Inhibition of HDAC activity directly reprograms murine embryonic stem cells to trophoblast stem cells.

Embryonic stem cells (ESCs) can differentiate into all cell types of the embryonic germ layers. ESCs can also generate totipotent 2C-like cells and trophectodermal cells. However, these latter transitions occur at low frequency due to epigenetic barriers, the nature of which is not fully understood. Here, we show that treating mouse ESCs with sodium butyrate (NaB) increases the population of 2C-like cells and enables direct reprogramming of ESCs into trophoblast stem cells (TSCs) without a transition through a 2C-like state. Mechanistically, NaB inhibits histone deacetylase activities in the LSD1-HDAC1/2 corepressor complex. This increases acetylation levels in the regulatory regions of both 2C- and TSC-specific genes, promoting their expression. In addition, NaB-treated cells acquire the capacity to generate blastocyst-like structures that can develop beyond the implantation stage in vitro and form deciduae in vivo. These results identify how epigenetics restrict the totipotent and trophectoderm fate in mouse ESCs.

Systematic evaluation of single-cell RNA-seq analyses performance based on long-read sequencing platforms.

INTRODUCTION: The rapid development of next-generation sequencing (NGS)-based single-cell RNA sequencing (scRNA-seq) allows for detecting and quantifying gene expression in a high-throughput manner, providing a powerful tool for comprehensively understanding cellular function in various biological processes. However, the NGS-based scRNA-seq only quantifies gene expression and cannot reveal the exact transcript structures (isoforms) of each gene due to the limited read length. On the other hand, the long read length of third-generation sequencing (TGS) technologies, including Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), enable direct reading of intact cDNA molecules. OBJECTIVES: Both ONT and PacBio have been used in conjunction with scRNA-seq, but their performance in single-cell analyses has not been systematically evaluated. METHODS: To address this, we generated ONT and PacBio data from the same single-cell cDNA libraries containing different amount of cells. RESULTS: Using NGS as a control, we assessed the performance of each platform in cell type identification. Additionally, the reliability in identifying novel isoforms and allele-specific gene/isoform expression by both platforms was verified, providing a systematic evaluation to design the sequencing strategies in single-cell transcriptome studies. CONCLUSION: Beyond gene expression analysis, which the NGS-based scRNA-seq only affords, TGS-based scRNA-seq achieved gene splicing analyses, identifying novel isoforms. Attribute to higher sequencing quality of PacBio, it outperforms ONT in accuracy of novel transcripts identification and allele-specific gene/isoform expression.

Categorizing Extrachromosomal Circular DNA as Biomarkers in Serum of Cancer.

Extrachromosomal circular DNA (eccDNA), a double-stranded circular DNA molecule found in multiple organisms, has garnered an increasing amount of attention in recent years due to its close association with the initiation, malignant progression, and heterogeneous evolution of cancer. The presence of eccDNA in serum assists in non-invasive tumor diagnosis as a biomarker that can be assessed via liquid biopsies. Furthermore, the specific expression patterns of eccDNA provide new insights into personalized cancer therapy. EccDNA plays a pivotal role in tumorigenesis, development, diagnosis, and treatment. In this review, we comprehensively outline the research trajectory of eccDNA, discuss its role as a diagnostic and prognostic biomarker, and elucidate its regulatory mechanisms in cancer. In particular, we emphasize the potential application value of eccDNA in cancer diagnosis and treatment and anticipate the development of novel tumor diagnosis strategies based on serum eccDNA in the future.

Single-cell analysis of isoform switching and transposable element expression during preimplantation embryonic development.

Alternative splicing is an essential regulatory mechanism for development and pathogenesis. Through alternative splicing one gene can encode multiple isoforms and be translated into proteins with different functions. Therefore, this diversity is an important dimension to understand the molecular mechanism governing embryo development. Isoform expression in preimplantation embryos has been extensively investigated, leading to the discovery of new isoforms. However, the dynamics of isoform switching of different types of transcripts throughout the development remains unexplored. Here, using single-cell direct isoform sequencing in over 100 single blastomeres from the mouse oocyte to blastocyst stage, we quantified isoform expression and found that 3-prime partial transcripts lacking stop codons are highly accumulated in oocytes and zygotes. These transcripts are not transcription by-products and might play a role in maternal to zygote transition (MZT) process. Long-read sequencing also enabled us to determine the expression of transposable elements (TEs) at specific loci. In this way, we identified 3,894 TE loci that exhibited dynamic changes along the preimplantation development, likely regulating the expression of adjacent genes. Our work provides novel insights into the transcriptional regulation of early embryo development.

