In Situ Capture of Chromatin Interactions by Biotinylated dCas9.

Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here, we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single-copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human ß-globin genes establishes evidence for composition-based hierarchical organization. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally regulated super-enhancers reveals spatial features that causally control gene transcription. Thus, comprehensive and unbiased analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.

AP2S1 regulates APP degradation through late endosome-lysosome fusion in cells and APP/PS1 mice.

AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic ß-amyloid peptide (Aß). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aß in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aß, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD.

Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells.

The study of enhancers has been hampered by the scarcity of methods to systematically quantify their endogenous activity. We develop Mosaic-seq to systematically perturb enhancers and measure their endogenous activities at single-cell resolution. Mosaic-seq uses a CRISPR barcoding system to jointly measure a cell's transcriptome and its sgRNA modulators, thus quantifying the effects of dCas9-KRAB-mediated enhancer repression in single cells. Applying Mosaic-seq to 71 constituent enhancers from 15 super-enhancers, our analysis of 51,448 sgRNA-induced transcriptomes finds that only a small number of constituents are major effectors of target gene expression. Binding of p300 and RNAPII are key features of these constituents. We determine two key parameters of enhancer activity in single cells: their penetrance in a population and their contribution to expression in these cells. Through combinatorial interrogation, we find that simultaneous repression of multiple weak constituents can alter super-enhancer activity in a manner greatly exceeding repression of individual constituents.

COMPLEX HEMODYNAMICS OF INTERVORTEX VEIN ANASTOMOSES IN HIGHLY MYOPIC EYES.

PURPOSE: To analyze the complex pattern of filling of the intervortex vein (IVV) anastomoses through large trunks in highly myopic eyes based on indocyanine green angiographic (ICGA) videos. METHODS: The medical records of 1,060 consecutive highly myopic eyes that had undergone ICGA were studied. IVV anastomoses were identified in the ICGA images, and the ICGA images and videos were analyzed comprehensively to characterize their hemodynamic features. RESULTS: Seven eyes with IVV anastomoses through large trunks were analyzed. In the ICGA videos of six eyes, laminar flow was observed in the IVV anastomotic vessels. The laminar flow started in the arterial phase in two eyes, with pulsatile fashion in 1 of them. The flow began in the early arteriovenous transition phase in four eyes. The laminar flow continued for a mean of 12.17 ± 3.06 seconds, and the remaining section was gradually filled slower than the surrounding veins. The anastomotic trunk for the remaining one eye was too narrow to be analyzed. Four eyes had longitudinal ICGA records, and two had significant attenuation and narrowing of the anastomotic vessels. CONCLUSION: The very early filling of part of the IVV anastomoses suggests that arteriovenous anastomoses are involved in the IVV of highly myopic eyes. However, this suggestion needs further study. There may be similar pathogenesis for IVV anastomoses either in thick or thin sclera.

MORPHOLOGIC CHARACTERISTICS AND PROGRESSION PATTERNS OF MACULAR NEOVASCULARIZATION-RELATED ATROPHY IN EYES WITH PATHOLOGIC MYOPIA.

PURPOSE: To investigate the development and progression patterns of macular neovascularization (MNV)-related atrophies in eyes with pathologic myopia. METHODS: Twenty-seven eyes of 26 patients with MNV followed from its onset to progression to macular atrophy were studied. A longitudinal series of autofluorescence and optical coherence tomography images were examined for the patterns of MNV-related atrophy. Changes in best-corrected visual acuity were determined for each pattern. RESULTS: The mean age was 67.2 ± 8.7 years. The mean axial length was 29.6 ± 1.5 mm. Three patterns of atrophy were identified: multiple-atrophic pattern, 63% of the eyes had small atrophies occurring at multiple sites around the MNV edge; single-atrophic pattern, 18.5% had atrophies occurring only on one side of the MNV edge; and exudation-related pattern, 18.5% had atrophy occurring within a previous serous exudation or hemorrhagic area and slightly away from the MNV edge. Eyes with atrophies in multiple-atrophic and exudation-related patterns progressed to large macular atrophies involving the central fovea and showed decrease in best-corrected visual acuity during the 3-year follow-up period. Eyes with single-atrophic pattern had a sparing of the fovea and had good recovery of the best-corrected visual acuity. CONCLUSION: There are three patterns of MNV-related atrophy in eyes with pathologic myopia with different courses of progression.

