Rapid and multi-target genotyping of Helicobacter pylori with digital microfluidics.

Helicobacter pylori (H. pylori) infection correlates closely with gastric diseases such as gastritis, ulcers, and cancer, influencing more than half of the world's population. Establishing a rapid, precise, and automated platform for H. pylori diagnosis is an urgent clinical need and would significantly benefit therapeutic intervention. Recombinase polymerase amplification (RPA)-CRISPR recently emerged as a promising molecular diagnostic assay due to its rapid detection capability, high specificity, and mild reaction conditions. In this work, we adapted the RPA-CRISPR assay on a digital microfluidics (DMF) system for automated H. pylori detection and genotyping. The system can achieve multi-target parallel detection of H. pylori nucleotide conservative genes (ureB) and virulence genes (cagA and vacA) across different samples within 30 min, exhibiting a detection limit of 10 copies/rxn and no false positives. We further conducted tests on 80 clinical saliva samples and compared the results with those derived from real-time quantitative polymerase chain reaction, demonstrating 100% diagnostic sensitivity and specificity for the RPA-CRISPR/DMF method. By automating the assay process on a single chip, the DMF system can significantly reduce the usage of reagents and samples, minimize the cross-contamination effect, and shorten the reaction time, with the additional benefit of losing the chance of experiment failure/inconsistency due to manual operations. The DMF system together with the RPA-CRISPR assay can be used for early detection and genotyping of H. pylori with high sensitivity and specificity, and has the potential to become a universal molecular diagnostic platform.

Post-infarction ventricular septal rupture complicated with cardiogenic shock and multiple organ hemorrhage: An autopsy case report.

Ventricular septal rupture (VSR) is a catastrophic mechanical complication of acute myocardial infarction (AMI) that can result in acute heart failure. Delaying operative intervention frequently leads to cardiogenic shock and multi-organ failure. Here we report a case of massive anterior MI complicated with VSR that was discovered through cardiac Doppler ultrasound and suspected multiple organ hemorrhage. The patient showed signs of rapid cardiogenic shock and eventually died. The morphological changes of VSR and MI were identified during necropsy, and microscopic examinations of the heart, brain, and kidney revealed multiple organ hemorrhage. This autopsy case suggested that the complication of VSR caused by AMI results in a reduction of oxygen and nutrient content of the circulating blood throughout the body and, eventually, functional failure of multiple organs. We provide clinical and pathological evidence elucidating changes in multiple organs under the severe condition of post-infarction VSR and demonstrate the consequences of a lack of immediate surgery and sufficient medical intervention for a patient suffering from AMI with VSR.

A fluorescent nano vector for early diagnosis and enhanced Interleukin-33 therapy of thoracic aortic dissection.

Thoracic aortic dissection (TAD) is the most devastating complication of vascular disease. The accuracy of the clinical diagnosis and treatment of TAD at the early stage is still limited. Herein, we report a nano-delivery strategy for early diagnosis and the first case of interleukin-33 (IL-33) based therapy for the effective intervention of TAD. A targeted fluorescent nano vector (FNV) is designed to co-assemble with IL-33, which protects IL-33 and prolongs its half-life. With specific targeting ability to the thoracic aorta, FNV can diagnose TAD at its early stage through fluorescent imaging. FNV@IL-33 nanocomplex presents better therapeutic effects on mice TAD progression compared with that of IL-33 alone by reducing smooth muscle apoptosis. Administration of FNV@IL-33 two weeks before onset, the development of TAD is greatly intervened. Our study provides a novel approach for early diagnosis and effective IL-33 therapy of TAD, which opens attractive opportunities for clinical prevention of cardiovascular diseases.

Machine Learning-Based Personalized Risk Prediction Model for Mortality of Patients Undergoing Mitral Valve Surgery: The PRIME Score.

