Early adolescent food routines: A photo-elicitation study.

Early adolescence (ages 10-14) encompasses a critical transition period in which food and nutrition decisions are shifting in important ways. Food routines are food-based activities that repeat across days, weeks, seasons, or lives. Examining routines can provide insight into how individuals are influenced in food choices. The objective of this study was to describe current influences on and experiences with food routines during early adolescence. In-depth interviews, using a photo-elicitation approach, were conducted with 30 participants (16 females; 14 males) in the United States. Participants took photos that were then used during the interview to describe food-related decisions and influences. Interviews were audio recorded and transcribed verbatim. Analysis was guided by a grounded theory approach to identify emergent themes related to routines and resulted in the development of a conceptual model for early adolescent food routines. Participants identified a wide range of routines and three main themes emerged: family, settings, and meals/foods consumed. Some had highly established routines throughout the week, while others described routines only for certain meals or days. Several participants described increased control or the ability to modify routines around some eating episodes such as snacks, lunches, and weekend breakfasts. Findings revealed how participants viewed eating routines and provided information about food-and nutrition-related behaviors that can inform future research and practice. Early adolescents appear to have complex food routines influenced by structures and different amounts of control.

Personal, proxy, and collective food agency among early adolescents.

Early adolescence is a critical time for health behavior development because agency increases during the transition from childhood to adolescence. This qualitative study sought to identify how early adolescent participants described food-related agency. One-on-one interviews were conducted with 30 early adolescents (10-13 years). Data analysis was guided by Bandura's three modes of agency: personal, proxy, and collective. Results suggest participants' food behaviors were informed by a growing knowledge about nutrition, household food rules, and school food environments. Participants described different modes of agency in four areas - grocery shopping, cooking, consumption decisions, and nutrition information seeking - with varying degrees of agency in each area. Understanding how each of the three modes operate and the interplay between them can information future research aimed at improving the nutrition behaviors of early adolescents.

Set4 is a chromatin-associated protein, promotes survival during oxidative stress, and regulates stress response genes in yeast.

The Set4 protein in the yeast Saccharomyces cerevisiae contains both a PHD finger and a SET domain, a common signature of chromatin-associated proteins, and shares sequence homology with the yeast protein Set3, the fly protein UpSET, and the human protein mixed-lineage leukemia 5 (MLL5). However, the biological role for Set4 and its potential function in chromatin regulation has not been well defined. Here, we analyzed yeast cell phenotypes associated with loss of Set4 or its overexpression, which revealed that Set4 protects against oxidative stress induced by hydrogen peroxide. Gene expression analysis indicated that Set4 promotes the activation of stress response genes in the presence of oxidative insults. Using ChIP analysis and other biochemical assays, we also found that Set4 interacts with chromatin and directly localizes to stress response genes upon oxidative stress. However, recombinant Set4 did not show detectable methyltransferase activity on histones. Our findings also suggest that Set4 abundance in the cell is balanced under normal and stress conditions to promote survival. Overall, these results suggest a model in which Set4 is a stress-responsive, chromatin-associated protein that activates gene expression programs required for cellular protection against oxidative stress. This work advances our understanding of mechanisms that protect cells during oxidative stress and further defines the role of the Set3-Set4 subfamily of SET domain-containing proteins in controlling gene expression in response to adverse environmental conditions.

SET domains and stress: uncovering new functions for yeast Set4.

Chromatin dynamics are central to the regulation of gene expression and genome stability, particularly in the presence of environmental signals or stresses that prompt rapid reprogramming of the genome to promote survival or differentiation. While numerous chromatin regulators have been implicated in modulating cellular responses to stress, gaps in our mechanistic understanding of chromatin-based changes during stress suggest that additional proteins are likely critical to these responses and the molecular details underlying their activities are unclear in many cases. We recently identified a role for the relatively uncharacterized SET domain protein Set4 in promoting cell survival during oxidative stress in Saccharomyces cerevisiae. Set4 is a member of the Set3 subfamily of SET domain proteins which are defined by the presence of a PHD finger and divergent SET domain sequences. Here, we integrate our new observations on the function of Set4 with known roles for other related family members, including yeast Set3, fly UpSET and mammalian proteins MLL5 and SETD5. We discuss outstanding questions regarding the molecular mechanisms by which these proteins control gene expression and their potential contributions to cellular responses to environmental stress.

CUL4B: trash talking at chromatin.

In this issue, Nakagawa and Xiong (2011) reveal a mechanism targeting WDR5 for proteolysis dependent on the X-linked mental retardation gene, CUL4B. This provides a link between the stability of a chromatin factor and gene expression implicated in neurological pathogenesis.

Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans.

The plasticity of ageing suggests that longevity may be controlled epigenetically by specific alterations in chromatin state. The link between chromatin and ageing has mostly focused on histone deacetylation by the Sir2 family, but less is known about the role of other histone modifications in longevity. Histone methylation has a crucial role in development and in maintaining stem cell pluripotency in mammals. Regulators of histone methylation have been associated with ageing in worms and flies, but characterization of their role and mechanism of action has been limited. Here we identify the ASH-2 trithorax complex, which trimethylates histone H3 at lysine 4 (H3K4), as a regulator of lifespan in Caenorhabditis elegans in a directed RNA interference (RNAi) screen in fertile worms. Deficiencies in members of the ASH-2 complex-ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2-extend worm lifespan. Conversely, the H3K4 demethylase RBR-2 is required for normal lifespan, consistent with the idea that an excess of H3K4 trimethylation-a mark associated with active chromatin-is detrimental for longevity. Lifespan extension induced by ASH-2 complex deficiency requires the presence of an intact adult germline and the continuous production of mature eggs. ASH-2 and RBR-2 act in the germline, at least in part, to regulate lifespan and to control a set of genes involved in lifespan determination. These results indicate that the longevity of the soma is regulated by an H3K4 methyltransferase/demethylase complex acting in the C. elegans germline.

The molecular mechanism of a cis-regulatory adaptation in yeast.

Despite recent advances in our ability to detect adaptive evolution involving the cis-regulation of gene expression, our knowledge of the molecular mechanisms underlying these adaptations has lagged far behind. Across all model organisms, the causal mutations have been discovered for only a handful of gene expression adaptations, and even for these, mechanistic details (e.g. the trans-regulatory factors involved) have not been determined. We previously reported a polygenic gene expression adaptation involving down-regulation of the ergosterol biosynthesis pathway in the budding yeast Saccharomyces cerevisiae. Here we investigate the molecular mechanism of a cis-acting mutation affecting a member of this pathway, ERG28. We show that the causal mutation is a two-base deletion in the promoter of ERG28 that strongly reduces the binding of two transcription factors, Sok2 and Mot3, thus abolishing their regulation of ERG28. This down-regulation increases resistance to a widely used antifungal drug targeting ergosterol, similar to mutations disrupting this pathway in clinical yeast isolates. The identification of the causal genetic variant revealed that the selection likely occurred after the deletion was already present at high frequency in the population, rather than when it was a new mutation. These results provide a detailed view of the molecular mechanism of a cis-regulatory adaptation, and underscore the importance of this view to our understanding of evolution at the molecular level.

Defining Biological and Biochemical Functions of Noncanonical SET Domain Proteins.

Within the SET domain superfamily of lysine methyltransferases, there is a well-conserved subfamily, frequently referred to as the Set3 SET domain subfamily, which contain noncanonical SET domains carrying divergent amino acid sequences. These proteins are implicated in diverse biological processes including stress responses, cell differentiation, and development, and their disruption is linked to diseases including cancer and neurodevelopmental disorders. Interestingly, biochemical and structural analysis indicates that they do not possess catalytic methyltransferase activity. At the molecular level, Set3 SET domain proteins appear to play critical roles in the regulation of gene expression, particularly repression and heterochromatin maintenance, and in some cases, via scaffolding other histone modifying activities at chromatin. Here, we explore the common and unique functions among Set3 SET domain subfamily proteins and analyze what is known about the specific contribution of the conserved SET domain to functional roles of these proteins, as well as propose areas of investigation to improve understanding of this important, noncanonical subfamily of proteins.

Clinical Education Data Management: Current Landscape in Entry-Level Physical Therapist Education.

BACKGROUND: Data management (DM) systems represent an opportunity for innovation in education and data-driven decision-making (DDDM) in allied health education. Understanding clinical education (CE) DM systems in entry-level physical therapy (PT) education programs could provide valuable insight into structure and operation and may represent opportunities to address CE challenges. The purpose of this study is to describe how PT programs are using CE DM systems to inform recommendations for CE DM and support knowledge sharing and DDDM. SUBJECTS: CE faculty and administrators were recruited from entry-level PT education programs to participate in a cross-sectional survey. METHODS: The authors designed a novel survey which included demographics and use of CE DM systems. Descriptive statistics and content analysis of narrative data were used to examine responses. RESULTS: The survey was distributed to 220 academic PT programs in June 2021 with 111 respondents (50% response rate). Respondents use multiple systems to complete CE tasks (e.g., placement process, on-boarding, agreement tracking, as a CE site database). Forty-three percent (n=47) use one system, 76% (n=35) of those use the same Software as a Service vendor. Eighty-six percent (n=96) are satisfied with their current CE DM system. Respondents enter data related to CE site information, CE environment, length of the CE experience, and accreditation-required clinical instructor information. Ninety-four percent (n=93) and 70% (n=70) extract data to make decisions about the placement process and curriculum, respectively. CONCLUSION: While variability across CE DM systems presents a challenge, survey respondents indicated common practices related to functionality, data entry, and extraction. Clinical education DM systems house critical data to address challenges in CE. Strategies to improve accessibility and use of this data to support DDDM should be explored.

