Predicting Invasiveness in Lepidic Pattern Adenocarcinoma of Lung: Analysis of Visual Semantic and Radiomic Features.

OBJECTIVES: To differentiate invasive lepidic predominant adenocarcinoma (iLPA) from adenocarcinoma in situ (AIS)/minimally invasive adenocarcinoma (MIA) of lung utilizing visual semantic and computer-aided detection (CAD)-based texture features on subjects initially diagnosed as AIS or MIA with CT-guided biopsy. MATERIALS AND METHODS: From 2011 to 2017, all patients with CT-guided biopsy results of AIS or MIA who subsequently underwent resection were identified. CT scan before the biopsy was used to assess visual semantic and CAD texture features, totaling 23 semantic and 95 CAD-based quantitative texture variables. The least absolute shrinkage and selection operator (LASSO) method or forward selection was used to select the most predictive feature and combination of semantic and texture features for detection of invasive lung adenocarcinoma. RESULTS: Among the 33 core needle-biopsied patients with AIS/MIA pathology, 24 (72.7%) had invasive LPA and 9 (27.3%) had AIS/MIA on resection. On CT, visual semantic features included 21 (63.6%) part-solid, 5 (15.2%) pure ground glass, and 7 (21.2%) solid nodules. LASSO selected seven variables for the model, but all were not statistically significant. "Volume" was found to be statistically significant when assessing the correlation between independent variables using the backward selection technique. The LASSO selected "tumor_Perc95", "nodule surround", "small cyst-like spaces", and "volume" when assessing the correlation between independent variables. CONCLUSIONS: Lung biopsy results showing noninvasive LPA underestimate invasiveness. Although statistically non-significant, some semantic features showed potential for predicting invasiveness, with septal stretching absent in all noninvasive cases, and solid consistency present in a significant portion of invasive cases.

TRAMP assembly alters the conformation and RNA binding of Mtr4 and Trf4-Air2.

The TRAMP complex contains two enzymatic activities essential for RNA processing upstream of the nuclear exosome. Within TRAMP, RNA is 3' polyadenylated by a sub-complex of Trf4/5 and Air1/2 and unwound 3' to 5' by Mtr4, a DExH helicase. The molecular mechanisms of TRAMP assembly and RNA shuffling between the two TRAMP catalytic sites are poorly understood. Here, we report solution hydrogen-deuterium exchange data with thermodynamic and functional assays to uncover these mechanisms for yeast TRAMP with Trf4 and Air2 homologs. We show that TRAMP assembly constrains RNA-recognition motifs that are peripheral to catalytic sites. These include the Mtr4 Arch and Air2 zinc knuckles 1, 2, and 3. While the Air2 Arch-interacting motif likely constrains the Mtr4 Arch via transient interactions, these do not fully account for the importance of the Mtr4 Arch in TRAMP assembly. We further show that tRNA binding by single active-site subunits, Mtr4 and Trf4-Air2, differs from the double active-site TRAMP. TRAMP has reduced tRNA binding on the Mtr4 Fist and RecA2 domains, offset by increased tRNA binding on Air2 zinc knuckles 2 and 3. Competition between these RNA-binding sites may drive tRNA transfer between TRAMP subunits. We identify dynamic changes upon TRAMP assembly and RNA-recognition motifs that transfer RNA between TRAMP catalytic sites.

Patterning optimization for device realization of patterned GaAsSbN nanowire photodetectors.

