Non-invasive screening of bladder cancer using digital microfluidics and FLIM technology combined with deep learning.

Non-invasive screening for bladder cancer is crucial for treatment and postoperative follow-up. This study combines digital microfluidics (DMF) technology with fluorescence lifetime imaging microscopy (FLIM) for urine analysis and introduces a novel non-invasive bladder cancer screening technique. Initially, the DMF was utilized to perform preliminary screening and enrichment of urine exfoliated cells from 54 participants, followed by cell staining and FLIM analysis to assess the viscosity of the intracellular microenvironment. Subsequently, a deep learning residual convolutional neural network was employed to automatically classify FLIM images, achieving a three-class prediction of high-risk (malignant), low-risk (benign), and minimal risk (normal) categories. The results demonstrated a high consistency with pathological diagnosis, with an accuracy of 91% and a precision of 93%. Notably, the method is sensitive for both high-grade and low-grade bladder cancer cases. This highly accurate non-invasive screening method presents a promising approach for bladder cancer screening with significant clinical application potential.

Enhancing the photodynamic effect of curcumin through modification with TiO2 nanoparticles and cationic polymers.

Curcumin (CUR), a natural compound extracted from turmeric, has shown potential as a photosensitizer in photodynamic therapy (PDT). The aim of this work was to enhance the efficacy of CUR by modifying it using titanium dioxide (TiO2) nanoparticles and a cationic polymer called Sofast to create a nanocomposite TiO2-CUR-Sofast (TCS). Compared to unmodified CUR, TCS exhibited a broadening toward longer wavelength in the absorption wavelength within the 400-550 nm range, leading to improved CUR absorption. Cellular uptake efficiency of TCS was also enhanced, and it demonstrated nearly 4.7-fold higher reactive oxygen species (ROS) generation than CUR. Furthermore, TCS displayed the ability to attach to the cell membrane and enter cells within a 30-min incubation period. Upon irradiation, TCS exhibited remarkable cytotoxicity, resulting in a significant reduction in the viability of various cancer cells. Autofluorescence lifetime imaging of intracellular reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) enzymes indicated that cancer cells treated with TCS and irradiation undergo a metabolic pathway shift from oxidative phosphorylation to glycolysis. These findings highlight the potential of TCS as an effective PDT agent for cancer treatment.

Clinical Features of Patients with Myelin Oligodendrocyte Glycoprotein Antibody-Associated Disease and Isolated Seizure Symptoms.

Background: Myelin oligodendrocyte glycoprotein (MOG) antibody-associated encephalitis is a new clinical phenotype of inflammatory demyelinating diseases. Some MOG antibody-positive patients with central nervous system demyelinating events present with isolated seizures. However, there are gaps in the epidemiological knowledge regarding seizures with MOG antibody-associated encephalitis in adults. This study characterized the clinical features and treatment of MOG antibody-positive patients with isolated seizures. Methods: We reviewed all the patients admitted to Tianjin Huanhu Hospital between Jan. 1st 2017 and Jan. 1st 2022, to screen the MOG antibody-positive patients with isolated seizures, and collected the concerned patients' information regarding epidemiology, clinical presentations, laboratory and radiological characteristics, electroencephalogram (EEG), treatments, and prognoses. Results: We collected six MOG antibody-positive adult patients who had isolated symptomatic seizures. The mean age of the patients was 33 years (range, 29-40 years), and five (83.3%) were men. All patients presented with motor seizures, five (83.3%) had cognitive dysfunction, and only one (16.7%) had status epilepticus. Five (83.3%) patients had a good response to immunotherapy and antiseizure medications; only one had a sequela. The cerebrospinal fluid or serum anti-MOG antibody test turned negative over time. Discussion: The most common seizure type in patients with MOG antibody-associated encephalitis with isolated seizures was focal to bilateral tonic-clonic seizures, and most patients had a good prognosis. Adding antiseizure medications were beneficial for MOG antibody-positive patients with seizures. Relapses and sequelae were associated with low-dose, short-time, or delayed therapy, and wide-range demyelinating brain damage.

