Androgen receptor-mediated CD8+ T cell stemness programs drive sex differences in antitumor immunity.

The incidence and mortality rates of many non-reproductive human cancers are generally higher in males than in females. However, the immunological mechanism underlying sexual differences in cancers remains elusive. Here, we demonstrated that sex-related differences in tumor burden depended on adaptive immunity. Male CD8+ T cells exhibited impaired effector and stem cell-like properties compared with female CD8+ T cells. Mechanistically, androgen receptor inhibited the activity and stemness of male tumor-infiltrating CD8+ T cells by regulating epigenetic and transcriptional differentiation programs. Castration combined with anti-PD-L1 treatment synergistically restricted tumor growth in male mice. In humans, fewer male CD8+ T cells maintained a stem cell-like memory state compared with female counterparts. Moreover, AR expression correlated with tumor-infiltrating CD8+ T cell exhaustion in cancer patients. Our findings reveal sex-biased CD8+ T cell stemness programs in cancer progression and in the responses to cancer immunotherapy, providing insights into the development of sex-based immunotherapeutic strategies for cancer treatment.

Monoterpenoid indole alkaloid dimers from the Melodinus axillaris induce G2/M phase arrest and apoptosis via p38 MAPK activation in HCT116 cells.

Four monoterpenoid indole alkaloid dimers (MIADs), axidimins A-D (1-4), which possesses unprecedented apidosperma-aspidosperma-type skeletons, along with twelve known MIAs were isolated from Melodinus axillaris. Their structures were established by comprehensive analysis of the HRESIMS, NMR, ECD calculation and DP4 + analysis. A possible biosynthetic pathway for axidimins A-D was proposed. In vitro, axidimins C and D exhibited significant cytotoxicities against HCT116 cells with IC50 values of 5.3 µM and 3.9 µM, respectively. The results obtained from flow cytometry and Western blot analysis clearly demonstrated that axidimins C and D significantly induced a reverse G2/M phase arrest and apoptosis of HCT116 cells. The potential mechanism of axidimins C and D on HCT116 cells were thoroughly discussed through the utilization of network pharmacology and molecular docking research. Subsequently, the selected targets were validated using Western blot and CETSA analysis, confirming that axidimins C and D exert its cytotoxic effects through the activation of the p38 MAPK pathway, ultimately leading to HCT116 cells death. This study provides evidence indicating that axidimins C and D have the potential to induce cell cycle arrest and apoptosis in HCT116 cells by modulating the p38 MAPK signaling pathway. These findings offer a novel perspective for the development of anti-colorectal cancer drugs.

Early Experience of Patients with left Bundle Branch Block Corrected through Left Bundle Branch Area Pacing Compared with Conventional Right Ventricular Pacing: A Single-Center Retrospective Study.

Background: Left bundle branch area pacing (LBBAP) has the advantages of narrow QRS duration, rapid peak left ventricular (LV) activation, and LV dyssynchrony correction with a low, stable pacing output. Here we report our experience with patients undergoing LBBAP with a left bundle branch block (LBBB) for clinically indicated pacemaker or cardiac resynchronization therapy implantation. We compared the initial follow-up data of these patients and patients undergoing conventional right ventricular pacing (RVP). Methods: This retrospective study was performed between January 2017 and December 2020 and recruited 19 consecutive patients (mean age: 63 years; 8 women, 11 men) who underwent LBBAP (13 LBBAP only and 6 LBBAP + LV pacing), and 14 consecutive patients (mean age: 75 years; 8 women, 6 men) who underwent RVP. Demographic data, QRS durations, and echocardiographic parameters were compared before and after the procedures. Results: LBBAP substantially shortened the QRS duration and improved LV dyssynchrony echocardiographic parameters. However, RVP was not significantly associated with prolonged QRS duration and worse LV dyssynchronization. LBBAP improved cardiac contractility in selected patients. We did not find adverse effects of LBBAP on patients with preserved systolic function, possibly due to the limited number of patients and follow-up time. However, two of the 11 patients with preserved systolic function at baseline who underwent conventional RVP developed heart failure after implantation. Conclusions: In our experience, LBBAP improves LBBB-related ventricular dyssynchrony. However, LBBAP requires greater skill, and doubts remain about lead extraction. LBBAP may be an option for patients with LBBB when performed by an experienced operator, however further studies are needed to verify our findings.

