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In recent years, major advances have been made in the understanding of the cellular and molecular mechanisms driving pulmonary vascular remodelling in various forms of pulmonary hypertension, including pulmonary arterial hypertension, pulmonary hypertension associated with left heart disease, pulmonary hypertension associated with chronic lung disease and hypoxia, and chronic thromboembolic pulmonary hypertension. However, the survival rates for these different forms of pulmonary hypertension remain unsatisfactory, underscoring the crucial need to more effectively translate innovative scientific knowledge into healthcare interventions. In these proceedings of the 7th World Symposium on Pulmonary Hypertension, we delve into recent developments in the field of pathology and pathophysiology, prioritising them while questioning their relevance to different subsets of pulmonary hypertension. In addition, we explore how the latest omics and other technological advances can help us better and more rapidly understand the myriad basic mechanisms contributing to the initiation and progression of pulmonary vascular remodelling. Finally, we discuss strategies aimed at improving patient care, optimising drug development, and providing essential support to advance research in this field., Competing Interests: Conflict of interest: C. Guignabert reports grants from Acceleron Pharma, MSD, Corteria Pharmaceuticals, Structure Therapeutics (ex-ShouTi) and Gossamer Bio, payment or honoraria for lectures, presentations, manuscript writing or educational events from MSD, and patents planned, issued or pending (WO/2024/023139, WO/2018/011376). J. Aman has no potential conflicts of interest to disclose. S. Bonnet reports grants from Morphic Therapeutic, Sunshine Bio and Janssen, consultancy fees from Morphic Therapeutic and Chiesi, and participation on a data safety monitoring board or advisory board with Morphic Therapeutic and Allienaire. P. Dorfmüller reports payment or honoraria for lectures, presentations, manuscript writing or educational events from AstraZeneca. A.J. Olschewski reports grants from Austrian Science Fund (FWF) (10.55776/I6299 and 10.55776/KLI1153), payment or honoraria for lectures, presentations, manuscript writing or educational events from MSD, patents pending (PCT/EP2017/055440), and stock (or stock options) with Bayer. S. Pullamsetti reports grants and consultancy fees from Gossamer Bio. M. Rabinovitch reports consultancy fees from Pfizer, Amgen, Merck and Tiakis, payment or honoraria for lectures, presentations, manuscript writing or educational events from NIH, patents planned, issued or pending (FK506: tacrolimus in pulmonary hypertension), participation on a data safety monitoring board or advisory board with Amgen and NIH, is associate editor for JACC BTS, receipt of equipment, materials, drugs, medical writing, gifts or other services from Tiakis (tiprelestat). R.T. Schermuly reports grants from Chiesi and Attgeno, and consultancy fees from Gossamer, Attgeno and Chiesi. M. Humbert reports grants from Gossamer and Merck, consultancy fees from 35 Pharma, Aerovate, AOP Orphan, Chiesi, Ferrer, Gossamer, Janssen, Keros, Liquidia, Merck, Novartis, Respira, Roivant and United Therapeutics, payment or honoraria for lectures, presentations, manuscript writing or educational events from Janssen and Merck, and participation on a data safety monitoring board or advisory board with 35 Pharma, Aerovate, Janssen, Keros, Merck, Novartis and United Therapeutics. K.R. Stenmark reports grants from NIH/NHLBI and DoD, and a leadership role with PVRI., (Copyright ©The authors 2024.)

Raynaud's syndrome is a dysautonomia where exposure to cold causes vasoconstriction and hypoxia, particularly in the extremities. We performed meta-analysis in four cohorts and discovered eight loci (ADRA2A, IRX1, NOS3, ACVR2A, TMEM51, PCDH10-DT, HLA, and RAB6C) where ADRA2A, ACVR2A, NOS3, TMEM51, and IRX1 co-localized with expression quantitative trait loci (eQTLs), particularly in distal arteries. CRISPR gene editing further showed that ADRA2A and NOS3 loci modified gene expression and in situ RNAscope clarified the specificity of ADRA2A in small vessels and IRX1 around small capillaries in the skin. A functional contraction assay in the cold showed lower contraction in ADRA2A-deficient and higher contraction in ADRA2A-overexpressing smooth muscle cells. Overall, our study highlights the power of genome-wide association testing with functional follow-up as a method to understand complex diseases. The results indicate temperature-dependent adrenergic signaling through ADRA2A, effects at the microvasculature by IRX1, endothelial signaling by NOS3, and immune mechanisms by the HLA locus in Raynaud's syndrome., Competing Interests: Declaration of interests J.M.E. is an inventor on patents and patent applications related to CRISPR screening technologies, has received materials from 10× Genomics unrelated to this study, and has received speaking honoraria from GSK plc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The discovery and replacement of lung surfactant have helped increase survival rates for neonatal respiratory distress syndrome in extremely premature infants.