Vertically grown nanowires (NWs) are a research interest in optoelectronics and photovoltaic applications due to their high surface to volume ratio and good light trapping capabilities. This study presents the effects of process and design parameters on self-catalyzed GaAsSbN NWs grown by plasma-assisted molecular beam epitaxy on patterned silicon substrates using electron beam lithography. Vertical alignment of the patterned NWs examined via scanning electron microscopy show the sensitivity of patterned NW growth to the parameters of NW diameter, pitch, dose time, etching techniques and growth plan. Diameters range from 90 nm to 250 nm. Pitch lengths of 200 nm, 400 nm, 600 nm, 800 nm, 1000 nm, and 1200 nm were examined. Dry etching of the oxide layer of the silicon substrate and PMMA coating is performed using reactive ion etching (RIE) for 20 s and 120 s respectively. Comparisons of different HF etch durations performed pre and post PMMA removal are presented. Additionally, the report of an observed surfactant effect in dilute nitride GaAsSbN NWs in comparison to non-nitride GaAsSb is presented. Optimizations to patterning, RIE, and HF etching are presented to obtain higher vertical yield of patterned GaAsSbN NWs, achieving ∼80% of the expected NWµm2. Room temperature and 4 K photoluminescence results show the effect of nitride incorporation for further bandgap tuning, and patterned pitch on the optical characteristics of the NWs which gives insights to the compositional homogeneity for NWs grown at each pitch length.

Structural and functional characterization of the IpaD π-helix reveals critical roles in DOC interaction, T3SS apparatus maturation, and Shigella virulence.

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella's T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein invasion plasmid antigen D (IpaD). Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.

Cosyntropin Stimulation Testing is More Selective than Postoperative Day 1 Basal Cortisol for Diagnosing Secondary Adrenal Insufficiency After Unilateral Adrenalectomy.

BACKGROUND: Secondary adrenal insufficiency (SAI) may occur in patients after unilateral adrenalectomy for adrenal-dependent hypercortisolism (HC) or primary aldosteronism (PA). This study aimed to assess whether postoperative day (POD) 1 basal cortisol was predictive of an abnormal cosyntropin stimulation test (CST) result and the need for glucocorticoid replacement (GR). METHODS: A retrospective review of consecutive patients who underwent unilateral adrenalectomy for HC, PA, or both between September 2014 and September 2022 was performed. On POD1, CST was performed for all the patients with HC, and before 2021 for all the patients with PA. The patients with an abnormal CST result were deemed at risk of SAI and discharged with GR. Receiver operating characteristic (ROC) curves were generated to evaluate the sensitivity (SN) and specificity (SP) of basal cortisol thresholds to predict an abnormal CST result. RESULTS: The patients underwent unilateral adrenalectomy for overt hypercortisolism (OH; n = 42), mild autonomous cortisol excess (MACE; n = 70), mixed PA/HC (n = 22), or PA (n = 73). On POD1, CST was performed for 152 patients (93% OH, 96% MACE,73% PA/HC, 41% PA), and 80 patients (53%) had SAI (67% OH, 55% MACE, 44% PA/HC, 33% PA). The SN and SP of a basal cortisol level of 10 µg/dL or lower to predict an abnormal CST were respectively 92% and 77% for OH, 94% and 73% for MACE, 100% and 85% for PA, and 100% and 67% for PA/HC. The optimal basal cortisol level for predicting an abnormal CST for patients with PA or PA/HC was 5 µg/dL or lower (SN/SP, 100%). CONCLUSIONS: After unilateral adrenalectomy for HC, PA, or mixed PA/HC, POD1 CST improved identification of patients at risk for SAI compared with basal cortisol levels alone. The authors recommend that POD1 CST be performed to determine the risk for SAI and the need for postoperative GR after unilateral adrenalectomy for patients with HC.

Molecular, clinical, and therapeutic determinants of outcome in NPM1-mutated AML.

ABSTRACT: Although NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcomes; however, only FLT3-internal tandem duplication (ITD) mutation and adverse karyotype are currently used for risk stratification because of inconsistent results and uncertainty about how other factors should influence treatment, particularly given the strong prognostic effect of postinduction measurable residual disease (MRD). Here, we analyzed a large group of patients with NPM1 mutations (NPM1mut) AML enrolled in prospective trials (National Cancer Research Institute [NCRI] AML17 and AML19, n = 1357) to delineate the impact of baseline molecular and clinical features, postinduction MRD status, and treatment intensity on the outcome. FLT3-ITD (hazard ratio [HR], 1.28; 95% confidence interval [CI], 1.01-1.63), DNMT3A (HR, 1.65; 95% CI, 1.32-2.05), WT1 (HR, 1.74; 95% CI, 1.27-2.38), and non-ABD NPM1mut (HR, 1.64; 95% CI, 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients who achieved MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD-negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin) regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the high-risk molecular subgroups.

