Investigating the effect of anatomical variations in the response of the neonatal brachial plexus to applied force: Use of a two-dimensional finite element model.

The brachial plexus is a set of nerves that innervate the upper extremity and may become injured during the birthing process through an injury known as Neonatal Brachial Plexus Palsy. Studying the mechanisms of these injuries on infant cadavers is challenging due to the justifiable sensitivity surrounding testing. Thus, these specimens are generally unavailable to be used to investigate variations in brachial plexus injury mechanisms. Finite Element Models are an alternative way to investigate the response of the neonatal brachial plexus to loading. Finite Element Models allow a virtual representation of the neonatal brachial plexus to be developed and analyzed with dimensions and mechanical properties determined from experimental studies. Using ABAQUS software, a two-dimensional brachial plexus model was created to analyze how stresses and strains develop within the brachial plexus. The main objectives of this study were (1) to develop a model of the brachial plexus and validate it against previous literature, and (2) to analyze the effect of stress on the nerve roots based on variations in the angles between the nerve roots and the spinal cord. The predicted stress for C5 and C6 was calculated as 0.246 MPa and 0.250 MPa, respectively. C5 and C6 nerve roots experience the highest stress and the largest displacement in comparison to the lower nerve roots, which correlates with clinical patterns of injury. Even small (+/- 3 and 6 degrees) variations in nerve root angle significantly impacted the stress at the proximal nerve root. This model is the first step towards developing a complete three-dimensional model of the neonatal brachial plexus to provide the opportunity to more accurately assess the effect of the birth process on the stretch within the brachial plexus and the impact of biological variations in structure and properties on the risk of Neonatal Brachial Plexus Palsy.

New synergistic combination therapy approaches with HDAC inhibitor quisinostat, cisplatin or PARP inhibitor talazoparib for urothelial carcinoma.

Urothelial carcinoma (UC) urgently requires new therapeutic options. Histone deacetylases (HDAC) are frequently dysregulated in UC and constitute interesting targets for the development of alternative therapy options. Thus, we investigated the effect of the second generation HDAC inhibitor (HDACi) quisinostat in five UC cell lines (UCC) and two normal control cell lines in comparison to romidepsin, a well characterized HDACi which was previously shown to induce cell death and cell cycle arrest. In UCC, quisinostat led to cell cycle alterations, cell death induction and DNA damage, but was well tolerated by normal cells. Combinations of quisinostat with cisplatin or the PARP inhibitor talazoparib led to decrease in cell viability and significant synergistic effect in five UCCs and platinum-resistant sublines allowing dose reduction. Further analyses in UM-UC-3 and J82 at low dose ratio revealed that the mechanisms included cell cycle disturbance, apoptosis induction and DNA damage. These combinations appeared to be well tolerated in normal cells. In conclusion, our results suggest new promising combination regimes for treatment of UC, also in the cisplatin-resistant setting.

Discovering single cannabidiol or synergistic antitumor effects of cannabidiol and cytokine-induced killer cells on non-small cell lung cancer cells.

Introduction: A multitude of findings from cell cultures and animal studies are available to support the anti-cancer properties of cannabidiol (CBD). Since CBD acts on multiple molecular targets, its clinical adaptation, especially in combination with cancer immunotherapy regimen remains a serious concern. Methods: Considering this, we extensively studied the effect of CBD on the cytokine-induced killer (CIK) cell immunotherapy approach using multiple non-small cell lung cancer (NSCLC) cells harboring diverse genotypes. Results: Our analysis showed that, a) The Transient Receptor Potential Cation Channel Subfamily V Member 2 (TRPV2) channel was intracellularly expressed both in NSCLC cells and CIK cells. b) A synergistic effect of CIK combined with CBD, resulted in a significant increase in tumor lysis and Interferon gamma (IFN-g) production. c) CBD had a preference to elevate the CD25+CD69+ population and the CD62L_CD45RA+terminal effector memory (EMRA) population in NKT-CIK cells, suggesting early-stage activation and effector memory differentiation in CD3+CD56+ CIK cells. Of interest, we observed that CBD enhanced the calcium influx, which was mediated by the TRPV2 channel and elevated phosphor-Extracellular signal-Regulated Kinase (p-ERK) expression directly in CIK cells, whereas ERK selective inhibitor FR180204 inhibited the increasing cytotoxic CIK ability induced by CBD. Further examinations revealed that CBD induced DNA double-strand breaks via upregulation of histone H2AX phosphorylation in NSCLC cells and the migration and invasion ability of NSCLC cells suppressed by CBD were rescued using the TRPV2 antagonist (Tranilast) in the absence of CIK cells. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. Conclusions: Taken together, CBD holds a great potential for treating NSCLC with CIK cell immunotherapy. In addition, we utilized NSCLC with different driver mutations to investigate the efficacy of CBD. Our findings might provide evidence for CBD-personized treatment with NSCLC patients.