Dyslipidemia and coronary artery calcium: From association to development of a risk-prediction nomogram.

BACKGROUND AND AIMS: The associations between dyslipidemia and coronary artery calcium (CAC) are controversial. We investigated their cross-sectional relationships and developed a predictive scoring system for prognostically significant coronary calcification (PSCC). METHODS AND RESULTS: This study evaluated the lipid profiles and the CAC score (CACS) measured through multidetector computed tomography (MDCT) among Taiwanese adult patients in a tertiary hospital between 2011 and 2016. Patients with CACS higher than 100 were classified as having PSCC. Dyslipidemia for each lipid component was defined based on the clinical cutoffs or the use of the lipid-lowering agents. Multivariable logistic regression was used to assess the association between dyslipidemia and PSCC and the model performance was assessed using calibration plot, discrimination, and a decision curve analysis. Of the 3586 eligible patients, 364 (10.2%) had PSCC. Increased age, male sex, higher body mass index (BMI), and higher level of triglyceride (TG) were associated with PSCC. The adjusted odds ratios (95% confidence intervals) of PSCC was 1.15 (0.90-1.47) for dyslipidemia defined by total cholesterol (TC) ≥200 mg/dL, 1.06 (0.83-1.35) for low-density-lipoprotein-cholesterol (LDL-C) ≥130 mg/dL, and 1.36 (1.06-1.75) for TG ≥ 200 mg/dL. The positive association between TG ≥ 200 mg/dL and PSCC was not modified by sex. Incorporating hypertriglyceridemia did not significantly improve the predictive performance of the base model comprising of age, sex, BMI, smoking, hypertension, diabetes, estimated glomerular filtration rate, and fasting glucose. CONCLUSIONS: Hypertriglyceridemia was significantly associated with the prevalent odds of PSCC. Our proposed predictive model may be a useful screening tool for PSCC.

Kansuinine A Ameliorates Atherosclerosis and Human Aortic Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Production and Suppressing IKKß/IκBα/NF-κB Signaling.

Reactive oxygen species (ROS)-induced vascular endothelial cell apoptosis is strongly associated with atherosclerosis progression. Herein, we aimed to examine whether Kansuinine A (KA), extracted from Euphorbia kansui L., prevents atherosclerosis development in a mouse model and inhibits cell apoptosis through oxidative stress reduction. Atherosclerosis development was analyzed in apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet (HFD) using Oil Red O staining and H&E staining. Human aortic endothelial cells (HAECs) were treated with KA, followed by hydrogen peroxide (H2O2), to investigate the KA-mediated inhibition of ROS-induced oxidative stress and cell apoptosis. Oil Red O staining and H&E staining showed that atherosclerotic lesion size was significantly smaller in the aortic arch of ApoE-/- mice in the HFD+KA group than that in the aortic arch of those in the HFD group. Further, KA (0.1-1.0 µM) blocked the H2O2-induced death of HAECs and ROS generation. The H2O2-mediated upregulation of phosphorylated IKKß, phosphorylated IκBα, and phosphorylated NF-κB was suppressed by KA. KA also reduced the Bax/Bcl-2 ratio and cleaved caspase-3 expression, preventing H2O2-induced vascular endothelial cell apoptosis. Our results indicate that KA may protect against ROS-induced endothelial cell apoptosis and has considerable clinical potential in the prevention of atherosclerosis and cardiovascular diseases.

IL-8 as a Potential Therapeutic Target for Periodontitis and Its Inhibition by Caffeic Acid Phenethyl Ester In Vitro.