LONGITUDINAL STUDY OF CHANGES IN INNER SCLERAL CURVATURE PATTERNS AND DEVELOPMENT OF POSTERIOR STAPHYLOMAS IN HIGHLY MYOPIC EYES OF CHILDREN AND ADOLESCENTS.

PURPOSE: To determine the longitudinal changes in patterns of inner scleral curvature and development of posterior staphylomas in the eyes of highly myopic youths. METHODS: A retrospective, longitudinal study. Ultra-widefield optic coherence tomographic (UWF-OCT) images from 47 eyes of 24 highly myopic patients with a follow-up period of 2 to 4 years were analyzed. Patients were divided into two groups, the children group younger than 10 years and the adolescents group aged 11 to 19 years. RESULTS: During the follow-up period, the scleral curvature patterns changed in either the horizontal or vertical optical coherence tomographic sections in 27 of the 46 eyes (58.7%) of both groups. The changes were significantly more frequent in children than adolescents. The change in the vertical section from symmetrical to asymmetrical in almost of children was correlated with an increase in the axial length and myopia. Four eyes had a staphyloma at the baseline. The staphyloma developed in six eyes of the remaining 43 eyes (14%). All staphyloma edges were found at or around the optic disc and were oriented in the horizontal direction. CONCLUSION: Our results showed that UWF-OCT images can be used to determine the process of new staphyloma formation and concurrent changes in scleral curvature patterns.

Stroke survivors', caregivers' and nurse coaches' perspectives on health coaching program towards hospital-to-home transition care: A qualitative descriptive process evaluation.

AIMS AND OBJECTIVES: To explore the perspectives of stroke survivors, caregivers and nurse coaches on a health coaching program during hospital-to-home transitional care. BACKGROUND: Stroke is a major public health problem that seriously affects the health and safety of people in China. Nurse-led health coaching is a promising support option in enabling smooth hospital-to-home transition for stroke survivors and family caregivers. A qualitative study is valuable for gaining insight into their perspectives on using this program. DESIGN: An exploratory, descriptive qualitative process evaluation was conducted parallel with a former randomised controlled trial. Data were obtained from 26 stroke survivors, 33 caregivers and four nurse coaches with semi-structured interviews. The inductive reflexive thematic analysis approach was used for data analysis. The COREQ checklist was followed in reporting this study. RESULTS: Seven themes were generated from the data: (1) the applicability of individualised health coaching sessions, (2) driving self-efficacy to establish self-care skills, (3) the key role of nurse coaches, (4) coordination among healthcare teams during the transition, (5) adequate community and social support, (6) insufficient rehabilitative services after discharge and (7) perceived extra workload for nurse coaches. CONCLUSIONS: The study captured perspectives on a nurse-led health coaching program towards hospital-to-home transition care from stroke survivors, caregivers and nurse coaches. Individualised health coaching sessions and driving self-efficacy were perceived as facilitators for empowering the self-care skills of stroke survivors and caregivers. The key role of nurse coaches in coordinating healthcare teams and adequate community and social support were detected as the power frame of the program's implementation. However, health system obstacles, such as insufficient rehabilitative services and the high workload of nurses, still need to be addressed to ensure the sustainability of health coaching intervention in transitional care. RELEVANCE TO CLINICAL PRACTICE: The study suggested the feasibility of implementing nurse-led health coaching to smooth post-stroke hospital-to-home transitional care. The findings also highlighted the importance of qualitative process evaluation when implementing evidence-based interventions in health care. TRIAL REGISTRATION: The trial was registered with the Australia New Zealand Clinical Trials Registry (ID: ACTRN12619000321145).