Background: Mitral valve surgery (MVS) is an effective treatment for mitral valve diseases. There is a lack of reliable personalized risk prediction models for mortality in patients undergoing mitral valve surgery. Our aim was to develop a risk stratification system to predict all-cause mortality in patients after mitral valve surgery. Methods: Different machine learning models for the prediction of all-cause mortality were trained on a derivation cohort of 1,883 patients undergoing mitral valve surgery [split into a training cohort (70%) and internal validation cohort (30%)] to predict all-cause mortality. Forty-five clinical variables routinely evaluated at discharge were used to train the models. The best performance model (PRIME score) was tested in an externally validated cohort of 220 patients undergoing mitral valve surgery. The model performance was evaluated according to the area under the curve (AUC). Net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were compared with existing risk strategies. Results: After a median follow-up of 2 years, there were 133 (7.063%) deaths in the derivation cohort and 17 (7.727%) deaths in the validation cohort. The PRIME score showed an AUC of 0.902 (95% confidence interval [CI], 0.849-0.956) in the internal validation cohort and 0.873 (95% CI: 0.769-0.977) in the external validation cohort. In the external validation cohort, the performance of the PRIME score was significantly improved compared with that of the existing EuroSCORE II (NRI = 0.550, [95% CI 0.001-1.099], P = 0.049, IDI = 0.485, [95% CI 0.230-0.741], P < 0.001). Conclusion: Machine learning-based model (the PRIME score) that integrate clinical, demographic, imaging, and laboratory features demonstrated superior performance for the prediction of mortality patients after mitral valve surgery compared with the traditional risk model EuroSCORE II. Clinical Trial Registration: [http://www.clinicaltrials.gov], identifier [NCT05141292].

MicroRNA-27b-3p down-regulates FGF1 and aggravates pathological cardiac remodelling.

AIMS: The heart undergoes pathological remodelling under increased stress and neuronal imbalance. MicroRNAs (miRNAs) are involved in post-transcriptional regulation of genes in cardiac physiology and pathology. However, the mechanisms underlying miRNA-mediated regulation of pathological cardiac remodelling remain to be studied. This study aimed to explore the function of endogenous microRNA-27b-3p (miR-27b-3p) in pathological cardiac remodelling. METHODS AND RESULTS: miR-27b-3p expression was elevated in the heart of a transverse aortic constriction (TAC)-induced cardiac hypertrophy mouse model. miR-27b-knockout mice showed significantly attenuated cardiac hypertrophy, fibrosis, and inflammation induced by two independent pathological cardiac hypertrophy models, TAC and Angiotensin II (Ang II) perfusion. Transcriptome sequencing analysis revealed that miR-27b deletion significantly down-regulated TAC-induced cardiac hypertrophy, fibrosis, and inflammatory genes. We identified fibroblast growth factor 1 (FGF1) as a miR-27b-3p target gene in the heart which was up-regulated in miR-27b-null mice. We found that both recombinant FGF1 (rFGF1) and inhibition of miR-27b-3p enhanced mitochondrial oxidative phosphorylation (OXPHOS) and inhibited cardiomyocyte hypertrophy. Importantly, rFGF1 administration inhibited cardiac hypertrophy and fibrosis in TAC- or Ang II-induced models and enhanced OXPHOS by activating PGC1α/ß. CONCLUSIONS: Our study demonstrated that miR-27b-3p induces pathological cardiac remodelling and suggests that inhibition of endogenous miR-27b-3p or administration of FGF1 might have the potential to suppress cardiac remodelling in a clinical setting.

Age-related decline of interferon-gamma responses in macrophage impairs satellite cell proliferation and regeneration.