Evaluating Differentiation Status of Mesenchymal Stem Cells by Label-Free Microscopy System and Machine Learning.

Mesenchymal stem cells (MSCs) play a crucial role in tissue engineering, as their differentiation status directly affects the quality of the final cultured tissue, which is critical to the success of transplantation therapy. Furthermore, the precise control of MSC differentiation is essential for stem cell therapy in clinical settings, as low-purity stem cells can lead to tumorigenic problems. Therefore, to address the heterogeneity of MSCs during their differentiation into adipogenic or osteogenic lineages, numerous label-free microscopic images were acquired using fluorescence lifetime imaging microscopy (FLIM) and stimulated Raman scattering (SRS), and an automated evaluation model for the differentiation status of MSCs was built based on the K-means machine learning algorithm. The model is capable of highly sensitive analysis of individual cell differentiation status, so it has great potential for stem cell differentiation research.

Anti-CXCR2 antibody-coated nanoparticles with an erythrocyte-platelet hybrid membrane layer for atherosclerosis therapy.

Atherosclerosis is the leading cause of mortality globally. RBC-platelet hybrid membrane-coated nanoparticles ([RBC-P]NPs), which biologically mimic platelets in vivo, display evidence of anti-atherosclerotic activity. The efficacy of a targeted RBC-platelet hybrid membrane-coated nanoparticles ([RBC-P]NP)-based approach was investigated as a primary preventive measure against atherosclerosis. A ligand-receptor interactome analysis conducted with circulating platelets and monocytes derived from CAD patients and healthy controls identified CXCL8-CXCR2 as a key platelet ligand-monocyte receptor dyad in CAD patients. Based on this analysis, a novel anti-CXCR2 [RBC-P]NP that specifically binds to CXCR2 and blocks the interaction between CXCL8 and CXCR2 was engineered and characterized. Administering anti-CXCR2 [RBC-P]NPs to Western diet-fed Ldlr-/- mice led to diminished plaque size, necrosis, and intraplaque macrophage accumulation relative to control [RBC-P]NPs or vehicle. Importantly, anti-CXCR2 [RBC-P]NPs demonstrated no adverse bleeding/hemorrhagic effects. A series of in vitro experiments was conducted to characterize anti-CXCR2 [RBC-P]NP's mechanism of action in plaque macrophages. Mechanistically, anti-CXCR2 [RBC-P]NPs inhibited p38α (Mapk14)-mediated, pro-inflammatory M1 skewing and corrected efferocytosis in plaque macrophages. This targeted [RBC-P]NP-based approach, in which the cardioprotective effects of anti-CXCR2 [RBC-P]NP therapy overweighs its bleeding/hemorrhagic risks, could potentially be used to proactively manage atherosclerotic progression in at-risk populations.

Stroke-induced hexokinase 2 in circulating monocytes exacerbates vascular inflammation and atheroprogression.

BACKGROUND: Stroke accelerates inflammatory monocyte recruitment to the endothelium and consequent atheroprogression via high-mobility group box 1-receptor for advanced glycation end products signaling. Notably, Hmgb1 interacts with multiple toll-like receptors (TLRs) and promotes TLR4-mediated proinflammatory myeloid cell activation. Therefore, TLR-associated mechanism(s) within monocytes may play a role in Hmgb1-driven poststroke atheroprogression. OBJECTIVES: We aimed to elucidate the TLR-associated mechanism(s) within monocytes that contribute to stroke-induced exacerbation of atherosclerotic disease. METHODS: A weighted gene coexpression network analysis on the whole blood transcriptomes of stroke model mice identified hexokinase 2 (HK2) as a key gene associated with TLR signaling in ischemic stroke. We conducted a cross-sectional analysis of monocyte HK2 levels in patients with ischemic stroke patients. We performed in vitro and in vivo studies using high-cholesterol diet-fed myeloid-specific Hk2-null ApoE-/- (ApoE-/-;Hk2ΔMφ) mice and ApoE-/-;Hk2fl/fl controls. RESULTS: We found markedly higher monocyte HK2 levels in patients with ischemic stroke patients during the acute and subacute phases poststroke. Similarly, stroke model mice displayed a profound increase in monocyte Hk2 levels. Using aortas and aortic valve samples collected from high-cholesterol diet-fed ApoE-/-;Hk2ΔMφ mice and ApoE-/-;Hk2fl/fl controls, we found that stroke-induced monocyte Hk2 upregulation enhanced poststroke atheroprogression and inflammatory monocyte recruitment to the endothelium. Stroke-induced monocyte Hk2 upregulation induced inflammatory monocyte activation, systemic inflammation, and atheroprogression via Il-1ß. Mechanistically, we demonstrated that stroke-induced monocyte Hk2 upregulation was dependent upon Hmgb1-driven p38-dependent hypoxia-inducible factor-1α stabilization. CONCLUSION: Stroke-induced monocyte Hk2 upregulation is a key mechanism underlying poststroke vascular inflammation and atheroprogression.