Computational identification of clonal cells in single-cell CRISPR screens.

BACKGROUND: Single-cell CRISPR screens are powerful tools to understand genome function by linking genetic perturbations to transcriptome-wide phenotypes. However, since few cells can be affordably sequenced in these screens, biased sampling of cells could affect data interpretation. One potential source of biased sampling is clonal cell expansion. RESULTS: Here, we identify clonal cells in single cell screens using multiplexed sgRNAs as barcodes. We find that the cells in each clone share transcriptional similarities and bear segmental copy number changes. These analyses suggest that clones are genetically distinct. Finally, we show that the transcriptional similarities of clonally expanded cells contribute to false positives in single-cell CRISPR screens. CONCLUSIONS: Experimental conditions that reduce clonal expansion or computational filtering of clonal cells will improve the reliability of single-cell CRISPR screens.

ANOMALIES OF CHOROIDAL VENOUS STRUCTURE IN HIGHLY MYOPIC EYES.

PURPOSE: To identify anomalies of choroidal venous structure in highly myopic (HM) eyes. METHODS: Widefield indocyanine green angiographic images of 175 HM eyes (refractive error ≤ -6.0D diopters or axial length >26.5 mm) and 100 control eyes taken between January 2014 and December 2018 were reviewed. RESULTS: There were no significant differences in age and gender between HM patients and controls. Three types of changes of large choroidal veins were found in 103 HM eyes (58.86%): Asymmetry of vortex veins in 44 eyes (25.14%), isolated long vein across the macula in 58 eyes (33.14%), and intervortex anastomoses in 25 eyes (14.29%). Similar changes in controls were found in 12 eyes (12%), 0 eye (0%), and 2 eyes (2%), respectively, which were significantly lower than those in the HM group (all P < 0.05). The patterns of asymmetry were affected by steeper staphyloma edges and anastomoses were observed through large trunks and terminal venules. In two eyes with large trunk anastomosis, attenuation of the less dominant vortex vein was observed afterward. CONCLUSION: Choroidal venous anomalies are more common in HM eyes than controls. Choroidal venous structure in HM eyes may be altering continuously, and such changes may underlie the development of myopic maculopathy.

PREVALENCE AND CHARACTERISTICS OF MULTIFOCAL CHOROIDITIS/PUNCTATE INNER CHOROIDOPATHY IN PATHOLOGIC MYOPIA EYES WITH PATCHY ATROPHY.

PURPOSE: To determine the prevalence and characteristics of multifocal choroiditis/punctate inner choroidopathy (MFC/PIC) in eyes with patchy atrophy because of pathologic myopia. METHODS: Five hundred eyes of 253 patients with patchy atrophy were examined between 2014 and 2020 at the Advanced Clinical Center for Myopia. The main outcome measures included the prevalence and characteristics of active MFC/PIC lesions diagnosed by optical coherence tomography. RESULTS: Fifty-five of the 500 eyes (11%) diagnosed with patchy atrophy had optical coherence tomography features of active MFC/PIC lesions, such as focal elevations of the retinal pigment epithelium filled with medium hyperreflectivity material, curvilinear scars (Schlaegel lines), and/or areas of outer retinal atrophy. At the time when the MFC/PIC was diagnosed, the mean age was 57.3 ± 12.0 years, and the mean axial length was 29.2 ± 1.8 mm. Macular neovascularization was found in 45 of eyes (81.8%) with MFC/PIC versus 151 eyes without such findings (33.9%; P < 0.001). In 25 of the 55 eyes (45.5%), active MFC/PIC lesions were found before the development of the patchy atrophy. The Bruch membrane defects were colocated with these lesions. CONCLUSION: Active MFC/PIC lesions were identified in a minority of eyes with pathologic myopia, and a subset of these lesions were observed to progress to findings indistinguishable from myopic patchy atrophy. Evidence of MFC/PIC in eyes with pathologic myopia appeared to be a risk factor for the development of macular neovascularization.