Computational scoring and experimental evaluation of enzymes generated by neural networks.

In recent years, generative protein sequence models have been developed to sample novel sequences. However, predicting whether generated proteins will fold and function remains challenging. We evaluate a set of 20 diverse computational metrics to assess the quality of enzyme sequences produced by three contrasting generative models: ancestral sequence reconstruction, a generative adversarial network and a protein language model. Focusing on two enzyme families, we expressed and purified over 500 natural and generated sequences with 70-90% identity to the most similar natural sequences to benchmark computational metrics for predicting in vitro enzyme activity. Over three rounds of experiments, we developed a computational filter that improved the rate of experimental success by 50-150%. The proposed metrics and models will drive protein engineering research by serving as a benchmark for generative protein sequence models and helping to select active variants for experimental testing.

Watch Me Play!: protocol for a feasibility study of a remotely delivered intervention to promote mental health resilience for children (ages 0-8) across UK early years and children's services.

BACKGROUND: Half of mental health problems are established by the age of 14 years and 75% by 24 years. Early intervention and prevention of mental ill health are therefore vitally important. However, increased demand over recent years has meant that access to child mental health services is often restricted to those in severest need. Watch Me Play! (WMP) is an early intervention designed to support caregiver attunement and attention to the child to promote social-emotional well-being and thereby mental health resilience. Originally developed in the context of a local authority mental health service for children in care, it is now also delivered online as a low intensity, scalable, preventative intervention. Although WMP shows promise and is already used in some services, we do not yet know whether it is effective. METHODS: A non-randomised single group feasibility study with embedded process evaluation. We propose to recruit up to 40 parents/carers of children aged 0-8 years who have been referred to early years and children's services in the UK. WMP involves a parent watching the child play and talking to their child about their play (or for babies, observing and following signals) for up to 20 min per session. Some sessions are facilitated by a trained practitioner who provides prompts where necessary, gives feedback, and discusses the child's play with the caregiver. Services will offer five facilitated sessions, and parents will be asked to do at least 10 additional sessions on their own with their child in a 5-week period. Feasibility outcomes examined are as follows: (i) recruitment, (ii) retention, (iii) adherence, (iv) fidelity of delivery, (v) barriers and facilitators of participation, (vi) intervention acceptability, (vii) description of usual care, and (viii) data collection procedures. Intervention mechanisms will be examined through qualitative interview data. Economic evaluation will be conducted estimating cost of the intervention and cost of service use for child and parents/carers quality-adjusted life years. DISCUSSION: This study will address feasibility questions associated with progression to a future randomised trial of WMP. TRIAL REGISTRATION: ISRCTN13644899 . Registered on 14th April 2023.

Sensitive remote homology search by local alignment of small positional embeddings from protein language models.

Accurately detecting distant evolutionary relationships between proteins remains an ongoing challenge in bioinformatics. Search methods based on primary sequence struggle to accurately detect homology between sequences with less than 20% amino acid identity. Profile- and structure-based strategies extend sensitive search capabilities into this twilight zone of sequence similarity but require slow pre-processing steps. Recently, whole-protein and positional embeddings from deep neural networks have shown promise for providing sensitive sequence comparison and annotation at long evolutionary distances. Embeddings are generally faster to compute than profiles and predicted structures but still suffer several drawbacks related to the ability of whole-protein embeddings to discriminate domain-level homology, and the database size and search speed of methods using positional embeddings. In this work, we show that low-dimensionality positional embeddings can be used directly in speed-optimized local search algorithms. As a proof of concept, we use the ESM2 3B model to convert primary sequences directly into the 3D interaction (3Di) alphabet or amino acid profiles and use these embeddings as input to the highly optimized Foldseek, HMMER3, and HH-suite search algorithms. Our results suggest that positional embeddings as small as a single byte can provide sufficient information for dramatically improved sensitivity over amino acid sequence searches without sacrificing search speed.