Surgical quality surrogates do not predict colon cancer survival in the setting of technical credentialing: a report from the prospective COST trial.

OBJECTIVE: We tested the hypothesis that the 12 lymph node (LN) count and other surgical variables would not predict survival in a setting where surgical techniques were standardized and surgeons were credentialed and audited. BACKGROUND: The National Quality Forum has adopted the 12-node minimum as a surgical quality metric due to the strong association between node count and survival. METHODS: We performed a secondary analysis of data from the Clinical Outcomes of Surgical Therapy (COST) multicenter randomized trial testing laparoscopic versus open colectomy. Surgeons were credentialed and video-audited for adherence to technical standards. Patients with noninvasive and stage IV disease were excluded, leaving 787 subjects (267 stage I, 284 stage II, and 236 stage III). Median age was 70 years and 50% were male. The overall 5-year survival was 77.2%. RESULTS: Five-year overall and disease-free survival was not influenced by LN count (< 12 vs ≥ 12), sex, tumor location (right vs left vs sigmoid), surgical technique (laparoscopic vs open), total bowel length, proximal margin, distal margin, radial margin, or mesenteric length (P > 0.05 for all). Univariate predictors of survival included age and cancer stage, and these remained significant in the multivariate model. Across all stages of disease, after adjusting for age and stage, LN count did not predict overall or disease-free survival (P = 0.60). CONCLUSIONS: Despite the known association between LN count and survival, we could not confirm an association between surgical surrogates and cancer outcomes. We postulate that standardization, credentialing, and monitoring may be more important than traditional surgical quality surrogates.

Set1 regulates telomere function via H3K4 methylation-dependent and -independent pathways and calibrates the abundance of telomere maintenance factors.

Set1 is an H3K4 methyltransferase that comprises the catalytic subunit of the COMPASS complex and has been implicated in transcription, DNA repair, cell cycle control, and numerous other genomic functions. Set1 also promotes proper telomere maintenance, as cells lacking Set1 have short telomeres and disrupted subtelomeric gene repression; however, the precise role for Set1 in these processes has not been fully defined. In this study, we have tested mutants of Set1 and the COMPASS complex that differentially alter H3K4 methylation status, and we have attempted to separate catalytic and noncatalytic functions of Set1. Our data reveal that Set1-dependent subtelomeric gene repression relies on its catalytic activity toward H3K4, whereas telomere length is regulated by Set1 catalytic activity but likely independent of the H3K4 substrate. Furthermore, we uncover a role for Set1 in calibrating the abundance of critical telomere maintenance proteins, including components of the telomerase holoenzyme and members of the telomere capping CST (Cdc13-Stn1-Ten1) complex, through both transcriptional and posttranscriptional pathways. Altogether, our data provide new insights into the H3K4 methylation-dependent and -independent roles for Set1 in telomere maintenance in yeast and shed light on possible roles for Set1-related methyltransferases in other systems.

The SMYD3-MAP3K2 signaling axis promotes tumor aggressiveness and metastasis in prostate cancer.

Aberrant activation of Ras/Raf/mitogen-activated protein kinase (MAPK) signaling is frequently linked to metastatic prostate cancer (PCa); therefore, the characterization of modulators of this pathway is critical for defining therapeutic vulnerabilities for metastatic PCa. The lysine methyltransferase SET and MYND domain 3 (SMYD3) methylates MAPK kinase kinase 2 (MAP3K2) in some cancers, causing enhanced activation of MAPK signaling. In PCa, SMYD3 is frequently overexpressed and associated with disease severity; however, its molecular function in promoting tumorigenesis has not been defined. We demonstrate that SMYD3 critically regulates tumor-associated phenotypes via its methyltransferase activity in PCa cells and mouse xenograft models. SMYD3-dependent methylation of MAP3K2 promotes epithelial-mesenchymal transition associated behaviors by altering the abundance of the intermediate filament vimentin. Furthermore, activation of the SMYD3-MAP3K2 signaling axis supports a positive feedback loop continually promoting high levels of SMYD3. Our data provide insight into signaling pathways involved in metastatic PCa and enhance understanding of mechanistic functions for SMYD3 to reveal potential therapeutic opportunities for PCa.