Simulation of the Childbirth Process in LS-DYNA.

Childbirth or labor, as the final phase of a pregnancy, is a biomechanical process that delivers the fetus from the uterus. It mainly involves two important biological structures in the mother, the uterus-generating the pushing force on the fetus-and the pelvis (bony pelvis and pelvic floor muscles)-resisting the movement of the fetus. The existing computational models developed in this field that simulate the childbirth process have focused on either the uterine expulsion force or the resistive structures of the pelvis, not both. An FEM model including both structures as a system was developed in this paper to simulate the fetus delivery process in LS-DYNA. Uterine active contraction was driven by contractile fiber elements using the Hill material model. The passive portion of the uterus and pelvic floor muscles were modeled with Neo Hookean and Mooney-Rivlin materials, respectively. The bony pelvis was modeled as a rigid body. The fetus was divided into three components: the head, neck, and body. Three uterine active contraction cycles were modeled. The model system was validated based on multiple outputs from the model, including the stress distribution within the uterus, the maximum Von Mises and principal stress on the pelvic floor muscles, the duration of the second stage of the labor, and the movement of the fetus. The developed model system can be applied to investigate the effects of pathomechanics related to labor, such as pelvic floor disorders and brachial plexus injury.

Contributions of mouse genetic strain background to age-related phenotypes in physically active HET3 mice.

We assessed aging hallmarks in skin, muscle, and adipose in the genetically diverse HET3 mouse, and generated a broad dataset comparing these to individual animal diagnostic SNPs from the 4 founding inbred strains of the HET3 line. For middle- and old-aged HET3 mice, we provided running wheel exercise to ensure our observations were not purely representative of sedentary animals, but age-related phenotypes were not improved with running wheel activity. Adipose tissue fibrosis, peripheral neuropathy, and loss of neuromuscular junction integrity were consistent phenotypes in older-aged HET3 mice regardless of physical activity, but aspects of these phenotypes were moderated by the SNP% contributions of the founding strains for the HET3 line. Taken together, the genetic contribution of founder strain SNPs moderated age-related phenotypes in skin and muscle innervation and were dependent on biological sex and chronological age. However, there was not a single founder strain (BALB/cJ, C57BL/6J, C3H/HeJ, DBA/2J) that appeared to drive more protection or disease-risk across aging in this mouse line, but genetic diversity in general was more protective.

Explaining the evolution of gossip.

Gossip, the exchange of personal information about absent third parties, is ubiquitous in human societies. However, the evolution of gossip remains a puzzle. The current article proposes an evolutionary cycle of gossip and uses an agent-based evolutionary game-theoretic model to assess it. We argue that the evolution of gossip is the joint consequence of its reputation dissemination and selfishness deterrence functions. Specifically, the dissemination of information about individuals' reputations leads more individuals to condition their behavior on others' reputations. This induces individuals to behave more cooperatively toward gossipers in order to improve their reputations. As a result, gossiping has an evolutionary advantage that leads to its proliferation. The evolution of gossip further facilitates these two functions of gossip and sustains the evolutionary cycle.

Body Dissatisfaction and Health Risk Behaviors Among Middle School Girls.