Early Detection of Cervical Cancer by Fluorescence Lifetime Imaging Microscopy Combined with Unsupervised Machine Learning.

Cervical cancer has high morbidity and mortality rates, affecting hundreds of thousands of women worldwide and requiring more accurate screening for early intervention and follow-up treatment. Cytology is the current dominant clinical screening approach, and though it has been used for decades, it has unsatisfactory sensitivity and specificity. In this work, fluorescence lifetime imaging microscopy (FLIM) was used for the imaging of exfoliated cervical cells in which an endogenous coenzyme involved in metabolism, namely, reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H], was detected to evaluate the metabolic status of cells. FLIM images from 71 participants were analyzed by the unsupervised machine learning method to build a prediction model for cervical cancer risk. The FLIM method combined with unsupervised machine learning (FLIM-ML) had a sensitivity and specificity of 90.9% and 100%, respectively, significantly higher than those of the cytology approach. One cancer recurrence case was predicted as high-risk several months earlier using this method as compared to using current clinical methods, implying that FLIM-ML may be very helpful for follow-up cancer care. This study illustrates the clinical applicability of FLIM-ML as a detection method for cervical cancer screening and a convenient tool for follow-up cancer care.

Label-free detection and quantification of ultrafine particulate matter in lung and heart of mouse and evaluation of tissue injury.

While it is known that air borne ultrafine particulate matter (PM) may pass through the pulmonary circulation of blood at the alveolar level between lung and heart and cross the air-blood barrier, the mechanism and effects are not completely clear. In this study the imaging method fluorescence lifetime imaging microscopy is adopted for visualization with high spatial resolution and quantification of ultrafine PM particles in mouse lung and heart tissues. The results showed that the median numbers of particles in lung of mice exposed to ultrafine particulate matter of diameter less than 2.5 µm was about 2.0 times more than that in the filtered air (FA)-treated mice, and about 1.3 times more in heart of ultrafine PM-treated mice than in FA-treated mice. Interestingly, ultrafine PM particles were more abundant in heart than lung, likely due to how ultrafine PM particles are cleared by phagocytosis and transport via circulation from lungs. Moreover, heart tissues showed inflammation and amyloid deposition. The component analysis of concentrated airborne ultrafine PM particles suggested traffic exhausts and industrial emissions as predominant sources. Our results suggest association of ultrafine PM exposure to chronic lung and heart tissue injuries. The current study supports the contention that industrial air pollution is one of the causative factors for rising levels of chronic pulmonary and cardiac diseases.

Single cell sorting of young yeast based on label-free fluorescence lifetime imaging microscopy.

Saccharomyces cerevisiae is an attractive organism used in the fermentation industry and is an important model organism for virus research. The ability to sort yeast cells is important for diverse applications. Replicative aging of Saccharomyces Cerevisiae is accompanied by metabolic changes that are related to an essential coenzyme, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H). Here, a single cell sorting method based on fluorescence lifetime imaging microscopy (FLIM) and laser-induced forward transfer (LIFT) was implemented for the first time. The aging level of yeast was determined based on the FLIM by NAD(P)H, which was a label-free and noninvasive method for studying individual cells. Then, young and active yeast cells were sorted by the LIFT system at the single cell level. During the entire experiment, a sterile and humid environment was maintained to ensure the activity of cells. The high viability of sorted cells was achieved by the LIFT combining with FLIM.