Postinduction molecular MRD identifies patients with NPM1 AML who benefit from allogeneic transplant in first remission.

ABSTRACT: Selection of patients with NPM1-mutated acute myeloid leukemia (AML) for allogeneic transplant in first complete remission (CR1-allo) remains controversial because of a lack of robust data. Consequently, some centers consider baseline FLT3-internal tandem duplication (ITD) an indication for transplant, and others rely on measurable residual disease (MRD) status. Using prospective data from the United Kingdom National Cancer Research Institute AML17 and AML19 studies, we examined the impact of CR1-allo according to peripheral blood NPM1 MRD status measured by quantitative reverse transcription polymerase chain reaction after 2 courses of induction chemotherapy. Of 737 patients achieving remission, MRD was positive in 19%. CR1-allo was performed in 46% of MRD+ and 17% of MRD- patients. We observed significant heterogeneity of overall survival (OS) benefit from CR1-allo according to MRD status, with substantial OS advantage for MRD+ patients (3-year OS with CR1-allo vs without: 61% vs 24%; hazard ratio [HR], 0.39; 95% confidence interval [CI], 0.24-0.64; P < .001) but no benefit for MRD- patients (3-year OS with CR1-allo vs without: 79% vs 82%; HR, 0.82; 95% CI, 0.50-1.33; P = .4). Restricting analysis to patients with coexisting FLT3-ITD, again CR1-allo only improved OS for MRD+ patients (3-year OS, 45% vs 18%; compared with 83% vs 76% if MRD-); no interaction with FLT3 allelic ratio was observed. Postinduction molecular MRD reliably identifies those patients who benefit from allogeneic transplant in first remission. The AML17 and AML19 trials were registered at www.isrctn.com as #ISRCTN55675535 and #ISRCTN78449203, respectively.

Highly Parallelized Construction of DNA from Low-Cost Oligonucleotide Mixtures Using Data-Optimized Assembly Design and Golden Gate.

Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically synthesized microarray-derived oligonucleotides. Here, we apply Data-optimized Assembly Design (DAD) to the synthesis of hundreds of codon-optimized genes in both constitutive and inducible vectors using Golden Gate Assembly. Starting from oligonucleotide pools, we synthesize genes in three simple steps: (1) amplification of parts belonging to individual assemblies in parallel from a single pool; (2) Golden Gate Assembly of parts for each construct; and (3) transformation. We construct genes from receiving DNA to sequence confirmed isolates in as little as 4 days. By leveraging the ligation fidelity afforded by T4 DNA ligase, we expect to be able to construct a larger breadth of sequences not currently supported by homology-based methods, which require stability of extensive single-stranded DNA overhangs.

Digital adaptation of the Standing up for Myself intervention in young people and adults with intellectual disabilities: the STORM feasibility study.