Multicenter, randomized controlled, open label evaluation of the efficacy and safety of arbidol hydrochloride tablets in the treatment of influenza-like cases.

OBJECTIVE: To study the efficacy and safety of arbidol hydrochloride tablets as a treatment for influenza-like diseases. METHODS: In this multicenter, randomized, controlled, open label study, a total of 412 influenza-like cases were collected from 14 hospitals in seven regions of Hebei Province from September 2021 to March 2022. Patients were randomly divided into two groups. The control group (n = 207) were administered oseltamivir phosphate capsules for five days and the experimental group (n = 205) were administered arbidol hydrochloride tablets for five days. The primary endpoint was the time to normal body temperature, and the secondary endpoints included the time to remission of influenza symptoms, incidence of influenza-like complications, and incidence of adverse reactions. RESULTS: Before treatment, there was no significant difference between the two groups in general conditions, blood routine, body temperature, or symptom severity. After treatment, there was no significant difference between the groups in the mean time to fever remission (59.24 h ± 25.21 vs. 61.05 h ± 29.47) or the mean time to remission of influenza symptoms (57.31 h ± 30.19 vs. 62.02 h ± 32.08). Survival analyses using Log-rank and Wilcoxon bilateral tests showed that there was no significant difference in fever relief time or influenza symptom relief time between the two groups. Regarding the incidence of complications and adverse events, there was only one case of tracheitis, one case of nausea, one case of vomiting, and one case of dizziness in the control group. In the experimental group, there was one case of nausea, one case of vomiting, and one case of drowsiness. In addition, one patient in the control group was hospitalized for urinary calculi. CONCLUSION: There was no significant difference between the patients with influenza-like cases treated with arbidol hydrochloride tablets and those treated with oseltamivir phosphate capsules. Further, the patients treated with arbidol hydrochloride tablets had fewer adverse reactions, and thus, the tablets were safe to use.

Single-cell third-generation sequencing-based multi-omics uncovers gene expression changes governed by ecDNA and structural variants in cancer cells.

BACKGROUND: Cancer cells often exhibit large-scale genomic variations, such as circular extrachromosomal DNA (ecDNA) and structural variants (SVs), which have been highly correlated with the initiation and progression of cancer. Currently, no adequate method exists to unveil how these variations regulate gene expression in heterogeneous cancer cell populations at a single-cell resolution. METHODS: Here, we developed a single-cell multi-omics sequencing method, scGTP-seq, to analyse ecDNA and SVs using long-read sequencing technologies. RESULTS AND CONCLUSIONS: We demonstrated that our method can efficiently detect ecDNA and SVs and illustrated how these variations affect transcriptomic changes in various cell lines. Finally, we applied and validated this method in a clinical sample of hepatocellular carcinoma (HCC), demonstrating a feasible way to monitor the evolution of ecDNA and SVs during cancer progression.

Protective effect of ulinastatin on myocardial injuries in children with severe pneumonia.