Salivary levels of interleukin-8 (IL-8) are elevated in patients with periodontitis. Caffeic acid phenethyl ester (CAPE) improves the periodontal status in subjects. However, whether CAPE can reduce IL-8 expression is unclear. We collected saliva to determine proinflammatory cytokine levels and used subgingival calculus and surrounding tissues from patients with periodontitis for oral microbiota analysis via 16s ribosomal RNA gene sequencing. THP-1 cells were stimulated with sterile-filtered saliva from patients, and target gene/protein expression was assessed. IL-8 mRNA expression was analyzed in saliva-stimulated THP-1 cells treated with CAPE and the heme oxygenase-1 (HO-1) inhibitor tin-protoporphyrin (SnPP). In 72 symptomatic individuals, IL-8 was correlated with periodontal inflammation (bleeding on probing, r = 0.45; p < 0.001) and disease severity (bleeding on probing, r = 0.45; p < 0.001) but not with the four oral microbiota species tested. Reduced salivary IL-8 secretion was correlated with effective periodontitis treatment (r = 0.37, p = 0.0013). In THP-1 cells, saliva treatment induced high IL-8 expression and IKK2 and nuclear factor-κB (NF-κB) phosphorylation. However, the IKK inhibitor BMS-345541, NF-κB inhibitor BAY 11-7082, and CAPE attenuated saliva-induced IL-8 expression. CAPE induced HO-1 expression and inhibited IKK2, IκBα, and NF-κB phosphorylation. Blocking HO-1 decreased the anti-inflammatory activity of CAPE. The targeted suppression of IL-8 production using CAPE reduces inflammation and periodontitis.

Regulation of Human Platelet Activation and Prevention of Arterial Thrombosis in Mice by Auraptene through Inhibition of NF-κB Pathway.

Platelets are major players in the occurrence of cardiovascular diseases. Auraptene is the most abundant coumarin derivative from plants, and it has been demonstrated to possess a potent capacity to inhibit platelet activation. Although platelets are anucleated cells, they also express the transcription factor, nuclear factor-κB (NF-κB), that may exert non-genomic functions in platelet activation. In the current study, we further investigated the inhibitory roles of auraptene in NF-κB-mediated signal events in platelets. MG-132 (an inhibitor of proteasome) and BAY11-7082 (an inhibitor of IκB kinase; IKK), obviously inhibited platelet aggregation; however, BAY11-7082 exhibited more potent activity than MG-132 in this reaction. The existence of NF-κB (p65) in platelets was observed by confocal microscopy, and auraptene attenuated NF-κB activation such as IκBα and p65 phosphorylation and reversed IκBα degradation in collagen-activated platelets. To investigate cellular signaling events between PLCγ2-PKC and NF-κB, we found that BAY11-7082 abolished PLCγ2-PKC activation; nevertheless, neither U73122 nor Ro31-8220 had effect on NF-κB activation. Furthermore, both auraptene and BAY11-7082 significantly diminished HO• formation in activated platelets. For in vivo study, auraptene prolonged the occlusion time of platelet plug in mice. In conclusion, we propose a novel inhibitory pathway of NF-κB-mediated PLCγ2-PKC activation by auraptene in human platelets, and further supported that auraptene possesses potent activity for thromboembolic diseases.

Spironolactone ameliorates endothelial dysfunction through inhibition of the AGE/RAGE axis in a chronic renal failure rat model.