BACKGROUND: Impaired muscle regeneration and increased muscle fibrosis are observed in aged muscle accompanied by progressive loss of muscle mass (sarcopenia). However, the underlying mechanism is still unclear. METHODS: The differentiated expressed genes in young and aged muscles after acute injury by cardiotoxin were identified by RNA-sequence analysis. Single-cell RNA-sequence analysis was used to identify cell clusters and functions in young muscle after acute injury, and flow cytometry analysis and sorting were used to validate the function. The proliferation and differentiation functions of satellite cells were accessed by immunostaining with 5-ethynyl-2'-deoxyuridine and embryonic myosin heavy chain (eMyHC), respectively. Muscle regeneration ability was accessed by histopathological and molecular biological methods. RESULTS: Gene expression patterns associated with responses to interferon-gamma (IFN-γ) (15 genes; false discovery rate < 0.001) were significantly down-regulated during muscle regeneration in aged mice (P = 2.25e-7). CD8+ T cells were the main source of increased IFN-γ after injury, adoptive transfer of wild-type CD8+ T cells to IFN-γ-deficient young mice resulted in 78% increase in cross-sectional areas (CSAs) of regenerated myofibres (P < 0.05) and 63% decrease in muscle fibrosis (P < 0.05) after injury. Single-cell RNA-sequence analysis identified a novel subset of macrophages [named as IFN-responsive macrophages (IFNRMs)] that specifically expressed IFN-responsive genes (Ifit3, Isg15, Irf7, etc.) in young mice at 3 days after injury, and the number of this macrophage subset was ~20% lower in aged mice at the same time (P < 0.05). IFNRMs secreted cytokine C-X-C motif chemokine 10 (CXCL10) that promoted the proliferation and differentiation of satellite cells via its receptor, CXCR3. Intramuscular recombinant CXCL10 treatment in aged mice rejuvenated the proliferation of satellite cells (80% increase in Ki-67+ Pax7+ cells, P < 0.01) and resulted in 27% increase in CSA of regenerated myofibres (P < 0.01) and 29% decrease in muscle fibrosis (P < 0.05). CONCLUSIONS: Our study indicates that decline in IFN-γ response in a novel subset of macrophage contributes to satellite cells dysfunctions in aged skeletal muscles and demonstrates that this mechanism can be targeted to restore age-associated myogenesis.

Effects of Extracellular Matrix Softening on Vascular Smooth Muscle Cell Dysfunction.

Vascular smooth muscle cells (VSMCs) shift from a physiological contractile phenotype to an adverse proliferative or synthetic state, which is a major event leading to aortic disease. VSMCs are exposed to multiple mechanical signals from their microenvironment including vascular extracellular matrix (ECM) stiffness and stretch which regulate VSMC contraction. How ECM stiffness regulates the function and phenotype of VSMCs is not well understood. In this study, we introduce in vitro and in vivo models to evaluate the impact of ECM stiffnesses on VSMC function. Through unbiased transcriptome sequencing analysis, we detected upregulation of synthetic phenotype-related genes including osteopontin, matrix metalloproteinases, and inflammatory cytokines in VSMCs cultured using soft matrix hydrogels in vitro, suggesting VSMC dedifferentiation toward a synthetic phenotype upon ECM softening. For the in vivo model, the lysyl oxidase inhibitor ß-aminopropionitrile monofumarate (BAPN) was administrated to disrupt the cross-linking of collagen to induce ECM softening. Consistently, decreased ECM stiffnesses promoted VSMC phenotypic switching to a synthetic phenotype as evidenced by upregulation of synthetic phenotype-related genes in the aortas of mice following BAPN treatment. Finally, BAPN-treated mice showed severe expansion and developed aortic dissection. Our study reveals the pivotal role of ECM softening in regulating the VSMC phenotype switch and provides a potential target for treating VSMC dysfunction and aortic dissection disease.

Reconstruction of a lncRNA-Associated ceRNA Network in Endothelial Cells under Circumferential Stress.

BACKGROUND: Numerous studies have highlighted that long noncoding RNA (lncRNA) can indirectly regulate the expression of mRNAs by binding to microRNA (miRNA). LncRNA-associated ceRNA networks play a vital role in the initiation and progression of several pathological mechanisms. However, the lncRNA-miRNA-mRNA ceRNA network in endothelial cells under cyclic stretch is seldom studied. METHODS: The miRNA, mRNA, and lncRNA expression profiles of 6 human umbilical vein endothelial cells (HUVECs) under circumferential stress were obtained by next-generation sequencing (NGS). We identified the differential expression of miRNAs, mRNAs, and lncRNAs using the R software package GDCRNATools. Cytoscape was adopted to construct a lncRNA-miRNA-mRNA ceRNA network. In addition, through GO and KEGG pathway annotations, we analyzed gene functions and their related pathways. We also adopted ELISA and TUNEL to investigate the effect of si-NEAT1 on endothelial inflammation and apoptosis. RESULTS: We recognized a total of 32978 lncRNAs, 1046 miRNAs, and 31958 mRNAs in 6 samples; among them, 155 different expressed lncRNAs, 74 different expressed miRNAs, and 960 different mRNAs were adopted. Based on the established theory, the ceRNA network was composed of 13 lncRNAs, 44 miRNAs, and 115 mRNAs. We constructed and visualized a lncRNA-miRNA-mRNA network, and the top 20 nodes are identified after calculating their degrees. The nodes with most degrees in three kinds of RNAs are hsa-miR-4739, NEAT1, and MAP3K2. Functional analysis showed that different biological processes enriched in biological regulation, response to stimulus and cell communication. Pathway analysis was mainly enriched in longevity regulating, cell cycle, mTOR, and FoxO signaling pathway. Circumferential stress can significantly downregulate NEAT1, and after transducing si-NEAT1 for 24 h, inflammatory cytokine IL-6 and MCP-1 were significantly increased; furthermore, fewer TUNEL-positive cells were found in the si-NEAT1 treated group. CONCLUSIONS: The establishing of a ceRNA network can help further understand the mechanism of vein graft failure. Our data demonstrated that NEAT1 may be a core factor among the mechanical stress factors and that cyclic stress can significantly reduce expression of NEAT1, give rise to inflammation in the early stage of endothelial dysfunction, and promote EC apoptosis, which may play an essential role in vein graft failure.