BACKGROUND: This study aimed to investigate the effect of ulinastatin on myocardial protection in children with severe pneumonia. METHODS: In this retrospective study, children with severe pneumonia were divided into two groups based on their treatment methods. The control group (n=39) received anti-infection therapy, while the experimental group (n=43) received anti-infection therapy combined with ulinastatin. The clinical treatment efficacy, levels of peripheral inflammatory factors, T lymphocyte subsets, QT dispersion and adverse reactions of the two groups were observed and compared before and after treatment. RESULTS: The clinical efficacy was improved after intervention (P<0.05), and the total response rate was 88.4% (38/43) in the experimental group and 64.1% in the control group. The post-treatment levels of interleukin-8 (IL-8), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) in the peripheral blood were lower than those before treatment, with significant differences (P<0.05). After treatment, the serum levels of CD3+, CD4+ and CD4+/CD8+ in the experimental group were significantly higher than those in the control group, whereas the levels of IL-6, IL-8 and hscRP in the peripheral blood were lower in the experimental group than those in the control group, with significant differences (P<0.05). The QT dispersion indexes, such as QTmax, QTmin, QTd, QTcmax, QTcd and QTcmin in the experimental group were shorter than those in the control group (P<0.05). CONCLUSION: Ulinastatin has significant therapeutic efficacy and safety in the clinical treatment of children with severe pneumonia, which may be related to inhibition of the release of inflammatory factors, shortened QT dispersion and the improvement of immune function of peripheral blood T lymphocyte subsets.

Characterization of mesenchymal stem cells in human fetal bone marrow by single-cell transcriptomic and functional analysis.

Bone marrow mesenchymal stromal/stem cells (MSCs) are a heterogeneous population that can self-renew and generate stroma, cartilage, fat, and bone. Although a significant progress has been made toward recognizing about the phenotypic characteristics of MSCs, the true identity and properties of MSCs in bone marrow remain unclear. Here, we report the expression landscape of human fetal BM nucleated cells (BMNCs) based on the single-cell transcriptomic analysis. Unexpectedly, while the common cell surface markers such as CD146, CD271, and PDGFRa used for isolating MSCs were not detected, LIFR+PDGFRB+ were identified to be specific markers of MSCs as the early progenitors. In vivo transplantation demonstrated that LIFR+PDGFRB+CD45-CD31-CD235a- MSCs could form bone tissues and reconstitute the hematopoietic microenvironment (HME) effectively in vivo. Interestingly, we also identified a subpopulation of bone unipotent progenitor expressing TM4SF1+CD44+CD73+CD45-CD31-CD235a-, which had osteogenic potentials, but could not reconstitute HME. MSCs expressed a set of different transcription factors at the different stages of human fetal bone marrow, indicating that the stemness properties of MSCs might change during development. Moreover, transcriptional characteristics of cultured MSCs were significantly changed compared with freshly isolated primary MSCs. Our cellular profiling provides a general landscape of heterogeneity, development, hierarchy, microenvironment of the human fetal BM-derived stem cells at single-cell resolution.

BIS feedback closed-loop target-controlled infusion of propofol or etomidate in elderly patients with spinal surgery.

OBJECTIVE: To investigate the safety of etomidate anesthesia induction combined with Bispectral index (BIS) feedback closed-loop target-controlled infusion of propofol for spinal surgery in elderly patients. METHODS: Clinical data of 90 elderly patients who underwent elective spinal surgery were retrospectively analyzed. The patients were assigned to an etomidate group (n=48) and a propofol group (n=42) according to the different anesthesia methods. The etomidate group was anesthetized with etomidate combined with BIS feedback closed-loop target-controlled infusion, and the propofol group was anesthetized with closed-loop target-controlled infusion induced by propofol. The mean arterial pressure (MAP) and heart rate (HR) of the two groups were statistically analyzed 5 min after admission to the operating room (T0), the moment of the intubation (T1), 3 min after intubation (T2), 1 min before prone position (T3), 3 min after prone position (T4), the end of suture skin (T5) and 3 min after supine position (T6). In addition, the vasoactive drug application, awakening time, tracheal tube extraction time and incidence of postoperative complications were compared between the two groups. RESULTS: There were significant changes in MAP and HR from T0 to T1 in both groups (MAP: etomidate group t=5.677, P<0.001, propofol group t=8.093, P<0.001; HR: etomidate group t=2.731, P=0.008, propofol group t=3.967, P<0.001). MAP changes in etomidate group from T0 to T1 were less (MAP: t=4.236, P<0.001; t=2.082, P=0.040), and there was no significant difference in HR between the two groups (P>0.05). There were fewer patients receiving vasoactive drugs in the etomidate group (χ2=5.070, P=0.024), but no significant difference was found in the incidence of complications between the two groups, χ2=3.670, P=0.055. CONCLUSION: Compared to propofol, the application of etomidate combined with BIS feedback closed-loop target-controlled infusion in spinal surgery anesthesia for elderly patients can keep hemodynamics in a stable state, without affecting postoperative resuscitation, showing high safety, so it is worthy of clinical application.