The Transcription Factor SUB1 Is a Master Regulator of the Macrophage TLR Response in Atherosclerosis.

Toll-like receptor 2 and 4 (TLR2, TLR4) signaling is implicated in atherosclerotic plaque formation. The two-stage master regulator Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis of macrophage TLR2 and TLR4 signature genes integrated with coexpression network genes derived from 371 patient-derived carotid specimens identifies activated RNA polymerase II transcriptional coactivator p15 (SUB1/Sub1, PC4) as a master regulon in the atherogenic TLR response. It is found that TLR2 and TLR4 signaling is proinflammatory and proatherosclerotic in chow-fed apolipoprotein E-deficient (ApoE-/- ) mice. Through transgenic myeloid-specific Sub1 knockout in ApoE-/- mice, it is discovered that these proatherosclerotic effects of TLR2 and TLR4 signaling are mediated by Sub1. Sub1 knockout in macrophages enhances anti-inflammatory M2 macrophage polarization and cholesterol efflux. Irradiated low density lipoprotein receptor-deficient (Ldlr-/- ) mice transplanted with Sub1-/- murine bone marrow display reduced atherosclerosis. Promoter analysis reveals Sub1-dependent activation of interferon regulatory factor 1 (Irf1) transcription in a casein kinase 2 (Ck2)-dependent manner, and Sub1-knockout macrophages display decreased Irf1 expression. Artificial Irf1 overexpression in Sub1-knockout macrophages enhances proinflammatory M1 skewing and lowers cholesterol clearance. In conclusion, the TLR master regulon Sub1, and its downstream effect on the transcription factor Irf1, promotes a proinflammatory M1 macrophage phenotype and enhances atherosclerotic burden in vivo.

The function of LncRNA-H19 in cardiac hypertrophy.

Cardiac hypertrophy, characterized by the enlargement of cardiomyocytes, is initially an adaptive response to physiological and pathological stimuli. Decompensated cardiac hypertrophy is related to fibrosis, inflammatory cytokine, maladaptive remodeling, and heart failure. Although pathological myocardial hypertrophy is the main cause of hypertrophy-related morbidity and mortality, our understanding of its mechanism is still poor. Long noncoding RNAs (lncRNAs) are noncoding RNAs that regulate various physiological and pathological processes through multiple molecular mechanisms. Recently, accumulating evidence has indicated that lncRNA-H19 is a potent regulator of the progression of cardiac hypertrophy. For the first time, this review summarizes the current studies about the role of lncRNA-H19 in cardiac hypertrophy, including its pathophysiological processes and underlying pathological mechanism, including calcium regulation, fibrosis, apoptosis, angiogenesis, inflammation, and methylation. The context within which lncRNA-H19 might be developed as a target for cardiac hypertrophy treatment is then discussed to gain better insight into the possible biological functions of lncRNA-H19 in cardiac hypertrophy.

Cardiac Rehabilitation Programs for Chronic Heart Disease: A Bayesian Network Meta-analysis.