BACKGROUND: Spironolactone can improve endothelial dysfunction in the setting of heart failure and diabetes models. However, its beneficial effect in the cardiovascular system is not clear in the setting of non-diabetic renal failure. We conducted this study to investigate whether spironolactone can ameliorate endothelial dysfunction in a 5/6 nephrectomy model, and to determine the underlying mechanism. METHODS: Twenty-four Sprague-Dawley rats were divided into four groups. A renal failure model was created using the 5/6 nephrectomy method. The four groups included: Sham-operation group (Group1), chronic kidney disease (CKD; Group2), CKD + ALT-711 (advanced glycation end products [AGEs] breaker; Group 3), and CKD + spironolactone group (Group4). Acetylcholine (Ach)-mediated vasodilatation responses were compared between the four groups. To investigate the underlying mechanism, we cultured human aortic endothelial cells (HAECs) for in-vitro assays. Differences between two groups were determined with the paired student's t test. Differences between three or more groups were determined through one-way analysis of variance (ANOVA) with post-hoc analysis with LSD method. RESULTS: Compared with Group 1, Group 2 has a significantly impaired Ach-mediated vasodilatation response. Group 3 and 4 exhibited improved vasoreactivity responses. To determine the underlying mechanism, we performed an in-vitro study using cultured HAECs. We noted significant sirtuin-3 (SIRT3) protein downregulation, reduced phosphorylation of endothelial nitric oxide synthase at serine 1177 (p-eNOS), and increased intracellular oxidative stress in cultured HAECs treated with AGEs (200 µg/mL). These effects were counter-regulated when cultured HAECs were pretreated with spironolactone (10 µM). Furthermore, the increased p-eNOS production by spironolactone was abrogated when the HAECs were pretreated with tenolvin (1 µM), a SIRT3 inhibitor. CONCLUSIONS: Spironolactone could ameliorate endothelial dysfunction in a 5/6 nephrectomy renal failure model through AGEs/Receptor for AGEs (RAGEs) axis inhibition, SIRT3 upregulation, and nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX-2) and its associated intracellular oxidative stress attenuation.

Plasma L5 levels are elevated in ischemic stroke patients and enhance platelet aggregation.

L5, the most electronegative and atherogenic subfraction of low-density lipoprotein (LDL), induces platelet activation. We hypothesized that plasma L5 levels are increased in acute ischemic stroke patients and examined whether lectin-like oxidized LDL receptor-1 (LOX-1), the receptor for L5 on endothelial cells and platelets, plays a critical role in stroke. Because amyloid ß (Aß) stimulates platelet aggregation, we studied whether L5 and Aß function synergistically to induce prothrombotic pathways leading to stroke. Levels of plasma L5, serum Aß, and platelet LOX-1 expression were significantly higher in acute ischemic stroke patients than in controls without metabolic syndrome (P < .01). In mice subjected to focal cerebral ischemia, L5 treatment resulted in larger infarction volumes than did phosphate-buffered saline treatment. Deficiency or neutralizing of LOX-1 reduced infarct volume up to threefold after focal cerebral ischemia in mice, illustrating the importance of LOX-1 in stroke injury. In human platelets, L5 but not L1 (the least electronegative LDL subfraction) induced Aß release via IκB kinase 2 (IKK2). Furthermore, L5+Aß synergistically induced glycoprotein IIb/IIIa receptor activation; phosphorylation of IKK2, IκBα, p65, and c-Jun N-terminal kinase 1; and platelet aggregation. These effects were blocked by inhibiting IKK2, LOX-1, or nuclear factor-κB (NF-κB). Injecting L5+Aß shortened tail-bleeding time by 50% (n = 12; P < .05 vs L1-injected mice), which was prevented by the IKK2 inhibitor. Our findings suggest that, through LOX-1, atherogenic L5 potentiates Aß-mediated platelet activation, platelet aggregation, and hemostasis via IKK2/NF-κB signaling. L5 elevation may be a risk factor for cerebral atherothrombosis, and downregulating LOX-1 and inhibiting IKK2 may be novel antithrombotic strategies.

Skin autofluorescence is associated with inappropriate left ventricular mass and diastolic dysfunction in subjects at risk for cardiovascular disease.