Hospital-based study of risk factors associated with development of myopic macular neovascularization in highly myopic eyes.

INTRODUCTION: Myopic macular neovascularization (MNV) is the most common cause of a reduction of central vision in eyes with pathologic myopia, and it can progress to macular atrophy in the long-term. The aim of this study was to determine the risk factors associated with the development of MNVs. METHODS: There were 17,198 follow-up records from 5,409 eyes of 2,784 highly myopic patients that were reviewed. The general information and ophthalmic information in the records were studied. The significance of the correlations of factors associated with the development and predicting the development of myopic MNV were determined. RESULTS: Being a woman (odds ratio [OR]: 0.727, P<0.001), having a longer axial length (OR = 0.948, P<0.001), a poorer baseline best-correct visual acuity (BCVA, OR = 2.098, P<0.001), having severe myopic maculopathy (overall: P<0.001), prior myopic MNV in the fellow eye (OR = 4.105, P<0.001), presence of patchy atrophy (overall P<0.001), lacquer cracks (OR = 1.718, P<0.001), prior foveal retinal detachment (RD, OR = 3.269, P<0.001), prior macular hole (MH, OR = 0.641, P <0.001), prior macular retinoschisis (OR = 1.533, P<0.001), and prior macular edema (OR = 1.508, P<0.001) were significantly correlated with the development of myopic MNV. Eyes with MNV and patchy atrophy would require an intensive follow-up examination for myopic patients as the fellow eye would have a risk of >70% for the development of myopic MNV in 3-years and nearly 80% in 5-years. CONCLUSIONS: Clinicians need to pay special attention to eyes with severe grades of myopic maculopathy, prior myopic MNV in the fellow eye, presence of patchy atrophy, and prior foveal retinal detachment to determine the onset of myopic MNV.

ROLE OF DILATED SUBFOVEAL CHOROIDAL VEINS IN EYES WITH MYOPIC MACULAR NEOVASCULARIZATION.

PURPOSE: To investigate the dilated choroidal veins (DCVs) at or around myopic macular neovascularizations (MNVs) and to determine whether there is a hemodynamic relationship between them. METHODS: Fifty-eight eyes of 57 patients with myopic MNVs were examined. Dilated choroidal veins were defined as choroidal veins whose diameter was 2X larger than adjacent veins. Indocyanine green angiography and swept-source optical coherence tomography images were reviewed to detect DCVs that crossed the subfoveal area. The filling sequence of the DCVs and MNVs was determined. RESULTS: Patients' mean age was 71.4 ± 10.6 years. The mean axial length was 29.3 ± 1.8 mm. Dilated choroidal veins below or around the MNV were found in 17 eyes (29.3%). Emissaries of the short posterior ciliary arteries were seen at or around MNVs in 8 of the 17 eyes. In these eyes, the short posterior ciliary artery was filled first or almost simultaneously with the filling of the MNV, followed by a laminar filling of the DCVs. In one eye, afferent arterioles from the short posterior ciliary arteries and efferent venules connected to DCVs were seen. CONCLUSION: Dilated choroidal veins are present below or around MNVs in about 30% of eyes with myopic MNVs. Our findings suggest that an MNV might be a vascular unit consisting of short posterior ciliary arteries, afferent arterioles, efferent venules, and DCVs.

ABRUPTLY EMERGING VESSELS IN EYES WITH MYOPIC PATCHY CHORIORETINAL ATROPHY.