Functional Nanocomplexes with Vascular Endothelial Growth Factor A/C Isoforms Improve Collateral Circulation and Cardiac Function.

Protein-based therapies are potential treatments for cancer, immunological, and cardiovascular diseases. However, effective delivery systems are needed because of their instability, immunogenicity, and so on. Crosslinked negatively charged heparin polysaccharide nanoparticle (HepNP) is proposed for protein delivery. HepNP can efficiently condense vascular endothelial growth factor (VEGF) because of the unique electronegative sulfonic acid and carboxyl domain of heparin. HepNP is then assembled with VEGF-C (Hep@VEGF-C) or VEGF-A (Hep@VEGF-A) protein for the therapy of myocardial infarction (MI) via intravenous (iv) injection. Hep@VEGF-A-mediated improvement of cardiac function by promoting angiogenesis is limited because of elevated vascular permeability, while Hep@VEGF-C effectively promotes lymphangiogenesis and reduces edema. On this basis, a graded delivery of VEGF-C (0.5-1 h post-MI) and VEGF-A (5 d post-MI) using HepNP is developed. At the dose ratio of 3:1 (Hep@VEGF-C vs Hep@VEGF-A), Hep@VEGF functional complexes substantially reduce the scar formation (≈-39%; p < 0.05) and improve cardiac function (≈+74%; p < 0.05). Such a HepNP delivery system provides a simple and effective therapeutic strategy for cardiovascular diseases by delivering functional proteins. Because of the unique binding ability of heparin with cytokines and growth factors, HepNP also has considerable application prospects in protein therapy for other serious diseases.

The value of plasma fibrillin-1 level in patients with spontaneous coronary artery dissection.

BACKGROUND: Spontaneous coronary artery dissection (SCAD) has emerged as an important etiology of myocardial infarction and sudden death, especially in young women. Early diagnosis is essential for appropriate management. OBJECTIVES: To explore the value of plasma fibrillin-1 (FBN1) levels in patients with SCAD. METHODS: 70 patients with non-atherosclerotic SCAD between January 2014 and September 2018 were age and sex matched with 70 patients with non-SCAD acute coronary syndrome (ACS) and 70 healthy controls. The plasma FBN1 level was measured and compared among three groups. The value of FBN1 for prognosis and treatment decision making was further explored. RESULTS: The plasma FBN1 level of SCAD group (58.44 ± 7.06 ng/mL) was higher than that of non-SCAD ACS group (52.39 ± 6.92 ng/mL, P < 0.001) or healthy controls (50.56 ± 4.48 ng/mL, P < 0.001). Compared with controls, significantly higher percentages of patients with SCAD were found in the highest compared with lowest quartile of FBN1 concentration. The area under the curve (AUC) for plasma FBN1 level to discriminate patients with SCAD from non-SCAD ACS was 0.81 (95% CI 0.74-0.88, P < 0.001). A cut-off value of 54.64 ng/mL was determined to differentiate SCAD from non-SCAD ACS with a sensitivity of 0.77 (95%CI: 0.66-0.86) and specificity of 0.76 (95%CI: 0.64-0.85). After a median follow-up of 28.35 (14.07 ± 44.69) months, 11 (15.7%) cases suffered from major adverse cardiac events (MACE). Higher FBN1 level was detected in patients with MACE (63.71 ± 7.49 vs. 57.45 ± 6.58 ng/mL) (P = 0.006). A cut-point of 58.14 was determined for SCAD patients to identify MACE. At this point, FBN1 might also have potential use for decision making in SCAD patients. CONCLUSION: Plasma FBN1 is a promising biomarker for aiding the diagnosis of SCAD and have potential value in prognosis prediction.