Spatiotemporal variations and driving factors for potential wind erosion on the Mongolian Plateau.

Wind erosion can cause desertification and sandstorms in arid and semiarid areas. However, quantitative studies of the dynamic changes in wind erosion over long time periods are relatively rare, and this knowledge gap hinders our understanding of desertification under the conditions of a changing climate. Here, we selected the Mongolian Plateau as the study area. Using the revised wind erosion equation (RWEQ) model, we assessed the spatial and temporal dynamics of wind erosion on the Mongolian Plateau from 1982 to 2018. Our results showed that the wind erosion intensity on the Mongolian Plateau increased from northeast to southwest. The annual mean wind erosion modulus was 46.5 t·ha-1 in 1982-2008, with a significant decline at a rate of -5.1 t·ha-1·10 yr-1. The intensity of wind erosion was the strongest in spring, followed by autumn and summer, and was weakest in winter. During 1982-2018, wind erosion showed a significant decreasing trend in all seasons except winter. The wind erosion contribution of spring to the total annual wind erosion significantly increased, while that of summer significantly decreased. These results can help decision-makers identify high-risk areas of soil erosion on the Mongolian Plateau and take effective measures to adapt to climate change.

The Dialectic and Coupling of Three Paradigms in the Process of Art Rural Construction: Environment, Culture, and Industry.

The development and construction of cities and villages are a complex process jointly promoted by multiple subjects such as government, residents, enterprises, planners, and community elites. Through the summary and analysis of the practice of art rural construction at home and abroad, it is found that its events show three main paradigms in the development process. The environmental creation paradigm reconstructs the environmental semantics of the countryside through the creation of environmental art works, showing the remarkable characteristics of "the presence of works." The cultural revival paradigm maintains the local culture through cooperation with residents in art and has the characteristics of "de-artization " and "de-heritage." The industrial development paradigm adjusts the industrial structure through the production of commodity art to achieve the improvement of the rural economic foundation. Under the new development trend, the three paradigms are gradually converging.

One Stone for Multiple Birds: A Versatile Cross-Linked Poly(dimethyl siloxane) Binder Boosts Cycling Life and Rate Capability of an NCM 523 Cathode at 4.6 V.

Increasing working voltage is a promising way to increase the energy density of lithium-ion batteries. Cycling and rate performance deteriorated due to excessive electrolyte decomposition and uncontrolled formation of a cathode-electrolyte interface (CEI) layer at a high voltage. A new concept is proposed to construct a high-voltage-stable electrode-electrolyte interface. An elastomeric poly(dimethyl siloxane) (PDMS) binder is incorporated into the electrode to modify the LiNi0.5Co0.2Mn0.3O2 (NCM 523) particle surface via an in situ cross-linking reaction between hydroxy-terminated PDMS and methyl trimethoxy silane promoted by moisture at ambient conditions (MPDMS). Improved electrochemical performance is achieved with the MPDMS binder in terms of reversible capacity (201 vs 185 mAh·g-1 at 0.2C), capacity retention (80 vs 68%, after 300 cycles at 1C), and rate performance (55.6% increase at 5C), as demonstrated by the NCM 523||Li half-cell. The NCM 523||graphite full-cell also shows improved performance at 4.6 V (147 vs 128 mAh·g-1, 82 vs 76%, after 200 cycles at 1C). The mechanism studies indicate that MPDMS exerts multiple effects, including cathode surface passivation, solvation structure tuning, electrolyte uptake enhancement, and mechanical stress relief. This work provides an inspiring route to realize high-voltage application of lithium-ion battery technology.

Integrated single-cell multiomics analysis reveals novel candidate markers for prognosis in human pancreatic ductal adenocarcinoma.