BACKGROUND: Previous studies demonstrate a relationship between body dissatisfaction and substance use and suicidal ideation among older adolescent girls and young women while less documentation exists for early adolescence. This study explored the relationship between reported weight loss attempts and substance use history and suicidal thoughts among younger female adolescents. METHODS: Participants (n = 1656) were middle school female students who participated in the 2019 Youth Behaviors Risk Survey. Participants were coded as "Trying to lose weight" and "Not trying to lose weight." Two hierarchal multiple binary logistic regressions were conducted, 1 for each of the dependent variables: (1) substance use history and (2) suicidality. RESULTS: Fifty-seven percent of the participants were trying to lose weight, 40% reported suicidal thoughts and 45% reported substance use history. Trying to lose weight was a significant predictor for both substance use (p < .01) and suicidality (p < .001). CONCLUSIONS: Body dissatisfaction and its association with risky health behaviors highlight the need for prevention education at earlier ages while reinforcing the need for availability of school counselors.

Comorbidity profiles in older patients last seen by mental health prior to suicide attempt.

OBJECTIVES: Suicide in late life is a public health concern. Determining profiles of psychiatric/medical comorbidity in those who attempt while engaged in mental health services may assist with prevention. We identified comorbidity profiles and their association with utilization, means, and fatality in a national sample who attempted suicide. METHODS: Using latent class analysis, all patients aged ≥ 65 from the Department of Veterans Affairs (VA) healthcare services (2012-2018) last seen in mental health prior to suicide attempt were included. Diagnoses and attempt data were obtained from VA and Center for Medicare & Medicaid Services, VA Suicide Prevention Applications Network, and VA National Mortality Data Repository. RESULTS: 2,269 patients were clustered into three profiles, all with high probability of depression. Profiles included minimal comorbidity (50.4%), high medical comorbidity (28.6%), and high (psychiatric/medical) comorbidity (21.0%). Over half (61.7%) attempted suicide within one week of their visit. The class with highest comorbidity had lowest proportion of fatal attempts, while minimal comorbidity class had highest proportion. CONCLUSIONS: Older patients last seen in mental health prior to suicide attempt were characterized by depression and varying additional comorbidity and attempt-related factors. Findings have implications for risk assessment and intervention in mental health settings, beyond depression.

Implementation Lessons Learned: Distress Behaviors in Dementia Intervention in Veterans Health Administration.

BACKGROUND AND OBJECTIVES: Evidence-based practices to manage distress behaviors in dementia (DBD) are not consistently implemented despite demonstrated effectiveness. The Veterans Health Administration (VA) trained teams to implement Staff Training in Assisted Living Residences (STAR)-VA, an intervention to manage DBD in VA nursing home settings, or Community Living Centers (CLCs). This paper summarizes multiyear formative evaluation results including challenges, adaptations, and lessons learned to support sustained integration into usual care across CLCs nationwide. RESEARCH DESIGN AND METHODS: STAR was selected as an evidence-based practice for DBD, adapted for and piloted in VA (STAR-VA), and implemented through a train-the-trainer program from 2013 to 2018. Training and consultation were provided to 92 CLC teams. Evaluation before and after training and consultation included descriptive statistics of measures of clinical impact and survey feedback from site teams regarding self-confidence, engagement, resource quality, and content analysis of implementation facilitators and challenges. RESULTS: STAR-VA training and consultation increased staff confidence and resulted in significant decreases in DBD, depression, anxiety, and agitation for Veterans engaged in the intervention. Implementation outcomes demonstrated feasibility and identified facilitators and barriers. Key findings were interpreted using implementation frameworks and informed subsequent modifications to sustain implementation. DISCUSSION AND IMPLICATIONS: STAR-VA successfully prepared teams to manage DBD and resulted in improved outcomes. Lessons learned include importance of behavioral health-nursing partnerships, continuous engagement, iterative feedback and adaptations, and sustainment planning. Evaluation of sustainment factors has informed selection of implementation strategies to address sustainment barriers. Lessons learned have implications for integrating team-based practices into system-level practice.

Norm Dynamics: Interdisciplinary Perspectives on Social Norm Emergence, Persistence, and Change.

Social norms are the glue that holds society together, yet our knowledge of them remains heavily intellectually siloed. This article provides an interdisciplinary review of the emerging field of norm dynamics by integrating research across the social sciences through a cultural-evolutionary lens. After reviewing key distinctions in theory and method, we discuss research on norm psychology-the neural and cognitive underpinnings of social norm learning and acquisition. We then overview how norms emerge and spread through intergenerational transmission, social networks, and group-level ecological and historical factors. Next, we discuss multilevel factors that lead norms to persist, change, or erode over time. We also consider cultural mismatches that can arise when a changing environment leads once-beneficial norms to become maladaptive. Finally, we discuss potential future research directions and the implications of norm dynamics for theory and policy.