Very Late Stent Thrombosis in Drug-Eluting Stents New Observations and Clinical Implications.

Despite the reduction in stent failure with newer-generation drug-eluting stents, very late stent thrombosis (VLST) remains an unpredictable and potentially catastrophic complication of coronary revascularization procedures and is associated with high morbidity and mortality. Here, we present an updated overview of the latest advances in understanding the causes of VLST. Clinical studies that revealed potential risk factors and pathophysiologic studies on the mechanisms of VLST are discussed. Importantly, novel insights from recent advances in intravascular imaging are included. To date, there is no clinical guideline for VLST treatment. We propose an evidence-based recommendation that an intravascular-imaging-informed percutaneous coronary intervention strategy combined with optimized antiplatelet therapy is the foundation for successful VLST clinical management. Moreover, the future of VLST prevention, such as improved patient risk stratification and advances in addressing late stent failure, are also discussed.

Association of Soluble ST2 Serum Levels With Outcomes in Pediatric Dilated Cardiomyopathy.

BACKGROUND: Prognosis in patients with pediatric dilated cardiomyopathy (PDCM) is urgently required to identify high-risk patients. Elevated soluble ST2 (sST2) is associated with prognosis in adult patients with heart failure. This study aimed to assess the prognostic value of sST2 in PDCM. METHODS: Ninety-four patients with PDCM were enrolled after admission from 2 centres in China and followed up for adverse events (death, cardiac transplantation, and heart-failure-related rehospitalization). B-type natriuretic peptide (BNP) and sST2 levels were measured. RESULTS: Over a median of 678 (interquartile range [IQR]: 533-785) days, 28 (29.8%) adverse events occurred. Patients in the highest tertile of sST2 levels had increased risk of short-term (< 6 months) (adjusted hazard ratio [HR]: 8.36, 95% confidence interval [CI], 1.02-73.52; P < 0.05) and long-term adverse events (2 years) (adjusted HR: 4.23; 95% CI, 1.32-13.60; P < 0.01) than those in lower tertiles. The C-statistic was increased with addition of sST2 to BNP from 0.697 (95% CI, 0.541-0.852) to 0.812 (95% CI, 0.697-0.939) for short-term and from 0.712 (95% CI, 0.604-0.819) to 0.798 (95% CI, 0.697-0.899) for prediction of long-term adverse events. An intermediate-risk subgroup was identified, and 24% had adverse events. When serial measurements were taken in a nested case-control subgroup, sST2 levels were constantly high in patients with late adverse events (> 6 months) but gradually decreased in nonadverse-event controls compared with 3-month and 6-month baseline levels. CONCLUSIONS: In patients with PDCM, serum sST2 levels are associated with adverse events and have robust prognostic value. Serial measurements of sST2 could help in managing patients for monitoring outcomes of treatment.

S100a8/a9 Signaling Causes Mitochondrial Dysfunction and Cardiomyocyte Death in Response to Ischemic/Reperfusion Injury.