The epigenomic abnormality of pancreatic ductal adenocarcinoma (PDAC) has rarely been investigated due to its strong heterogeneity. Here, we used single-cell multiomics sequencing to simultaneously analyze the DNA methylome, chromatin accessibility and transcriptome in individual tumor cells of PDAC patients. We identified normal epithelial cells in the tumor lesion, which have euploid genomes, normal patterns of DNA methylation, and chromatin accessibility. Using all these normal epithelial cells as controls, we determined that DNA demethylation in the cancer genome was strongly enriched in heterochromatin regions but depleted in euchromatin regions. There were stronger negative correlations between RNA expression and promoter DNA methylation in cancer cells compared to those in normal epithelial cells. Through in-depth integrated analyses, a set of novel candidate biomarkers were identified, including ZNF667 and ZNF667-AS1, whose expressions were linked to a better prognosis for PDAC patients by affecting the proliferation of cancer cells. Our work systematically revealed the critical epigenomic features of cancer cells in PDAC patients at the single-cell level.

White Matter Metabolite Relaxation and Diffusion Abnormalities in First-Episode Psychosis: A Longitudinal Study.

Microstructural abnormalities in the white matter (WM) are implicated in the pathophysiology of psychosis. In vivo magnetic resonance spectroscopy (MRS) can probe the brain's intracellular microenvironment through the measurement of transverse relaxation and diffusion of neurometabolites and possibly provide cell-specific information. In our previous studies, we observed differential metabolite signal abnormalities in first episode and chronic stages of psychosis. In the present work, longitudinal data were presented for the first time on white matter cell-type specific abnormalities using a combination of diffusion tensor spectroscopy (DTS), T2 MRS, and diffusion tensor imaging (DTI) from a group of 25 first episode psychosis patients and nine matched controls scanned at baseline and one and two years of follow-up. We observed significantly reduced choline ADC in the year 1 of follow-up (0.194 µm2/ms) compared to baseline (0.229 µm2/ms), followed by a significant increase in NAA ADC in the year 2 follow-up (0.258 µm2/ms) from baseline (0.222 µm2/ms) and year 1 follow-up (0.217 µm2/ms). In contrast, NAA T2 relaxation, reflecting a related but different aspect of microenvironment from diffusion, was reduced at year 1 follow-up (257 ms) compared to baseline (278 ms). These abnormalities were observed in the absence of any abnormalities in water relaxation and diffusion at any timepoint. These findings indicate that abnormalities are seen in in glial-enriched (choline) signals in early stages of psychosis, followed by the subsequent emergence of neuronal-enriched (NAA) diffusion abnormalities, all in the absence of nonspecific water signal abnormalities.

Heterogeneity in endothelial cells and widespread venous arterialization during early vascular development in mammals.

Arteriogenesis rather than unspecialized capillary expansion is critical for restoring effective circulation to compromised tissues in patients. Deciphering the origin and specification of arterial endothelial cells during embryonic development will shed light on the understanding of adult arteriogenesis. However, during early embryonic angiogenesis, the process of endothelial diversification and molecular events underlying arteriovenous fate settling remain largely unresolved in mammals. Here, we constructed the single-cell transcriptomic landscape of vascular endothelial cells (VECs) during the time window for the occurrence of key vasculogenic and angiogenic events in both mouse and human embryos. We uncovered two distinct arterial VEC types, the major artery VECs and arterial plexus VECs, and unexpectedly divergent arteriovenous characteristics among VECs that are located in morphologically undistinguishable vascular plexus intra-embryonically. Using computational prediction and further lineage tracing of venous-featured VECs with a newly developed Nr2f2CrexER mouse model and a dual recombinase-mediated intersectional genetic approach, we revealed early and widespread arterialization from the capillaries with considerable venous characteristics. Altogether, our findings provide unprecedented and comprehensive details of endothelial heterogeneity and lineage relationships at early angiogenesis stages, and establish a new model regarding the arteriogenesis behaviors of early intra-embryonic vasculatures.

Single-cell multi-omics sequencing and its applications in studying the nervous system.