Rationale: Unraveling immune-driven vascular pathology in pulmonary arterial hypertension (PAH) requires a comprehensive understanding of the immune cell landscape. Although patients with hereditary (H)PAH and bone morphogenetic protein receptor type 2 (BMPR2) mutations have more severe pulmonary vascular pathology, it is not known whether this is related to specific immune cell subsets. Objectives: This study aims to elucidate immune-driven vascular pathology by identifying immune cell subtypes linked to severity of pulmonary arterial lesions in PAH. Methods: We used cutting-edge multiplexed ion beam imaging by time of flight to compare pulmonary arteries (PAs) and adjacent tissue in PAH lungs (idiopathic [I]PAH and HPAH) with unused donor lungs, as controls. Measurements and Main Results: We quantified immune cells' proximity and abundance, focusing on those features linked to vascular pathology, and evaluated their impact on pulmonary arterial smooth muscle cells (SMCs) and endothelial cells. Distinct immune infiltration patterns emerged between PAH subtypes, with intramural involvement independently linked to PA occlusive changes. Notably, we identified monocyte-derived dendritic cells within PA subendothelial and adventitial regions, influencing vascular remodeling by promoting SMC proliferation and suppressing endothelial gene expression across PAH subtypes. In patients with HPAH, pronounced immune dysregulation encircled PA walls, characterized by heightened perivascular inflammation involving T cell immunoglobulin and mucin domain-3 (TIM-3) +  T cells. This correlated with an expanded DC subset expressing indoleamine 2,3-dioxygenase 1, TIM-3, and SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1, alongside increased neutrophils, SMCs, and alpha-smooth muscle actin (ACTA2) + endothelial cells, reinforcing the heightened severity of pulmonary vascular lesions. Conclusions: This study presents the first architectural map of PAH lungs, connecting immune subsets not only with specific PA lesions but also with heightened severity in HPAH compared with IPAH. Our findings emphasize the therapeutic potential of targeting monocyte-derived dendritic cells, neutrophils, cellular interactions, and immune responses to alleviate severe vascular pathology in IPAH and HPAH.

Hemodynamic loading is known to contribute to the development and progression of pulmonary arterial hypertension (PAH). This loading drives changes in mechanobiological stimuli that affect cellular phenotypes and lead to pulmonary vascular remodeling. Computational models have been used to simulate mechanobiological metrics of interest, such as wall shear stress, at single time points for PAH patients. However, there is a need for new approaches that simulate disease evolution to allow for prediction of long-term outcomes. In this work, we develop a framework that models the pulmonary arterial tree through adaptive and maladaptive responses to mechanical and biological perturbations. We coupled a constrained mixture theory-based growth and remodeling framework for the vessel wall with a morphometric tree representation of the pulmonary arterial vasculature. We show that non-uniform mechanical behavior is important to establish the homeostatic state of the pulmonary arterial tree, and that hemodynamic feedback is essential for simulating disease time courses. We also employed a series of maladaptive constitutive models, such as smooth muscle hyperproliferation and stiffening, to identify critical contributors to development of PAH phenotypes. Together, these simulations demonstrate an important step toward predicting changes in metrics of clinical interest for PAH patients and simulating potential treatment approaches., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pulmonary arterial hypertension (PAH) is a progressive disease in which pulmonary arterial (PA) endothelial cell (EC) dysfunction is associated with unrepaired DNA damage. BMPR2 is the most common genetic cause of PAH. We report that human PAEC with reduced BMPR2 have persistent DNA damage in room air after hypoxia (reoxygenation), as do mice with EC-specific deletion of Bmpr2 (EC-Bmpr2 -/- ) and persistent pulmonary hypertension. Similar findings are observed in PAEC with loss of the DNA damage sensor ATM, and in mice with Atm deleted in EC (EC-Atm -/- ). Gene expression analysis of EC-Atm -/- and EC-Bmpr2 -/- lung EC reveals reduced Foxf1, a transcription factor with selectivity for lung EC. Reducing FOXF1 in control PAEC induces DNA damage and impaired angiogenesis whereas transfection of FOXF1 in PAH PAEC repairs DNA damage and restores angiogenesis. Lung EC targeted delivery of Foxf1 to reoxygenated EC-Bmpr2 -/- mice repairs DNA damage, induces angiogenesis and reverses pulmonary hypertension., (© 2023. The Author(s).)