BACKGROUND: Enhanced advanced glycation end products deposition within myocardial tissue may cause diastolic dysfunction. However, whether this is related to left ventricular hypertrophy or inappropriate left ventricular mass remains unclear. METHODS: We prospectively enrolled 139 subjects at risk for cardiovascular diseases. We used echocardiography for measurements of left ventricular mass and cardiac systolic and diastolic functional parameters. An advanced glycation end product reader was applied for measurements of skin autofluorescence values. Comparisons of left ventricular mass and echocardiographic parameters between the higher and lower skin autofluorescence groups were analyzed. RESULTS: Compared with the lower skin autofluorescence group, left ventricular mass index and the ratio of observed left ventricular mass/predicted left ventricular mass (oLVM/pLVM) was significantly higher in the higher skin autofluorescence group (61.22 ± 17.76 vs. 47.72 ± 11.62, P < 0.01, 1.62 ± 0.38 vs. 1.21 ± 0.21, P < 0.01). After adjustment for potential confounding factors, skin autofluorescence was an independent factor for left ventricular mass index (ß = 0.32, P < 0.01) and the ratio of oLVM/pLVM (ß = 0.41, P < 0.01). Skin autofluorescence ≥2.35 arbitrary unit predicted left ventricular hypertrophy at a sensitivity of 58.8%, and a specificity of 73.0% (P < 0.01). Skin autofluorescence ≥2.25 arbitrary unit predicted inappropriate left ventricular mass at a sensitivity of 71.1%, and a specificity of 83.9% (P < 0.01). Skin autofluorescence was positively correlated with E/E', an indicator for diastolic dysfunction (r = 0.21, P = 0.01). CONCLUSIONS: Skin autofluorescence is a useful tool for detecting left ventricular hypertrophy, inappropriate left ventricular mass and diastolic dysfunction.

[Decreasing the Output of Biomedical Waste in the Intensive Care Unit].

BACKGROUND & PROBLEMS: Advancing healthcare technologies have increased the use of disposable supplies that are made with PVC (polyvinyl chloride). Furthermore, biomedical effluents are steadily increasing due to severe patient treatment requirements in intensive care units. If these biomedical wastes are not properly managed and disposed, they will cause great harm to the environment and to public health. The statistics from an intensive care unit at one medical center in northern Taiwan show that the per-person biomedical effluents produced in 2014 increased 8.51% over 2013 levels. The main reasons for this increase included the low accuracy of classification of the contents of biomedical effluent collection buckets and of personnel effluents in the intensive care unit and the generally poor selection and designation of appropriate containers. PURPOSE: Improvement measures were implemented in order to decrease the per-day weight of biomedical effluents by 10% per person (-0.22 kg/person/day). METHODS: The project team developed various strategies, including creating classification-related slogans and posting promotional posters, holding education and training using actual case studies, establishing an "environmental protection pioneer" team, and promoting the use of appropriate containers. RESULTS: The implementation of the project decreased the per-day weight of biomedical effluents by 13.2% per person. CONCLUSIONS: Implementation of the project effectively reduced the per-person daily output of biological wastes and improved the waste separation behavior of healthcare personnel in the unit, giving patients and their families a better healthcare environment and helping advance the cause of environmental protection worldwide.

Nobiletin, a Polymethoxylated Flavone, Inhibits Glioma Cell Growth and Migration via Arresting Cell Cycle and Suppressing MAPK and Akt Pathways.

Nobiletin, a bioactive polymethoxylated flavone (5,6,7,8,3(') ,4(') -hexamethoxyflavone), is abundant in citrus fruit peel. Although nobiletin exhibits antitumor activity against various cancer cells, the effect of nobiletin on glioma cells remains unclear. The aim of this study was to determine the effects of nobiletin on the human U87 and Hs683 glioma cell lines. Treating glioma cells with nobiletin (20-100 µm) reduced cell viability and arrested the cell cycle in the G0/G1 phase, as detected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide (PI) staining, respectively; however, nobiletin did not induce cell apoptosis according to PI-annexin V double staining. Data from western blotting showed that nobiletin significantly attenuated the expression of cyclin D1, cyclin-dependent kinase 2, cyclin-dependent kinase 4, and E2 promoter-binding factor 1 (E2F1) and the phosphorylation of Akt/protein kinase B and mitogen-activated protein kinases, including p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. Our data also showed that nobiletin inhibited glioma cell migration, as detected by both functional wound healing and transwell migration assays. Altogether, the present results suggest that nobiletin inhibits mitogen-activated protein kinase and Akt/protein kinase B pathways and downregulates positive regulators of the cell cycle, leading to subsequent suppression of glioma cell proliferation and migration. Our findings evidence that nobiletin may have potential for treating glioblastoma multiforme.

