle dysmorphia (MD) may be a better ED index for this population. However, little research has investigated relationships between ED symptoms, MD symptoms, and suicidality among men. This study investigated relationships between ED/MD symptoms and suicidality among 272 men. Results may inform clinical assessment, treatment, and classification of MD.

Within-person reciprocal associations between peer victimization and need for approval.

Prior research links need for approval (NFA; the extent to which self-worth is contingent on peer approval or disapproval) to critical developmental outcomes, but little is known about how NFA develops over time or within social contexts. To address this gap, the present study used a sophisticated analytic approach (autoregressive latent trajectory modeling with standardized residuals) to examine dynamic associations between one salient social experience-peer victimization-and two dimensions of NFA, conceptualized in terms of approach motivation (NFAapproach; enhanced self-worth based on peer approval) and avoidance motivation (NFAavoid; depleted self-worth based on peer disapproval). Following 636 youth (338 girls; Mage = 7.96 years at Wave 1; 66.7% White; 35.0% subsidized school lunch) from second to seventh grade, analyses revealed that peer victimization predicts subsequent increases in NFAavoid, which in turn predicts subsequent increases in victimization. Findings also revealed that although mean levels of NFAavoid decrease during childhood, increases or decreases in NFA become more entrenched. Thus, childhood peer victimization may disrupt normative decreases in NFAavoid and contribute to a cycle in which negative peer judgments increasingly foster low self-worth and further peer difficulties. Preventing this cycle may require encouraging peer-victimized youth to base their self-worth on internal standards rather than peer feedback while helping them develop positive relationships that promote self-worth. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Dystrophin missense mutations alter focal adhesion tension and mechanotransduction.

Dystrophin is an essential muscle protein that contributes to cell membrane stability by mechanically linking the actin cytoskeleton to the extracellular matrix via an adhesion complex called the dystrophin-glycoprotein complex. The absence or impaired function of dystrophin causes muscular dystrophy. Focal adhesions (FAs) are also mechanosensitive adhesion complexes that connect the cytoskeleton to the extracellular matrix. However, the interplay between dystrophin and FA force transmission has not been investigated. Using a vinculin-based bioluminescent tension sensor, we measured FA tension in transgenic C2C12 myoblasts expressing wild-type (WT) dystrophin, a nonpathogenic single nucleotide polymorphism (SNP) (I232M), or two missense mutations associated with Duchenne (L54R), or Becker muscular dystrophy (L172H). Our data revealed cross talk between dystrophin and FAs, as the expression of WT or I232M dystrophin increased FA tension compared to dystrophin-less nontransgenic myoblasts. In contrast, the expression of L54R or L172H did not increase FA tension, indicating that these disease-causing mutations compromise the mechanical function of dystrophin as an FA allosteric regulator. Decreased FA tension caused by these mutations manifests as defective migration, as well as decreased Yes-associated protein 1 (YAP) activation, possibly by the disruption of the ability of FAs to transmit forces between the extracellular matrix and cytoskeleton. Our results indicate that dystrophin influences FA tension and suggest that dystrophin disease-causing missense mutations may disrupt a cellular tension-sensing pathway in dystrophic skeletal muscle.

Does social withdrawal inhibit defending bullied peers and do perceived injunctive norms mitigate those effects?

Although a number of social-cognitive and contextual correlates of defending against bullying have been identified, research on the personality traits associated with defending have yielded weak and inconsistent results. The current study provided a novel examination as to whether a tendency toward social withdrawal is associated with less frequent defending and whether perceived injunctive norms for defending and aggression minimize the impact of social withdrawal on defending behaviors. A sample of 1,564 children (760 girls; Mage = 10.05; 55.0% White; 36.1% Black) were followed in the fall, winter, and spring of a school year. Defending was measured with self-reports and peer-reports. Social withdrawal was measured using teacher-reports. Perceived injunctive norms were estimated by calculating within-person correlations between participants' ratings of peers' popularity and defending and between peers' popularity and aggression. Results revealed that social withdrawal was associated with less peer-reported defending in the fall, and this effect was sustained over the school year. For boys, lower levels of social withdrawal in the fall were associated with less peer-reported defending when they viewed popular peers as unlikely to defend. A temporary (i.e., fall) association was found between viewing defenders as popular and self-reported defending, and children became less likely to self-report defending over the school year if they viewed popular children as aggressive. These findings underscore the need to examine how temperamental traits and perceived contextual norms cocontribute to bystanders' behavior when witnessing bullying. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Emotion mindsets and depressive symptoms in adolescence: The role of emotion regulation competence.

Recent theories posit that emotion mindsets (i.e., the extent to which individuals believe emotions are malleable or fixed) play a crucial role in experiences of emotion and influence emotion regulation (ER) processes. Drawing from mindset theory, this study examined the hypothesis that fixed emotion mindsets (FEMs) would predict depressive symptoms via compromised ER competence in adolescence, a period when many first episodes of depression occur. Results supported these hypotheses across two studies assessing participants in midadolescence (ages 14-18; M age = 16.17) and late adolescence (ages 18-21; M age = 18.52). Using a comprehensive approach to assessing ER, results demonstrated that FEMs were associated with less voluntary engagement and more disengagement and emotion dysregulation. In turn, higher voluntary engagement was associated with lower depressive symptoms, whereas higher disengagement and emotion dysregulation were associated with higher depressive symptoms. These findings highlight that one understudied pathway from FEMs to depressive symptoms may be the manner in which individuals respond to their emotions, implicating emotion mindsets as one target for efforts to improve clinical outcomes during adolescence. (PsycInfo Database Record (c) 2022 APA, all rights reserved).