Deficiency of iron‑sulfur (FeS) clusters promotes metabolic rewiring of the endothelium and the development of pulmonary hypertension (PH) in vivo. Joining a growing number of FeS biogenesis proteins critical to pulmonary endothelial function, recent data highlighted that frataxin (FXN) reduction drives Fe-S-dependent genotoxic stress and senescence across multiple types of pulmonary vascular disease. Trinucleotide repeat mutations in the FXN gene cause Friedreich's ataxia, a disease characterized by cardiomyopathy and neurodegeneration. These tissue-specific phenotypes have historically been attributed to mitochondrial reprogramming and oxidative stress. Whether FXN coordinates both nuclear and mitochondrial processes in the endothelium is unknown. Here, we aim to identify the mitochondria-specific effects of FXN deficiency in the endothelium that predispose to pulmonary hypertension. Our data highlight an Fe-S-driven metabolic shift separate from previously described replication stress whereby FXN knockdown diminished mitochondrial respiration and increased glycolysis and oxidative species production. In turn, FXN-deficient endothelial cells had increased vasoconstrictor production (EDN1) and decreased nitric oxide synthase expression (NOS3). These data were observed in primary pulmonary endothelial cells after pharmacologic inhibition of FXN, mice carrying a genetic endothelial deletion of FXN, and inducible pluripotent stem cell-derived endothelial cells from patients with FXN mutations. Altogether, this study indicates FXN is an upstream driver of pathologic aberrations in metabolism and genomic stability. Moreover, our study highlights FXN-specific vasoconstriction in vivo, prompting future studies to investigate available and novel PH therapies in contexts of FXN deficiency., Competing Interests: Declaration of Competing Interest S.Y.C. has served as a consultant for Merck and United Therapeutics. S.Y.C. has held research grants from United Therapeutics and Bayer. S.Y.C. is a director, officer, and shareholder of Synhale Therapeutics. S.Y.C. and T.B. hold patents and have filed patent applications regarding the therapeutic targeting of metabolism and senescence in pulmonary hypertension., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Mutations in BMPR2 (bone morphogenetic protein receptor 2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Our goal was to determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH., Methods: SMC-specific Bmpr2 -/- mice ( BKO SMC ) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKO SMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism., Results: BKO SMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary arteries. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of pERK1/2 (phosphorylated extracellular signal related kinase 1/2)-pP38-pSMAD2/3 mediating elevation in ARRB2 (β-arrestin2), pAKT (phosphorylated protein kinase B) inactivation of GSK3-beta, CTNNB1 (β-catenin) nuclear translocation and reduction in RHOA (Ras homolog family member A) and RAC1 (Ras-related C3 botulinum toxin substrate 1). Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension., Conclusions: Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.