Highly electronegative LDL from patients with ST-elevation myocardial infarction triggers platelet activation and aggregation.

Platelet activation and aggregation underlie acute thrombosis that leads to ST-elevation myocardial infarction (STEMI). L5-highly electronegative low-density lipoprotein (LDL)-is significantly elevated in patients with STEMI. Thus, we examined the role of L5 in thrombogenesis. Plasma LDL from patients with STEMI (n = 30) was chromatographically resolved into 5 subfractions (L1-L5) with increasing electronegativity. In vitro, L5 enhanced adenosine diphosphate-stimulated platelet aggregation twofold more than did L1 and induced platelet-endothelial cell (EC) adhesion. L5 also increased P-selectin expression and glycoprotein (GP)IIb/IIIa activation and decreased cyclic adenosine monophosphate levels (n = 6, P < .01) in platelets. In vivo, injection of L5 (5 mg/kg) into C57BL/6 mice twice weekly for 6 weeks shortened tail bleeding time by 43% (n = 3; P < .01 vs L1-injected mice) and increased P-selectin expression and GPIIb/IIIa activation in platelets. Pharmacologic blockade experiments revealed that L5 signals through platelet-activating factor receptor and lectin-like oxidized LDL receptor-1 to attenuate Akt activation and trigger granule release and GPIIb/IIIa activation via protein kinase C-α. L5 but not L1 induced tissue factor and P-selectin expression in human aortic ECs (P < .01), thereby triggering platelet activation and aggregation with activated ECs. These findings indicate that elevated plasma levels of L5 may promote thrombosis that leads to STEMI.

Sesamol reduces the atherogenicity of electronegative L5 LDL in vivo and in vitro.

Highly electronegative low-density lipoprotein (LDL) L5 induces endothelial cell (EC) apoptosis, which leads to the development of atherosclerosis. We examined the effects of sesamol (1), a natural organic component of sesame oil, on plasma L5 levels and atherosclerosis development in a rodent model and on the L5-induced apoptosis of ECs. Syrian hamsters, which have an LDL profile similar to that of humans, were fed a normal chow diet (control), a high-fat diet (HFD), or a HFD supplemented with the administration of 50 or 100 mg/kg of 1 via oral gavage (HFD+1) for 16 weeks (n = 8 per group). Hamsters in the HFD+1 groups had reduced plasma L5 levels when compared with the HFD group. Oil Red O staining showed that atherosclerotic lesion size was markedly reduced in the aortic arch of hamsters in the HFD+1 groups when compared with that in the HFD group. In human aortic ECs, 0.3-3 µM 1 blocked L5-induced apoptosis in a dose-dependent manner. Further mechanistic studies showed that 1 inhibited the L5-induced lectin-like oxidized LDL receptor-1 (LOX-1)-dependent phosphorylation of p38 MAPK and activation of caspase-3 and increased phosphorylation of eNOS and Akt. Our findings suggest that sesamol (1) protects against atherosclerosis by reducing L5-induced atherogenicity.

Gender disparity in LDL-induced cardiovascular damage and the protective role of estrogens against electronegative LDL.