Everybody's welcome: The big tent approach to epigenetic drug discovery.

The rapid expansion of epigenetics research is fueled by the increasing understanding that epigenetic processes are critical to regulating cellular development and dysfunction of epigenetic programs is responsible for a diverse set of human pathologies, including cancer, autoimmune and neurodegenerative diseases. The expansive set of components contributing to epigenetic disease mechanisms and the often reversible nature of epigenetic lesions provide prime opportunities for the development of novel therapeutic strategies. Here, we provide an overview of epigenetics and its relationship to disease, discuss current epigenetics-based therapies and suggest new avenues for the identification of therapies targeting deregulated epigenetic programs in disease.

Features associated with successful recruitment of diverse patients onto cancer clinical trials: report from the American College of Surgeons Oncology Group.

BACKGROUND: The clinical trials mechanism of standardized treatment and follow-up for cancer patients with similar stages and patterns of disease is the most powerful approach available for evaluating the efficacy of novel therapies, and clinical trial participation should protect against delivery of care variations associated with racial/ethnic identity and/or socioeconomic status. Unfortunately, disparities in clinical trial accrual persist, with African Americans (AA) and Hispanic/Latino Americans (HA) underrepresented in most studies. STUDY DESIGN: We evaluated the accrual patterns for 10 clinical trials conducted by the American College of Surgeons Oncology Group (ACOSOG) 1999-2009, and analyzed results by race/ethnicity as well as by study design. RESULTS: Eight of 10 protocols were successful in recruiting AA and/or HA participants; three of four randomized trials were successful. Features that were present among all of the successfully recruiting protocols were: (1) studies designed to recruit patients with regional or advanced-stage disease (2 of 2 protocols); and (2) studies that involved some investigational systemic therapy (3 of 3 protocols). DISCUSSION: AA and HA cancer patients can be successfully accrued onto randomized clinical trials, but study design affects recruitment patterns. Increased socioeconomic disadvantages observed within minority-ethnicity communities results in barriers to screening and more advanced cancer stage distribution. Improving cancer early detection is critical in the effort to eliminate outcome disparities but existing differences in disease burden results in diminished eligibility for early-stage cancer clinical trials among minority-ethnicity patients.

Immunoaffinity purification of endogenous proteins from S. cerevisiae for post-translational modification and protein interaction analysis.

Protein regulation by post-translational modifications and protein-protein interactions is critical to controlling molecular pathways. Here, we describe an immunoaffinity purification approach in Saccharomyces cerevisiae. The protocol uses an endogenously-expressed epitope-tagged protein and can be applied to the identification of post-translational modifications or protein binding partners. The lysine methyltransferase Set5 is used as an example here to purify phosphorylated Set5 and identify phosphosites; however, this approach can be applied to a diverse set of proteins in yeast. For complete details on the use and execution of this protocol, please refer to Jaiswal et al. (2020).

Set4 regulates stress response genes and coordinates histone deacetylases within yeast subtelomeres.

The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status identified a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, where the loss of Set4 decreases cell fitness and cell wall integrity. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for Set4 in regulating repressive, heterochromatin-like environments.

Dipstick tests for secondhand smoke exposure.

INTRODUCTION: We tested TobacAlert and NicAlert immunochromatographic strips for use as indicators of secondhand smoke (SHS) exposure. METHODS: Urine samples collected from SHS-unexposed volunteers were spiked with cotinine to concentrations of 5, 8, 13, and 23 ng cotinine/ml urine. One sample was not spiked and used as a control. According to manufacturer's instructions, 45 NicAlert and 45 TobacAlert dipsticks were prepared. The exercise was repeated once. Cotinine levels in urine samples were measured using liquid chromatography/mass spectrometry (LC/MS). STATA was used for statistical analyses. RESULTS: Ninety NicAlert and 90 TobacAlert dipsticks were tested. Each strip was read by 3 different readers, for 270 NicAlert and 270 TobacAlert readings; 98/270 (36%) NicAlert and 104/270 (39%) TobacAlert readings agreed with the readings predicted by LC/MS-determined cotinine levels. Spearman's rho for the NicAlert strips was .13 and for the TobacAlert strips .23. Both were statistically significant. Using a dichotomous scheme to interpret any strip reading >or=1 as "positive," indicating SHS exposure, NicAlert strips were 94% sensitive and 31% specific, while TobacAlert strips were 89% sensitive and 60% specific. DISCUSSION: NicAlert and TobacAlert strips performed poorly at low cotinine levels. While the strips could be used to prescreen samples prior to more accurate testing, their use in the clinical or research setting to indicate SHS exposure should be restricted to carefully selected scenarios.