BACKGROUND: Cardiac rehabilitation is a medically supervised program after coronary events that involves exercise and dietary modification. We evaluated the comparative benefits and harms of cardiac rehabilitation strategies via a network meta-analysis. METHODS: We followed a pre-specified protocol (PROSPERO: CRD42018094998). We searched Embase, MEDLINE, and Cochrane Central Register of Randomized Trials databases for randomized controlled trials that evaluated cardiac rehabilitation vs a second form of rehabilitation or standard/usual care in adults after myocardial infarction, coronary artery bypass grafting, percutaneous coronary intervention, or angiography. Risk of bias and evidence quality was evaluated using the Cochrane tool and Grading of Recommendations Assessment, Development and Evaluation (GRADE), respectively. Pairwise and Bayesian network meta-analyses were performed for 11 clinical outcomes. RESULTS: We included 134 randomized controlled trials involving 62,322 participants. Compared with standard care, exercise-only cardiac rehabilitation reduced the odds of cardiovascular mortality (odds ratio [OR], 0.70; 95% credibility interval [CrI], 0.51-0.96; moderate-quality evidence), major adverse cardiovascular events (OR, 0.57; 95% CrI, 0.40-0.78; low-quality evidence), nonfatal myocardial infarction (OR, 0.71; 95% CrI, 0.54-0.93; moderate-quality evidence), all-cause hospitalization (OR, 0.74; 95% CrI, 0.54-0.98; moderate-quality evidence), and cardiovascular hospitalization (OR, 0.69; 95% CrI, 0.51-0.88; moderate-quality evidence). Exercise-only cardiac rehabilitation was associated with lower cardiovascular hospitalization risk relative to cardiac rehabilitation without exercise (OR, 0.68; 95% CrI, 0.48-0.97; moderate-quality evidence). CONCLUSIONS: Cardiac rehabilitation programs containing exercise might provide broader cardiovascular benefits compared with those without exercise.

The complete plastomes of two flowering epiparasites (Phacellaria glomerata and P. compressa): Gene content, organization, and plastome degradation.

A plant parasite obligately parasitizing another plant parasite is referred to as epiparasite, which is extremely rare in angiosperms, and their complete plastome sequences have not been characterized to date. In this study, the complete plastomes of two flowering epiparasites: Phacellaria compressa and P. glomerata (Amphorogynaceae, Santalales) were sequenced. The plastomes of both species are of similar size, structure, gene content, and arrangement of genes to other hemiparasites in Santalales. Their plastomes were characterized by the functional loss of plastid-encoded NAD(P)H-dehydrogenase and infA genes, which strongly coincides with the general pattern of plastome degradation observed in Santalales hemiparasites. Our study demonstrates that the relatively higher level of nutritional reliance on the host plants and the reduced vegetative bodies of P. compressa and P. glomerata do not appear to cause any unique plastome degradation compared with their closely related hemiparasites.

Macrophage Paired Immunoglobulin-Like Receptor B Deficiency Promotes Peripheral Atherosclerosis in Apolipoprotein E-Deficient Mice.

Background: Peripheral atherosclerotic disease (PAD) is the narrowing or blockage of arteries that supply blood to the lower limbs. Given its complex nature, bioinformatics can help identify crucial genes involved in the progression of peripheral atherosclerosis. Materials and Methods: Raw human gene expression data for 462 PAD arterial plaque and 23 normal arterial samples were obtained from the GEO database. The data was analyzed using an integrated, multi-layer approach involving differentially-expressed gene analysis, KEGG pathway analysis, GO term enrichment analysis, weighted gene correlation network analysis, and protein-protein interaction analysis. The monocyte/macrophage-expressed leukocyte immunoglobulin-like receptor B2 (LILRB2) was strongly associated with the human PAD phenotype. To explore the role of the murine LILRB2 homologue PirB in vivo, we created a myeloid-specific PirB-knockout Apoe -/- murine model of PAD (PirB MΦKO) to analyze femoral atherosclerotic burden, plaque features of vulnerability, and monocyte recruitment to femoral atherosclerotic lesions. The phenotypes of PirB MΦKO macrophages under various stimuli were also investigated in vitro. Results: PirB MΦKO mice displayed increased femoral atherogenesis, a more vulnerable plaque phenotype, and enhanced monocyte recruitment into lesions. PirB MΦKO macrophages showed enhanced pro-inflammatory responses and a shift toward M1 over M2 polarization under interferon-γ and oxidized LDL exposure. PirB MΦKO macrophages also displayed enhanced efferocytosis and reduced lipid efflux under lipid exposure. Conclusion: Macrophage PirB reduces peripheral atherosclerotic burden, stabilizes peripheral plaque composition, and suppresses macrophage accumulation in peripheral lesions. Macrophage PirB inhibits pro-inflammatory activation, inhibits efferocytosis, and promotes lipid efflux, characteristics critical to suppressing peripheral atherogenesis.

Mitofusin 2 but not mitofusin 1 mediates Bcl-XL-induced mitochondrial aggregation.