PURPOSE: To determine the prevalence and characteristics of abruptly emerging vessels (AEVs) within patchy atrophy (PA) in myopic eyes. METHODS: We studied 160 highly myopic eyes of 144 patients between March and November in 2016. Fundus photographs and swept-source optical coherence tomography images were analyzed. RESULTS: Patient mean age was 67.1 ± 10.5 years. Mean axial length was 30.9 ± 2.0 mm. The mean size of the 264 PAs was 5.6 ± 8.3 mm. Abruptly emerging vessels were detected in 69 (43.1%) eyes and were located within or near PA edge in fundus photographs. Swept-source optical coherence tomography showed that the AEVs were continuous with perforating scleral vessels and were observed on the inner surface of the sclera at the site where they appeared in fundus photographs. A slight bowing of sclera around the AEVs was observed in 41 (59%) eyes. Patchy atrophy with AEVs was significantly larger (10.7 ± 11.3 mm) than PA without AEVs (3.4 ± 5.1 mm). CONCLUSION: Abruptly emerging vessels are commonly found in eyes with myopic PA. The sclera surrounding the AEVs is slightly bowed. Further studies are needed to determine whether the penetrating site of AEVs is structurally more fragile and leads to Bruch membrane defects or AEVs are secondarily involved during PA progression.

Unusual architecture of the p7 channel from hepatitis C virus.

The hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations. We wanted to examine the structural solution that the viroporin adopts in order to achieve selective cation conduction, because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The activity of p7 can be inhibited by amantadine and rimantadine, which are potent blockers of the influenza M2 channel and licensed drugs against influenza infections. The adamantane derivatives have been used in HCV clinical trials, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here we determine the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbours, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also points to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter, whereas an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording shows that these residues are critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide a molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

Effect of statins and the clinical nursing characteristics in patients with acute myocardial infarction.

Statins are widely used in clinical practice because of their effectiveness and evidence-based medical evidence. In this paper, the effect of different kinds of statins on the treatment of patients with acute myocardial infarction has analyzed. By analyzing the clinical data of patients with acute myocardial infarction in our hospital, the authors summarized the characteristics of clinical nursing of statins. At the same time, this study retrospectively investigates the female patients with acute myocardial infarction treated in our hospital. Compared with the standard regimen, the enhanced statin lipid-lowering regimen is protective in reducing mortality and cardiovascular events for patients with diagnosed acute coronary syndrome (ACS). The morbidity of female patients with acute myocardial infarction is lower than the male, it also has larger concealment. The nursing staff should have a rapid identification and triage to female patients with acute myocardial infarction and have a health education of knowledge of acute myocardial infarction.

IFN-λ4 desensitizes the response to IFN-α treatment in chronic hepatitis C through long-term induction of USP18.

The recently discovered interferon lambda 4 (IFN-λ4) is a new member of the human type III interferons which could induce a strong antiviral effect through the JAK-STAT cascade. However, hepatitis C virus (HCV) patients who are capable of expressing IFN-λ4 usually have poor response to IFN-α treatment, and the mechanism behind this paradox remains unknown. Here, we reported that IFN-λ4 desensitized IFN-α-stimulated JAK-STAT signalling. Microarray analysis revealed that IFN-λ4 could induce ubiquitin specific peptidase 18 (USP18), a known inhibitor of the type I IFN signalling pathway, in a more sustained pattern compared with type I interferon induction. Moreover, only HCV genotype 1b but not 2a replicon cells pretreated with IFN-λ4 had an attenuated response to type I IFN treatment, which might be due to the different level of USP18 expression. Consistently, knockdown of USP18 in HCV genotype 1b-containing replicon cells reversed the resistance induced by IFN-λ4 and promoted viral clearance. Finally, IFN-λ4 is also strongly associated with the poor response to IFN-α in a Chinese HCV genotype 1b cohort. In conclusion, these data indicate that IFN-λ4 attenuates the response of HCV genotype 1b to IFN-α therapy and inhibits the JAK-STAT signalling pathway by inducing USP18 expression.

The ORF4a protein of human coronavirus 229E functions as a viroporin that regulates viral production.