Cancer registries: a novel alternative to long-term clinical trial follow-up based on results of a comparative study.

BACKGROUND: Data collection and review were identified as major contributors to the cost of randomized clinical trials (RCTs). PURPOSE: We proposed and assessed a novel alternative for long-term clinical trial follow-up based on the data captured through an accredited Cancer Registry (CR) that is part of the National Cancer Database (NCDB). METHODS: Patients from Mayo Clinic, Rochester, enrolled in the North Central Cancer Treatment Group N934653 (COST) trial (98 patients) and the American College of Surgeons Oncology Group Z0030 trial (55 patients) were included in the study. Demographic, treatment, and long-term outcome data were compared between the hospital-based CR and the RCTs' databases. Concordances were used to estimate the agreement between two databases. Kaplan-Meier curves were plotted to examine the consistency of time-to-event long-term outcomes of the CR and RCT databases. RESULTS: High concordances (>95%) were observed for most demographic and treatment variables between the CR data and RCT data. The vital status concordances were 100% and 94.5% between the CR and COST and Z0030 databases, respectively. Three discrepant death dates were observed, one in the COST trial and two in the Z0030 trial. The concordances of disease-free status between the CR and RCT databases were 99.0% and 87.3%, and 15 discrepant disease recurrence cases were identified: 4 for COST and 11 for Z0030. LIMITATIONS: The analysis has been focused on patients from a single site, Mayo Clinic, Rochester, enrolled in two large RCT evaluating surgical treatments. The findings herein need to be confirmed in a broader setting, such as multi-center, multi-registry including nonsurgical trials. CONCLUSIONS: CR data were nearly identical to data from two randomized phase III trials in different disease types and conducted by two different cooperative groups. The NCDB Cancer Registries represent a feasible alternative for obtaining long-term follow-up data for large clinical trials.

Using Yeast to Define the Regulatory Role of Protein Lysine Methylation.

The post-translational modifications (PTM) of proteins are crucial for cells to survive under diverse environmental conditions and to respond to stimuli. PTMs are known to govern a broad array of cellular processes including signal transduction and chromatin regulation. The PTM lysine methylation has been extensively studied within the context of chromatin and the epigenetic regulation of the genome. However, it has also emerged as a critical regulator of non-histone proteins important for signal transduction pathways. While the number of known non-histone protein methylation events is increasing, the molecular functions of many of these modifications are not yet known. Proteomic studies of the model system Saccharomyces cerevisiae suggest lysine methylation may regulate a diversity of pathways including transcription, RNA processing, translation, and signal transduction cascades. However, there has still been relatively little investigation of lysine methylation as a broad cellular regulator beyond chromatin and transcription. Here, we outline our current state of understanding of non-histone protein methylation in yeast and propose ways in which the yeast system can be leveraged to develop a much more complete picture of molecular mechanisms through which lysine methylation regulates cellular functions.

Function of the MYND Domain and C-Terminal Region in Regulating the Subcellular Localization and Catalytic Activity of the SMYD Family Lysine Methyltransferase Set5.

SMYD lysine methyltransferases target histones and nonhistone proteins for methylation and are critical regulators of muscle development and implicated in neoplastic transformation. They are characterized by a split catalytic SET domain and an intervening MYND zinc finger domain, as well as an extended C-terminal domain. Saccharomyces cerevisiae contains two SMYD proteins, Set5 and Set6, which share structural elements with the mammalian SMYD enzymes. Set5 is a histone H4 lysine 5, 8, and 12 methyltransferase, implicated in the regulation of stress responses and genome stability. While the SMYD proteins have diverse roles in cells, there are many gaps in our understanding of how these enzymes are regulated. Here, we performed mutational analysis of Set5, combined with phosphoproteomics, to identify regulatory mechanisms for its enzymatic activity and subcellular localization. Our results indicate that the MYND domain promotes Set5 chromatin association in cells and is required for its role in repressing subtelomeric genes. Phosphoproteomics revealed extensive phosphorylation of Set5, and phosphomimetic mutations enhance Set5 catalytic activity but diminish its ability to interact with chromatin in cells. These studies uncover multiple regions within Set5 that regulate its localization and activity and highlight potential avenues for understanding mechanisms controlling the diverse roles of SMYD enzymes.