Studying the Effects of Shoulder Dystocia and Neonate-Focused Delivery Maneuvers on Brachial Plexus Strain: A Computational Study.

The purpose of this computational study was to investigate the effects of neonate-focused clinical delivery maneuvers on brachial plexus (BP) during shoulder dystocia. During shoulder dystocia, the anterior shoulder of the neonate is obstructed behind the symphysis pubis of the maternal pelvis, postdelivery of the neonate's head. This is managed by a series of clinical delivery maneuvers. The goal of this study was to simulate these delivery maneuvers and study their effects on neonatal BP strain. Using madymo models of a maternal pelvis and a 90th-percentile neonate, various delivery maneuvers and positions were simulated including the lithotomy position alone of the maternal pelvis, delivery with the application of various suprapubic pressures (SPPs), neonate in an oblique position, and during posterior arm delivery maneuver. The resulting BP strain (%) along with the required maternal delivery force was reported in these independently simulated scenarios. The lithotomy position alone served as the baseline. Each of the successive maneuvers reported a decrease in the required delivery force and resulting neonatal BP strain. As the applied SPP force increased (three scenarios simulated), the required maternal delivery force and neonatal BP strain decreased. A further decrease in both delivery force and neonatal BP strain was observed in the oblique position, with the lowest delivery force and neonatal BP strain reported during the posterior arm delivery maneuver. Data obtained from the improved computational models in this study enhance our understanding of the effects of clinical maneuvers on neonatal BP strain during complicated birthing scenarios such as shoulder dystocia.

Racial Hate at the Intersection of Online and Offline Worlds: The Joint Impact of Online and Offline Racism on the Mental Health of Racially Minoritized Individuals.

With the everyday influence of social media and online platforms, racially minoritized individuals face the risk of experiencing racial discrimination in both offline and online contexts. Yet, there is scant research on how online and offline racism could jointly impact the mental health of racially minoritized individuals. Thus, the study examined online racism as a moderator in the link between offline racism and mental health issues. Using data from 651 racially minoritized emerging adults (Mage = 22.15, SD = 3.39), a moderation analysis was conducted using the PROCESS macro with bias-corrected bootstrapping (10,000 resamples). Experiencing more offline racism was significantly associated with greater mental health issues. Online racism was a significant moderator in the link between offline racism and mental health issues. The link between offline racism and mental health issues was not significant at low levels of online racism. However, the link was significant and strengthened at mean to high levels of online racism. The results suggest complex interactions in understanding the compounding impact of online and offline racism on mental health costs among racially minoritized individuals. Implications for future research on understanding the interplay between online and offline racism are discussed.

Paths Forward for Clinicians Amidst the Rise of Unregulated Clinical Decision Support Software: Our Perspective on NarxCare.

Amidst the US overdose epidemic, policymakers, law enforcement agencies, and healthcare institutions have contributed to a decrease in opioid prescribing, assuming reduced mortality would result-an assumption we now understand was oversimplified. At this intersection between public health and public safety domains as they relate to opioid prescribing, unregulated and proprietary clinical decision support tools have emerged without rigorous external validation or public data sharing. In the following piece, we discuss challenges facing clinicians practicing medicine amidst unregulated clinical decision support tools, using the case of Bamboo Health's NarxCare-a prescription drug monitoring program-based analytics platform marketed as a clinical decision support tool-that is already positioned to impact over 1 billion patient encounters annually. We argue that sufficient evidence does not yet exist to support NarxCare's wide implementation, and that clinical decision support tools like NarxCare have flourished in recent years due to a lack of federal regulatory oversight and shielding by their proprietary formulas, which have facilitated their unchecked and outsized influence on patient care. Finally, we suggest specific actions by federal regulatory agencies, healthcare institutions, individual clinicians, and researchers, as well as academic journals, to mitigate potential harms associated with unregulated clinical decision support tools.

Tumorigenic effects of human mesenchymal stromal cells and fibroblasts on bladder cancer cells.