Background: Pulmonary vascular abnormalities pose a risk for severe life-threatening hemodynamic disturbances following surgical repair of congenital cardiac communications (CCC s ). In the distal lung, small airways and vessels share a common microenvironment, where biological crosstalks take place. Because respiratory cells infected by viruses express a number of molecules with potential impact on airway and vascular remodeling, we decided to test the hypothesis that CCC patients carrying viral genomes in the airways might be at a higher risk for pulmonary (and systemic) hemodynamic disturbances postoperatively., Methods: Sixty patients were prospectively enrolled (age 11 [7-16] months, median with interquartile range). Preoperative pulmonary/systemic mean arterial pressure ratio (PAP/SAP) was 0.78 (0.63-0.88). The presence or absence of genetic material for respiratory viruses in nasopharyngeal and tracheal aspirates was investigated preoperatively in the absence of respiratory symptoms using real-time polymerase chain reaction (kit for detection of 19 pathogens). Post-cardiopulmonary bypass (CPB) inflammatory reaction was analyzed by measuring serum levels of 36 inflammatory proteins (immunoblotting) 4 h after its termination. Postoperative hemodynamics was assessed using continuous recording of PAP and SAP with calculation of PAP/SAP ratio., Results: Viral genomes were detected in nasopharynx and the trachea in 64% and 38% of patients, respectively. Rhinovirus was the most prevalent agent. The presence of viral genomes in the trachea was associated with an upward shift of postoperative PAP curve (p = 0.011) with a PAP/SAP of 0.44 (0.36-0.50) in patients who were positive versus 0.34 (0.30-0.45) in those who were negative (p = 0.008). The presence or absence of viral genomes in nasopharynx did not help predict postoperative hemodynamics. Postoperative PAP/SAP was positively correlated with post-CPB levels of interleukin-1 receptor antagonist (p = 0.026), macrophage migration inhibitory factor (p = 0.019) and monocyte chemoattractant protein-1 (p = 0.031), particularly in patients with virus-positive tracheal aspirates., Conclusions: Patients with CCC s carrying respiratory viral genomes in lower airways are at a higher risk for postoperative pulmonary hypertension, thus deserving special attention and care. Preoperative exposure to respiratory viruses and post-CPB inflammatory reaction seem to play a combined role in determining the postoperative behavior of the pulmonary circulation., (© 2023. The Author(s).)

Rationale: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. Objectives: To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Methods: Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Measurements and Main Results: Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated β1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Conclusions: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.

Physiologic laminar shear stress (LSS) induces an endothelial gene expression profile that is vasculo-protective. In this report, we delineate how LSS mediates changes in the epigenetic landscape to promote this beneficial response. We show that under LSS, KLF4 interacts with the SWI/SNF nucleosome remodeling complex to increase accessibility at enhancer sites that promote the expression of homeostatic endothelial genes. By combining molecular and computational approaches we discover enhancers that loop to promoters of KLF4- and LSS-responsive genes that stabilize endothelial cells and suppress inflammation, such as BMPR2, SMAD5, and DUSP5. By linking enhancers to genes that they regulate under physiologic LSS, our work establishes a foundation for interpreting how non-coding DNA variants in these regions might disrupt protective gene expression to influence vascular disease., (© 2022. The Author(s).)

Leishmania parasites use elaborate virulence mechanisms to invade and thrive in macrophages. These virulence mechanisms inhibit host cell defense responses and generate a specialized replicative niche, the parasitophorous vacuole. In this work, we performed a genome-wide RNAi screen in Drosophila macrophage-like cells to identify the host factors necessary for Leishmania amazonensis infection. This screen identified 52 conserved genes required specifically for parasite entry, including several components of the SUMOylation machinery. Further studies in mammalian macrophages found that L. amazonensis infection inhibited SUMOylation within infected macrophages and this inhibition enhanced parasitophorous vacuole growth and parasite proliferation through modulation of multiple genes especially ATP6V0D2 , which in turn affects CD36 expression and cholesterol levels. Together, these data suggest that parasites actively sabotage host SUMOylation and alter host transcription to improve their intracellular niche and enhance their replication., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Introduction: Adherence to therapeutic guidelines in psychiatry is anchored and facilitated by rating scales. However, they are rarely used in routine care, particularly for psychotic disorders. Consequently, adherence to treatment guidelines are not ideal and patient outcomes are often sub-optimal. In this study, we used the clinician-rated Clinical Global Impressions Scale (CGI) to implement a measurement-based care (MBC) approach and derive indices of quality of care at a first episode psychosis (FEP) program., Methods: At the individual level, an algorithm was created using CGI scores and their changes over time to define the concept of Patient Requiring Clinical Attention (PRCA) that encompasses several categories (e.g. episode of severity, treatment inertia, or treatment resistance). At the service level, CGI scores were used to derive several indices of quality of care: severity of illness and its change over time, conformity to the use of low doses of antipsychotic medications, and clozapine offer index., Results: 135 Patients were included in this study of whom 19 patients were identified as PRCA. Of these, 12 (63%) received timely medication, and 7 (37%) were suspected cases of therapeutic inertia. Additionally, 15 patients met criteria for treatment resistance of whom 7 were offered clozapine (47%). At the service level, the average CGI improved by 2 points from baseline to month 1 and average doses of antipsychotic medications prescribed were in line with prescription guidelines for FEP patients., Conclusion: The proposed CGI-based treatment algorithm and service evaluation strategy can help to optimize quality care and services for patients., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Introduction: Measurement-based care (MBC) is an evidence-based practice wherein clinical decisions are informed by patient data collected throughout treatment. MBC has yielded superior patient outcomes compared to standard care. However, the implementation of MBC in the day-to-day practice, particularly in psychotic disorders, poses several challenges. This study evaluates the clinician-rated Clinical Global Impressions Scale of Severity (CGI-S), for MBC implementation at a first-episode psychosis program., Methods: The CGI-S was evaluated in the context of routine care on fidelity to practice, inter-rater reliability among psychiatrists and concurrent validity with scales measuring different domains of psychopathology (SAPS, SANS, GAF, BPRS, PANSS-6)., Results: A high fidelity to practice (67%) and inter-rater reliability was found (r wg  = 0.92). CGI-S correlations were significant and strongest with BPRS (r = 0.55; p < 0.01), GAF (r = 0.53; p < 0.01), SAPS (r = 0.52, p < 0.01), and PANSS-6 (r = 0.41; p < 0.05) scores. However, correlations with SANS and PANSS-6 Negative sub-scale were weak., Conclusion: Findings suggest the CGI may be used to overcome important barriers towards MBC implementation within the context of first episode psychosis. However, as suggested by data, further improvements in capturing negative symptoms by rating clinicians are needed., Twitter: A novel strategy for measurement-based care to optimize treatment for individuals with first episode psychosis and related psychotic disorders., (Copyright © 2022. Published by Elsevier B.V.)