Background: Stigma contributes to the negative social conditions persons with intellectual disabilities are exposed to, and it needs tackling at multiple levels. Standing Up for Myself is a psychosocial group intervention designed to enable individuals with intellectual disabilities to discuss stigmatising encounters in a safe and supportive setting and to increase their self-efficacy in managing and resisting stigma. Objectives: To adapt Standing Up for Myself to make it suitable as a digital intervention; to evaluate the feasibility and acceptability of Digital Standing Up for Myself and online administration of outcome measures in a pilot; to describe usual practice in the context of the coronavirus disease 2019 pandemic to inform future evaluation. Design: Adaptation work followed by a single-arm pilot of intervention delivery. Setting and participants: Four third and education sector organisations. Individuals with mild-to-moderate intellectual disabilities, aged 16+, members of existing groups, with access to digital platforms. Intervention: Digital Standing Up for Myself intervention. Adapted from face-to-face Standing Up for Myself intervention, delivered over four weekly sessions, plus a 1-month follow-up session. Outcomes: Acceptability and feasibility of delivering Digital Standing Up for Myself and of collecting outcome and health economic measures at baseline and 3 months post baseline. Outcomes are mental well-being, self-esteem, self-efficacy in rejecting prejudice, reactions to discrimination and sense of social power. Results: Adaptation to the intervention required changes to session duration, group size and number of videos; otherwise, the content remained largely the same. Guidance was aligned with digital delivery methods and a new group member booklet was produced. Twenty-two participants provided baseline data. The intervention was started by 21 participants (four groups), all of whom were retained at 3 months. Group facilitators reported delivering the intervention as feasible and suggested some refinements. Fidelity of the intervention was good, with over 90% of key components observed as implemented by facilitators. Both facilitators and group members reported the intervention to be acceptable. Group members reported subjective benefits, including increased confidence, pride and knowing how to deal with difficult situations. Digital collection of all outcome measures was feasible and acceptable, with data completeness ≥ 95% for all measures at both time points. Finally, a picture of usual practice has been developed as an intervention comparator for a future trial. Limitations: The pilot sample was small. It remains unclear whether participants would be willing to be randomised to a treatment as usual arm or whether they could be retained for 12 months follow-up. Conclusions: The target number of groups and participants were recruited, and retention was good. It is feasible and acceptable for group facilitators with some training and supervision to deliver Digital Standing Up for Myself. Further optimisation of the intervention is warranted. Future work: To maximise the acceptability and reach of the intervention, a future trial could offer the adapted Digital Standing Up for Myself, potentially alongside the original face-to-face version of the intervention. Study registration: This study was registered as ISRCTN16056848. Funding: This award was funded by the National Institute for Health and Care Research (NIHR) Public Health Research programme (NIHR award ref: 17/149/03) and is published in full in Public Health Research; Vol. 12, No. 1. See the NIHR Funding and Awards website for further award information.

People with intellectual disabilities (or 'learning disabilities' in United Kingdom language) are more likely to experience poor physical and mental health than the general population. Stigma (negative stereotypes, prejudice and discrimination) has been linked to lower self-esteem, quality of life, and mental and physical ill health. Efforts to empower people with intellectual disabilities themselves to challenge stigma with a view to improving well-being, health and self-esteem are lacking. In 2017, we developed Standing Up for Myself, a brief group-based programme for people with mild-to-moderate intellectual disabilities aged 16+ to address this gap. As this study got underway, face-to-face meetings were suspended due to the coronavirus disease 2019 pandemic. We used the opportunity to assess whether Standing Up for Myself could be delivered through web-based meetings. We adapted Standing Up for Myself for digital delivery, with close input from advisors with intellectual disabilities and experienced group facilitators. We then tested the digital version in charity and education settings to evaluate if Digital Standing Up for Myself could be delivered as planned and how acceptable it was to group facilitators and participants. Four groups, with a total of 22 members, signed up to try Digital Standing Up for Myself. One participant dropped out before starting Standing Up for Myself, and the other 21 continued until the end of the programme. Retention and attendance were good; participants on average attended four of the five sessions. Ninety per cent of the core programme requirements were fully delivered as detailed in the Digital Standing Up for Myself manual. Problems with technology were manageable, although facilitators found using the Standing Up for Myself Wiki platform (an online platform for storage and sharing of resources) difficult, particularly when sharing video content. Facilitators felt acceptable levels of privacy were achieved and there were no reports of undue distress. All facilitators and many group members said they would recommend Digital Standing Up for Myself to others. Group members shared how the programme benefitted them, noting increased awareness about disabilities, and for some increased confidence, pride and independence. Some had learnt how to stand up for themselves and manage difficult situations and took pride in this. Completing outcome and health cost measures via web-based meetings was acceptable and data were largely fully complete and useable.

Discovery of cytosine deaminases enables base-resolution methylome mapping using a single enzyme.