Background: Patients with muscle-invasive bladder cancer face a poor prognosis due to rapid disease progression and chemoresistance. Thus, there is an urgent need for a new therapeutic treatment. The tumor microenvironment (TME) has crucial roles in tumor development, growth, progression, and therapy resistance. TME cells may also survive standard treatment of care and fire up disease recurrence. However, whether specific TME components have tumor-promoting or tumor-inhibitory properties depends on cell type and cancer entity. Thus, a deeper understanding of the interaction mechanisms between the TME and cancer cells is needed to develop new cancer treatment approaches that overcome therapy resistance. Little is known about the function and interaction between mesenchymal stromal cells (MSC) or fibroblasts (FB) as TME components and bladder cancer cells. Methods: We investigated the functional impact of conditioned media (CM) from primary cultures of different donors of MSC or FB on urothelial carcinoma cell lines (UCC) representing advanced disease stages, namely, BFTC-905, VMCUB-1, and UMUC-3. Underlying mechanisms were identified by RNA sequencing and protein analyses of cancer cells and of conditioned media by oncoarrays. Results: Both FB- and MSC-CM had tumor-promoting effects on UCC. In some experiments, the impact of MSC-CM was more pronounced. CM augmented the aggressive phenotype of UCC, particularly of those with epithelial phenotype. Proliferation and migratory and invasive capacity were significantly increased; cisplatin sensitivity was reduced. RNA sequencing identified underlying mechanisms and molecules contributing to the observed phenotype changes. NRF2 and NF-κB signaling was affected, contributing to improved cisplatin detoxification. Likewise, interferon type I signaling was downregulated and regulators of epithelial mesenchymal transition (EMT) were increased. Altered protein abundance of CXCR4, hyaluronan receptor CD44, or TGFß-signaling was induced by CM in cancer cells and may contribute to phenotypical changes. CM contained high levels of CCL2/MCP-1, MMPs, and interleukins which are well known for their impact on other cancer entities. Conclusions: The CM of two different TME components had overlapping tumor-promoting effects and increased chemoresistance. We identified underlying mechanisms and molecules contributing to the aggressiveness of bladder cancer cells. These need to be further investigated for targeting the TME to improve cancer therapy.

Queer Faculty in the Academy: Is It Getting Better?

This paper weaves the author's personal experience as an out lesbian researcher of LGBTQ health with contemporary research on LGBTQ faculty members' experiences. It also draws from the literature on other marginalized faculty members' experiences (women, faculty of color) to identify common themes that prevent the full inclusion of diverse faculty in higher education. Structural oppression is often invisible and university's focus on values of meritocracy, individual effort, competition, and elitism create unspoken barriers to faculty success. The paper discusses three general themes: the paradox of visibility (including being out on faculty, doing LGBTQ research, and being an advocate/activist), curricular issues, and internalizing of oppression. The final section outlines strategies for overcoming some of the barriers, including LGBTQ-specific organizing and community building and creating coalitions across marginalized faculty groups.

The Way They See Us: Examining the Content, Accuracy, and Bias of Metaperceptions Held by Syrian Refugees About the Communities That Host Them.

Discourse about people seeking refuge from conflict varies considerably. To understand what components of this discourse reach refugees the most, we examined refugees' perceptions of how their host communities perceive them (i.e., intergroup metaperceptions). We sampled refugees who fled Syria to Jordan, Lebanon, Germany, and the Netherlands. Focus groups with 102 Syrian refugees revealed that the most prevalent metaperception discussed by refugees was that they thought their host communities saw them as threatening (Study 1). Surveys with 1,360 Syrian refugees and 1,441 members of the host communities (Study 2) found that refugees' metaperceptions tracked the perceptions held by their host communities (i.e., they were accurate), but there was also a significant mean difference, indicating that they were positively biased. Analyses further tested the roles of evaluative concern and group salience on metaperception accuracy, as well as differences in accuracy and bias across country and perception domain.

Epigenetic Priming and Development of New Combination Therapy Approaches.