Untreated ventricular septal defects (VSDs) can lead to pulmonary arterial hypertension (PAH) characterized by elevated pulmonary artery (PA) pressure and vascular remodeling, known as PAH associated with congenital heart disease (PAH-CHD). Though previous studies have investigated hemodynamic effects on vascular mechanobiology in late-stage PAH, hemodynamics leading to PAH-CHD initiation have not been fully quantified. We hypothesize that abnormal hemodynamics from left-to-right shunting in early stage VSDs affects PA biomechanical properties leading to PAH initiation. To model PA hemodynamics in healthy, small, moderate, and large VSD conditions prior to the onset of vascular remodeling, computational fluid dynamics simulations were performed using a 3D finite element model of a healthy 1-year-old's proximal PAs and a body-surface-area-scaled 0D distal PA tree. VSD conditions were modeled with increased pulmonary blood flow to represent degrees of left-to-right shunting. In the proximal PAs, pressure, flow, strain, and wall shear stress (WSS) increased with increasing VSD size; oscillatory shear index decreased with increasing VSD size in the larger PA vessels. WSS was higher in smaller diameter vessels and increased with VSD size, with the large VSD condition exhibiting WSS >100 dyn/cm[Formula: see text], well above values typically used to study dysfunctional mechanotransduction pathways in PAH. This study is the first to estimate hemodynamic and biomechanical metrics in the entire pediatric PA tree with VSD severity at the stage leading to PAH initiation and has implications for future studies assessing effects of abnormal mechanical stimuli on endothelial cells and vascular wall mechanics that occur during PAH-CHD initiation and progression., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited treatment options. Despite endothelial cells (ECs) comprising 30% of the lung cellular composition, the role of EC dysfunction in pulmonary fibrosis (PF) remains unclear. We hypothesize that sterol regulatory element-binding protein 2 (SREBP2) plays a critical role in the pathogenesis of PF via EC phenotypic modifications. Transcriptome data demonstrate that SREBP2 overexpression in ECs led to the induction of the TGF, Wnt, and cytoskeleton remodeling gene ontology pathways and the increased expression of mesenchymal genes, such as snail family transcriptional repressor 1 (snai1), α-smooth muscle actin, vimentin, and neural cadherin. Furthermore, SREBP2 directly bound to the promoter regions and transactivated these mesenchymal genes. This transcriptomic change was associated with an epigenetic and phenotypic switch in ECs, leading to increased proliferation, stress fiber formation, and ECM deposition. Mice with endothelial-specific transgenic overexpression of SREBP2 (EC-SREBP2[N]-Tg mice) that were administered bleomycin to induce PF demonstrated exacerbated vascular remodeling and increased mesenchymal transition in the lung. SREBP2 was also found to be markedly increased in lung specimens from patients with IPF. These results suggest that SREBP2, induced by lung injury, can exacerbate PF in rodent models and in human patients with IPF.