Deaminases have important uses in modification detection and genome editing. However, the range of applications is limited by the small number of characterized enzymes. To expand the toolkit of deaminases, we developed an in vitro approach that bypasses a major hurdle with their toxicity in cells. We assayed 175 putative cytosine deaminases on a variety of substrates and found a broad range of activity on double- and single-stranded DNA in various sequence contexts, including CpG-specific deaminases and enzymes without sequence preference. We also characterized enzyme selectivity across six DNA modifications and reported enzymes that do not deaminate modified cytosines. The detailed analysis of diverse deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed SEM-seq, a non-destructive single-enzyme methylation sequencing method using a modification-sensitive double-stranded DNA deaminase. The streamlined protocol enables accurate, base-resolution methylome mapping of scarce biological material, including cell-free DNA and 10 pg input DNA.

Epidemiology of Gender Diversity.

In the United States, approximately 1.6 million individuals identify as transgender and gender diverse (TGD), encompassing a wide range of identities and experiences. Despite progress in visibility and acceptance, TGD people continue to face health care and societal disparities, especially affecting racial minorities. Although legal advancements have been achieved, the key to addressing these persistent health care disparities lies in implementing comprehensive and culturally sensitive health care practices and supportive policies. With a growing number of TGD people seeking gender-affirming care, it is imperative that health care practitioners understand the unique challenges faced by this community and provide tailored services with sensitivity and expertise.

Conserved Residues at the Mtr4 C-Terminus Coordinate Helicase Activity and Exosome Interactions.

Mtr4 is an essential RNA helicase involved in nuclear RNA processing and degradation and is a member of the Ski2-like helicase family. Ski2-like helicases share a common core architecture that includes two RecA-like domains, a winged helix, and a helical bundle (HB) domain. In Mtr4, a short C-terminal tail immediately follows the HB domain and is positioned at the interface of the RecA-like domains. The tail ends with a SLYΦ sequence motif that is highly conserved in a subset of Ski2-like helicases. Here, we show that this sequence is critical for Mtr4 function. Mutations in the C-terminus result in decreased RNA unwinding activity. Mtr4 is a key activator of the RNA exosome complex, and mutations in the SLYΦ motif produce a slow growth phenotype when combined with a partial exosome defect in S. cerevisiae, suggesting an important role of the C-terminus of Mtr4 and the RNA exosome. We further demonstrate that C-terminal mutations impair RNA degradation activity by the major RNA exosome nuclease Rrp44 in vitro. These data demonstrate a role for the Mtr4 C-terminus in regulating helicase activity and coordinating Mtr4-exosome interactions.

Protease-Activated Receptor 2 Controls Vascular Smooth Muscle Cell Proliferation in Cyclic AMP-Dependent Protein Kinase/Mitogen-Activated Protein Kinase Kinase 1/2-Dependent Manner.

INTRODUCTION: Cardiovascular disorders are characterized by vascular smooth muscle (VSM) transition from a contractile to proliferative state. Protease-activated receptor 2 (PAR2) involvement in this phenotypic conversion remains unclear. We hypothesized that PAR2 controls VSM cell proliferation in phenotype-dependent manner and through specific protein kinases. METHODS: Rat clonal low (PLo; P3-P6) and high passage (PHi; P10-P15) VSM cells were established as respective models of quiescent and proliferative cells, based on reduced PKG-1 and VASP. Western blotting determined expression of cytoskeletal/contractile proteins, PAR2, and select protein kinases. DNA synthesis and cell proliferation were measured 24-72 h following PAR2 agonism (SLIGRL; 100 nM-10 µm) with/without PKA (PKI; 10 µm), MEK1/2 (PD98059; 10 µm), and PI3K (LY294002; 1 µm) blockade. RESULTS: PKG-1, VASP, SM22α, calponin, cofilin, and PAR2 were reduced in PHi versus PLo cells. Following PAR2 agonism, DNA synthesis and cell proliferation increased in PLo cells but decreased in PHi cells. Western analyses showed reduced PKA, MEK1/2, and PI3K in PHi versus PLo cells, and kinase blockade revealed PAR2 controls VSM cell proliferation through PKA/MEK1/2. DISCUSSION: Findings highlight PAR2 and PAR2-driven PKA/MEK1/2 in control of VSM cell growth and provide evidence for continued investigation of PAR2 in VSM pathology.