Muscle-invasive urothelial carcinoma of the bladder (MIBC) has been treated with cisplatin-based chemotherapy for over 30 years. With the advent of immune checkpoint inhibitors, antibody drug conjugates and FGFR3 inhibitors new therapeutic options have been approved for patients with urothelial carcinoma (UC) and are still under investigation regarding association between patients' response and recently defined molecular subtypes. Unfortunately, similar to chemotherapy, only a fraction of UC patients responds to these new treatment approaches. Thus, either further new efficacious therapeutic options for treatment of individual subtypes or new approaches to overcome treatment resistance and to increase patients' response to standard of care treatment are needed.Epigenetic modifications of DNA and chromatin are known to mediate cellular plasticity or treatment resistance, and the responsible epigenetic regulators are frequently mutated or aberrantly expressed in UC. Thus, these enzymes provide targets for novel drug combination therapies to "episensitize" toward approved standard therapies by epigenetic priming. In general, these epigenetic regulators comprise writers and erasers like DNA methyltransferases and DNA demethylases (for DNA methylation), histone methyltransferases and histone demethylases (for histone methylation), as well as acetyl transferases and histone deacetylases (for histone and nonhistone acetylation). Such modifications, e.g., acetyl groups, are recognized by further epigenetic reader proteins, e.g., like the bromodomain and extra-terminal domain (BET) family proteins that often interact in multi-protein complexes and finally regulate chromatin conformation and transcriptional activity.Concurringly, epigenetic regulators target a plethora of cellular functions. Their pharmaceutical inhibitors often inhibit enzymatic activity of more than one isoenzyme or may have further noncanonical cytotoxic effects. Thus, analysis of their functions in UC pathogenesis as well as of the antineoplastic capacity of corresponding inhibitors alone or in combination with other approved drugs should follow a multidimensional approach. Here, we present our standard approach to analyze cellular effects of new epigenetic inhibitors on UC cells alone to define their potency and to conclude on putative reasonable combination therapy partners. We further describe our approach to identify efficacious synergistic combination therapies (e.g., with cisplatin or PARP inhibitors) that may have reduced normal toxicity through dose reduction, which can then be further analyzed in animal experiments. This approach may also serve as prototype for the preclinical evaluation of other epigenetic treatment approaches.

COVID-19 anti-Asian racism, perceived burdensomeness, thwarted belongingness, and suicidal ideation among Asian American Emerging Adults.

Since the start of the COVID-19 pandemic, there has been a significant surge in COVID-19 related anti-Asian racism and hate crimes. Given the ostracising and dehumanising narrative of COVID-19 related anti-Asian hate, we examined whether COVID-19 anti-Asian racism would be associated with suicidal ideation through increased thwarted belongingness and perceived burdensomeness among Asian American emerging adults. With data from 139 participants (Mage = 23.04), we conducted a path analysis of COVID-19 anti-Asian racism (four items) predicting suicidal ideation (item nine; Patient Health Questionnaire-9) via perceived burdensomeness and thwarted belongingness (Interpersonal Needs Questionnaire). COVID-19 anti-Asian racism significantly predicted suicidal ideation. The indirect effect through perceived burdensomeness was significant but not through thwarted belongingness; greater COVID-19 anti-Asian racism was associated with greater perceived burdensomeness, which in turn was associated with greater suicidal ideation. The significance of perceived burdensomeness was substantiated given the non-significant direct effect. The results suggest that the ongoing COVID-19 anti-Asian racism may be an alarming risk factor for suicidal ideation for Asian American emerging adults.

Social and moral psychology of COVID-19 across 69 countries.

The COVID-19 pandemic has affected all domains of human life, including the economic and social fabric of societies. One of the central strategies for managing public health throughout the pandemic has been through persuasive messaging and collective behaviour change. To help scholars better understand the social and moral psychology behind public health behaviour, we present a dataset comprising of 51,404 individuals from 69 countries. This dataset was collected for the International Collaboration on Social & Moral Psychology of COVID-19 project (ICSMP COVID-19). This social science survey invited participants around the world to complete a series of moral and psychological measures and public health attitudes about COVID-19 during an early phase of the COVID-19 pandemic (between April and June 2020). The survey included seven broad categories of questions: COVID-19 beliefs and compliance behaviours; identity and social attitudes; ideology; health and well-being; moral beliefs and motivation; personality traits; and demographic variables. We report both raw and cleaned data, along with all survey materials, data visualisations, and psychometric evaluations of key variables.