In addition to a set of canonical genes, coronaviruses encode additional accessory proteins. A locus located between the spike and envelope genes is conserved in all coronaviruses and contains a complete or truncated open reading frame (ORF). Previously, we demonstrated that this locus, which contains the gene for accessory protein 3a from severe acute respiratory syndrome coronavirus (SARS-CoV), encodes a protein that forms ion channels and regulates virus release. In the current study, we explored whether the ORF4a protein of HCoV-229E has similar functions. Our findings revealed that the ORF4a proteins were expressed in infected cells and localized at the endoplasmic reticulum/Golgi intermediate compartment (ERGIC). The ORF4a proteins formed homo-oligomers through disulfide bridges and possessed ion channel activity in both Xenopus oocytes and yeast. Based on the measurement of conductance to different monovalent cations, the ORF4a was suggested to form a non-selective channel for monovalent cations, although Li(+) partially reduced the inward current. Furthermore, viral production decreased when the ORF4a protein expression was suppressed by siRNA in infected cells. Collectively, this evidence indicates that the HCoV-229E ORF4a protein is functionally analogous to the SARS-CoV 3a protein, which also acts as a viroporin that regulates virus production. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

Cerebral furin deficiency causes hydrocephalus in mice.

Furin is a pro-protein convertase that moves between the trans-Golgi network and cell surface in the secretory pathway. We have previously reported that cerebral overexpression of furin promotes cognitive functions in mice. Here, by generating the brain-specific furin conditional knockout (cKO) mice, we investigated the role of furin in brain development. We found that furin deficiency caused early death and growth retardation. Magnetic resonance imaging showed severe hydrocephalus. In the brain of furin cKO mice, impaired ciliogenesis and the derangement of microtubule structures appeared along with the down-regulated expression of RAB28, a ciliary vesicle protein. In line with the widespread neuronal loss, ependymal cell layers were damaged. Further proteomics analysis revealed that cell adhesion molecules including astrocyte-enriched ITGB8 and BCAR1 were altered in furin cKO mice; and astrocyte overgrowth was accompanied by the reduced expression of SOX9, indicating a disrupted differentiation into ependymal cells. Together, whereas alteration of RAB28 expression correlated with the role of vesicle trafficking in ciliogenesis, dysfunctional astrocytes might be involved in ependymal damage contributing to hydrocephalus in furin cKO mice. The structural and molecular alterations provided a clue for further studying the potential mechanisms of furin.

Barriers and facilitators to the formation of professional identity among nursing students: A four-year longitudinal qualitative study.

BACKGROUND: Nursing professional identity (NPI) is essential for nurses to develop their nursing profession. It reflects the competencies consistent with the professional practices of nurses and contributes to them providing better healthcare and public health. The formation process of NPI started with undergraduate nursing education and continued throughout the nursing career. OBJECTIVE: To explore nursing students' perceptions of facilitators and barriers to the formation of NPI during their study. METHODS: A 4-year longitudinal, qualitative research design with yearly semi-structured interviews undertaken from 2019 to 2022. The reflexive thematic analysis methodology was applied for the data analysis. RESULTS: Ninety-three nursing students were recruited, joining a group or individual interview. The four-year nursing baccalaureate program revealed a dynamic formation process of NPI: "Outsider of nursing", "Entering the nursing courses", "Building nursing competence", and "Thinking and acting like a nurse". A total of 12 themes were identified to present the barriers and facilitators to the NPI formation at different stages. Specifically, the six barriers include conflict between their ideals and reality, sociocultural stereotypes about nursing, the negative impact of COVID-19, the pre-internship concerns, struggling to meet expectations, and potential danger and discrimination in the healthcare settings. The enablers were: self-motivation and inner belief towards the nursing profession, the power of role models, the improvement of nursing capacity, well integration into the healthcare professional teams, understanding of the clinical environment, and recognition and encouragement from others. CONCLUSIONS: The formation of nursing students' NPI is an ever-changing process, with various intrinsic and extrinsic influences during their four-year study. Nursing educators are suggested to prepare and develop students' professional comportment in their theoretical and clinical practice to develop their professional identity as a nurse.