Bcl-2 family proteins, as central players of the apoptotic program, participate in regulation of the mitochondrial network. Here, a quantitative live-cell fluorescence resonance energy transfer (FRET) two-hybrid assay was used to confirm the homo-/hetero-oligomerization of mitofusins 2 and 1 (MFN2 and MFN1), and also demonstrate the binding of MFN2 to MFN1 with 1:1 stoichiometry. A FRET two-hybrid assay for living cells co-expressing CFP-labeled Bcl-XL (an anti-apoptotic Bcl-2 family protein encoded by BCL2L1) and YFP-labeled MFN2 or MFN1 demonstrated the binding of MFN2 or MFN1 to Bcl-XL with 1:1 stoichiometry. Neither MFN2 nor MFN1 bound with monomeric Bax in healthy cells, but both MFN2 and MFN1 bind to punctate Bax (pro-apoptotic Bcl-2 family protein) during apoptosis. Oligomerized Bak (also known as BAK1; a pro-apoptotic Bcl-2 family protein) only associated with MFN1 but not MFN2. Moreover, co-expression of Bcl-XL with MFN2 or MFN1 had the same anti-apoptotic effect as the expression of Bcl-XL alone to staurosporine-induced apoptosis, indicating the Bcl-XL has its full anti-apoptotic ability when complexed with MFN2 or MFN1. However, knockdown of MFN2 but not MFN1 reduced mitochondrial aggregation induced by overexpression of Bcl-XL, indicating that MFN2 but not MFN1 mediates Bcl-XL-induced mitochondrial aggregation.

The Complete Plastomes of Five Hemiparasitic Plants (Osyris wightiana, Pyrularia edulis, Santalum album, Viscum liquidambaricolum, and V. ovalifolium): Comparative and Evolutionary Analyses Within Santalales.

Most species of Santalales (the sandalwood order) are hemiparasites, including both facultative and obligate hemiparasites. Despite its rich diversity, only a small fraction of the species in the sandalwood order have sequenced plastomes. The evolution of parasitism-associated plastome reduction in Santalales remains under-studied. Here, we report the complete plastomes of three facultative hemiparasites (Pyrularia edulis, Cervantesiaceae; Osyris wightiana, and Santalum album, Santalaceae), and two obligate hemiparasites (Viscum liquidambaricolum and Viscum ovalifolium, Viscaceae). Coupled with publicly available data, we investigated the dynamics of plastome degradation in Santalales hemiparasites. Our results indicate that these hemiparasites can be characterized by various degrees of plastome downsizing, structural rearrangement, and gene loss. The loss or pseudogenization of ndh genes was commonly observed in Santalales hemiparasites, which may be correlated to the lifestyle shift from photoautotroph to hemiparasitism. However, the obligate hemiparasites did not exhibit a consistently higher level of gene loss or pseudogenization compared to facultative hemiparasites, which suggests that the degree of plastome reduction is not correlated with the trophic level facultative or obligate hemiparasitism. Instead, closely related taxa tend to possess highly similar plastome size, structure, and gene content. This implies the parasitism-associated plastome degradation in Santalales may evolve in a lineage-specific manner.

Co-expression of Mcl-1 and Bak induces mitochondrial swelling.

We here used fluorescence imaging to explore the effect of co-overexpression of Mcl-1 and Bak/BH3-only proteins on mitochondrial morphology. The cells co-expressing CFP-Mcl-1 and YFP-Bak/BimL/Puma/tBid showed co-localization of Mcl-1 with Bak/Puma/BimL/tBid and also showed the inhibitory action of Mcl-1 on the Bak-, BimL-, Puma- or tBid-mediated cell death. Co-expression of Mcl-1 and Bak but not BH3-only proteins induced time-dependent mitochondrial swelling. Fluorescence resonance energy transfer (FRET) imaging proved the direct binding of Mcl-1 to Bak, BimL, Puma and tBid, respectively. In addition, Mcl-1 prevented Bak oligomerization by retrotranslocating Bak from mitochondria into cytoplasm. Moreover, Mcl-1-Bak complex exhibited a good co-localization with mitochondria, and co-expression of Mcl-1 and Bak for more than 24 h not only induced mitochondrial swelling but also impaired mitochondrial membrane potential. Collectively, co-expression of Mcl-1 and Bak but not BH3-only proteins significantly induced mitochondrial swelling and subsequent loss of mitochondrial membrane potential.

miR-532-3p-CSF2RA Axis as a Key Regulator of Vulnerable Atherosclerotic Plaque Formation.