BACKGROUND: Myocardial ischemia-reperfusion (MI/R) injury is a significant clinical problem without effective therapy. Unbiased omics approaches may reveal key MI/R mediators to initiate MI/R injury. METHODS: We used a dynamic transcriptome analysis of mouse heart exposed to various MI/R periods to identify S100a8/a9 as an early mediator. Using loss/gain-of-function approaches to understand the role of S100a8/a9 in MI/R injury, we explored the mechanisms through transcriptome and functional experiment. Dynamic serum S100a8/a9 levels were measured in patients with acute myocardial infarction before and after percutaneous coronary intervention. Patients were prospectively followed for the occurrence of major adverse cardiovascular events. RESULTS: S100a8/a9 was identified as the most significantly upregulated gene during the early reperfusion stage. Knockout of S100a9 markedly decreased cardiomyocyte death and improved heart function, whereas hematopoietic overexpression of S100a9 exacerbated MI/R injury. Transcriptome/functional studies revealed that S100a8/a9 caused mitochondrial respiratory dysfunction in cardiomyocytes. Mechanistically, S100a8/a9 downregulated NDUF gene expression with subsequent mitochondrial complex I inhibition via Toll-like receptor 4/Erk-mediated Pparg coactivator 1 alpha/nuclear respiratory factor 1 signaling suppression. Administration of S100a9 neutralizing antibody significantly reduced MI/R injury and improved cardiac function. Finally, we demonstrated that serum S100a8/a9 levels were significantly increased 1 day after percutaneous coronary intervention in patients with acute myocardial infarction, and elevated S100a8/a9 levels were associated with the incidence of major adverse cardiovascular events. CONCLUSIONS: Our study identified S100a8/a9 as a master regulator causing cardiomyocyte death in the early stage of MI/R injury via the suppression of mitochondrial function. Targeting S100a8/a9-intiated signaling may represent a novel therapeutic intervention against MI/R injury. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03752515.

Relationships between circulating branched chain amino acid concentrations and risk of adverse cardiovascular events in patients with STEMI treated with PCI.

The incidence of in-hospital cardiovascular adverse events (AEs) in patients with ST-segment elevation myocardial infarction (STEMI) following primary percutaneous coronary intervention (PCI) is relatively high. Identification of metabolic markers could improve our understanding of the underlying pathological changes in these patients. We aimed to identify associations between concentrations of plasma metabolites on admission and development of in-hospital AEs in post-PCI patients with STEMI. We used targeted mass spectrometry to measure plasma concentrations of 26 amino acid metabolites on admission in 96 patients with STEMI who subsequently developed post-PCI AEs and in 96 age- and sex-matched patients without post-PCI cardiovascular AEs. Principal component analysis (PCA) revealed that PCA-derived factors, including branched chain amino acids (BCAAs), were associated with increased risks of all three pre-specified outcomes: cardiovascular mortality/acute heart failure (AHF), cardiovascular mortality, and AHF. Addition of BCAA to the Global Registry of Acute Coronary Events risk score increased the concordance C statistic from 0.702 to 0.814 (p < 0.001), and had a net reclassification index of 0.729 (95% confidence interval, 0.466-0.992, p < 0.001). These findings demonstrate that high circulating BCAA concentrations on admission are associated with subsequent in-hospital AEs after revascularization in patients with STEMI.

Unlockable Nanocomplexes with Self-Accelerating Nucleic Acid Release for Effective Staged Gene Therapy of Cardiovascular Diseases.

Nucleic acid (NA)-based therapy is proposed to address serious diseases such as cardiovascular diseases (CVDs). Powerful NA delivery vehicles are essential for effective gene therapy. Herein, a novel type of delivery vehicle, an unlockable core-shell nanocomplex (Hep@PGEA) with self-accelerating NA release, is structurally designed. Hep@PGEA is composed of disulfide-bridged heparin nanoparticle (HepNP) core and low-toxicity PGEA cationic shell. In comparison with NA, heparin, a negatively charged polysaccharide macromolecule, exhibits stronger interactions with cationic species. Upon the breakdown of redox-responsive HepNP cores, unlocked heparin would interact with the outer cationic shells and replace the condensed NA to facilitate NA release. Such unique Hep@PGEA is successfully explored for effective miRNA-pDNA staged gene therapy of myocardial infarction (MI), one of the most serious CVDs. With the progression of MI, glutathione amounts in heart tissues increase. MiR-499 (for the inhibition of cardiomyocyte apoptosis) and plasmid encoding vascular endothelial growth factor (for the promotion of angiogenesis) are sequentially delivered for systemic treatment of MI. Such treatment produces impressive results in restoring heart function and suppressing cardiac hypertrophy. Due to the wide existence of redox agents in cells, the proposed unlockable delivery nanovehicle and staged therapy strategy can provide new methods to effectively treat different serious diseases.