Single-cell sequencing has become one of the most powerful and popular techniques in dissecting molecular heterogeneity and modeling the cellular architecture of a biological system. During the past twenty years, the throughput of single-cell sequencing has increased from hundreds of cells to over tens of thousands of cells in parallel. Moreover, this technology has been developed from sequencing transcriptome to measure different omics such as DNA methylome, chromatin accessibility, and so on. Currently, multi-omics which can analyze different omics in the same cell is rapidly advancing. This work advances the study of many biosystems, including the nervous system. Here, we review current single-cell multi-omics sequencing techniques and describe how they improve our understanding of the nervous system. Finally, we discuss the open scientific questions in neural research that may be answered through further improvement of single-cell multi-omics sequencing technology.

Nuclear peripheral chromatin-lamin B1 interaction is required for global integrity of chromatin architecture and dynamics in human cells.

The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.

Review of Metabolomics-Based Biomarker Research for Parkinson's Disease.

Parkinson's disease (PD), as the second most common neurodegenerative disease, is seriously affecting the life quality of the elderly. However, there is still a lack of efficient medical methods to diagnosis PD before apparent symptoms occur. In recent years, clinical biomarkers including genetic, imaging, and tissue markers have exhibited remarkable benefits in assisting PD diagnoses. Due to the advantages of high-throughput detection of metabolites and almost non-invasive sample collection, metabolomics research of PD is widely used for diagnostic biomarker discovery. However, there are also a few shortages for those identified biomarkers, such as the scarcity of verifications regarding the sensitivity and specificity. Thus, reviewing the research progress of PD biomarkers based on metabolomics techniques is of great significance for developing PD diagnosis. To comprehensively clarify the progress of current metabolic biomarker studies in PD, we reviewed 20 research articles regarding the discovery and validation of biomarkers for PD diagnosis from three mainstream academic databases (NIH PubMed, ISI Web of Science, and Elsevier ScienceDirect). By analyzing those materials, we summarized the metabolic biomarkers identified by those metabolomics studies and discussed the potential approaches used for biomarker verifications. In conclusion, this review provides a comprehensive and updated overview of PD metabolomics research in the past two decades and particularly discusses the validation of disease biomarkers. We hope those discussions might provide inspiration for PD biomarker discovery and verification in the future.

Relationship between non-culprit lesion plaque characteristics changes and in-stent neoatherosclerosis formation: 1-year follow-up optical coherence tomography study.

The relationship between the in-stent neoatherosclerosis (ISNA) formation and the plaque's characteristic changes in the non-culprit lesion is unclear. We aim to investigate the plaque characteristics changes at non-culprit lesions between patients with ISNA and without ISNA formation at 1-year follow-up. We retrospectively enrolled patients who had DES implantation in de novo lesion and underwent immediately after stenting and 1-year follow-up optical coherence tomography (OCT) examination. OCT-defined ISNA was defined as the presence of lipid-laden neointima or calcification within the culprit stent with a longitudinal extension of ≥1 mm. Non-culprit lesions were divided into two groups: ISNA group (with ISNA) and non-ISNA group (without ISNA). Plaque characteristics of non-culprit lesions were evaluated at baseline and 1-year follow-up. In total, 89 patients with 89 non-culprit lesions (ISNA: n = 37; non-ISNA: n = 52) were included in the analyses. The lesions in the ISNA group show a smaller minimum lumen area compared to the non-ISNA group at 1-year follow-up (2.57 ± 1.08 mm2 versus 3.20 ± 1.62 mm2, p = 0.044). The lesions of the ISNA group show a significant decrease in minimum lumen area changes percent (-7.25% versus 6.46%, p = 0.039). And there are more lesions with minimum lumen area (64.9% versus 38.5%, p = 0.014) and minimum lumen diameter (64.9% versus 40.4%, p = 0.023) decrease in the ISNA group. Furthermore, the lesions in ISNA group have more plaques with lipid core length increase (25.0% versus 10.0%, p = 0.040), more plaques with FCT decrease (50.0% versus 74.0%, p = 0.027) and less TCFA change to non-TCFA (33.3% versus 87.5%, p = 0.010). The plaque characteristic changes in non-culprit lesions are closely related to ISNA formation. The ISNA formation may accompany by a tardier plaque stabilization process in non-culprit lesions.