BACKGROUND: The most dangerous atherosclerotic plaques, referred to as "vulnerable," are most likely to trigger acute atherothrombotic events such as myocardial infarction (heart attack) and stroke. Our goal was to uncover the molecular drivers of vulnerable plaque formation. METHODS: To elucidate the functional gene modules that drive vulnerable plaque formation, we performed a weighted gene coexpression network analysis integrated with a protein-protein interaction network analysis in human atherosclerotic carotid samples, which identified the candidate gene granulocyte-macrophage colony-stimulating factor 2 (GM-CSF) receptor alpha subunit (CSF2RA). Follow-up in vitro experiments were performed to elucidate the regulatory relationship between CSF2RA and the microRNA miR-532-3p as well as modifiers of macrophagic miR-532-3p-CSF2RA axis expression. Microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) studies elucidated the effect of statins on carotid miR-532-3p-CSF2RA axis expression in patients with carotid atherosclerotic disease. Apoe-/-, Ldlr-/-, and Csf2ra mutant Apoe-/- mouse models of atherosclerosis were employed to assess the effects of agomiR-532-3p therapy in vivo. RESULTS: The integrated weighted gene coexpression network analysis/protein-protein interaction network analysis revealed that the macrophagic GM-CSF receptor CSF2RA is significantly upregulated in macrophage-rich vulnerable plaques. Follow-up analysis identified the miR-532-3p-CSF2RA axis, as miR-532-3p downregulates CSF2RA via binding to CSF2RA's 3'UTR. Macrophagic miR-532-3p-CSF2RA dysregulation was enhanced via modified low-density lipoprotein or tumor necrosis factor α exposure in vitro. Moreover, this miR-532-3p-CSF2RA dysregulation was observed in human vulnerable plaques and Apoe-/- mouse plaques, effects rescued by statin therapy. In vivo, agomiR-532-3p therapy suppressed murine plaque formation and promoted plaque stabilization in a Csf2ra-dependent manner. CONCLUSION: Macrophagic miR-532-3p-CSF2RA axis dysregulation is a key driver in vulnerable plaque formation.

Anti-apoptotic capacity of Mcl-1Δ127.

The anti-apoptotic ability of Mcl-1Δ127, a caspase cleavage product of Mcl-1, is debated. We here used fluorescence imaging to assess the anti-apoptotic capacity of Mcl-1Δ127 in living cells. Fluorescence imaging of living cells expressing CFP-Mcl-1Δ127 showed that Mcl-1Δ127 existed mainly in cytoplasm. Fluorescence imaging of living cells co-expressing CFP-Mcl-1Δ127 and YFP-Bak, CFP-Mcl-1Δ127 and YFP-BimL, CFP-Mcl-1Δ127 and YFP-Puma or CFP-Mcl-1Δ127 and YFP-tBid showed that Mcl-1Δ127 markedly inhibited the oligomerization of Bak, BimL, Puma and tBid on mitochondria and also inhibited the Bak-, BimL-, Puma- or tBid-mediated cell death, resulting in their partial localization in cytoplasm. Fluorescence resonance energy transfer (FRET) imaging proved that Mcl-1Δ127 bound to Bak, BimL, Puma and tBid, respectively. Fluorescence loss in photobleaching (FLIP) analyses showed that Mcl-1Δ127 did prevent Bak oligomerization by retrotranslocating Bak from mitochondria into cytoplasm. Collectively, Mcl-1Δ127 has the same anti-apoptotic capacity as Mcl-1, and prevents apoptosis by sequestering BH3-only or Bak proteins, thus inhibiting their oligomerization on mitochondria.