BACKGROUND: Increased levels of the most electronegative type of LDL, L5, have been observed in the plasma of patients with metabolic syndrome (MetS) and ST-segment elevation myocardial infarction and can induce endothelial dysfunction. Because men have a higher predisposition to developing coronary artery disease than do premenopausal women, we hypothesized that LDL electronegativity is increased in men and promotes endothelial damage. METHODS: L5 levels were compared between middle-aged men and age-matched, premenopausal women with or without MetS. We further studied the effects of gender-influenced LDL electronegativity on aortic cellular senescence and DNA damage in leptin receptor-deficient (db/db) mice by using senescence-associated-ß-galactosidase and γH2AX staining, respectively. We also studied the protective effects of 17ß-estradiol and genistein against electronegative LDL-induced senescence in cultured bovine aortic endothelial cells (BAECs). RESULTS: L5 levels were higher in MetS patients than in healthy subjects (P < 0.001), particularly in men (P = 0.001). LDL isolated from male db/db mice was more electronegative than that from male or female wild-type mice. In addition, LDL from male db/db mice contained abundantly more apolipoprotein CIII and induced more BAEC senescence than did female db/db or wild-type LDL. In the aortas of db/db mice but not wild-type mice, we observed cellular senescence and DNA damage, and the effect was more significant in male than in female db/db mice. Pretreatment with 17ß-estradiol or genistein inhibited BAEC senescence induced by male or female db/db LDL and downregulated the expression of lectin-like oxidized LDL receptor-1 and tumor necrosis factor-alpha protein. CONCLUSION: The gender dichotomy of LDL-induced cardiovascular damage may underlie the increased propensity to coronary artery disease in men.

Undescribed compounds from Clinopodium chinense (Benth.) O. Kuntze and their coagulation activity studies.

Four rare compounds (1-4), including one 1,4-epoxy-benzoxepane derivative and one ringed prenylated naphthoquinoid skeleton, as well as one isopimarane-type diterpenoid and one megastigmane-type glycoside, along with three known megastigmane-type glycosides (5-7) were isolated from the ethanol extracts of C. chinense. Their structures were determined on the basis of 1D, 2D NMR, HR-ESI-MS and DP4+ analysis. Meanwhile, the in vitro evaluation indicated that compound 2 and 6 exhibited excellent procoagulant activities, which can significantly shorten prothrombin time (PT) and activated partial thromboplastin time (APTT), respectively.

Identification of Hypoxia-ALCAMhigh Macrophage- Exhausted T Cell Axis in Tumor Microenvironment Remodeling for Immunotherapy Resistance.

Although hypoxia is known to be associated with immune resistance, the adaptability to hypoxia by different cell populations in the tumor microenvironment and the underlying mechanisms remain elusive. This knowledge gap has hindered the development of therapeutic strategies to overcome tumor immune resistance induced by hypoxia. Here, bulk, single-cell, and spatial transcriptomics are integrated to characterize hypoxia associated with immune escape during carcinogenesis and reveal a hypoxia-based intercellular communication hub consisting of malignant cells, ALCAMhigh macrophages, and exhausted CD8+ T cells around the tumor boundary. A hypoxic microenvironment promotes binding of HIF-1α complex is demonstrated to the ALCAM promoter therefore increasing its expression in macrophages, and the ALCAMhigh macrophages co-localize with exhausted CD8+ T cells in the tumor spatial microenvironment and promote T cell exhaustion. Preclinically, HIF-1ɑ inhibition reduces ALCAM expression in macrophages and exhausted CD8+ T cells and potentiates T cell antitumor function to enhance immunotherapy efficacy. This study reveals the systematic landscape of hypoxia at single-cell resolution and spatial architecture and highlights the effect of hypoxia on immunotherapy resistance through the ALCAMhigh macrophage-exhausted T cell axis, providing a novel immunotherapeutic strategy to overcome hypoxia-induced resistance in cancers.

Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults.

High-density lipoprotein (HDL) is regarded as atheroprotective because it provides antioxidant and anti-inflammatory benefits and plays an important role in reverse cholesterol transport. In this paper, we outline a novel methodology for studying the heterogeneity of HDL. Using anion-exchange chromatography, we separated HDL from 6 healthy individuals into five subfractions (H1 through H5) with increasing charge and evaluated the composition and biologic activities of each subfraction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that apolipoprotein (apo) AI and apoAII were present in all 5 subfractions; apoCI was present only in H1, and apoCIII and apoE were most abundantly present in H4 and H5. HDL-associated antioxidant enzymes such as lecithin-cholesterol acyltransferase, lipoprotein-associated phospholipase A2, and paraoxonase 1 were most abundant in H4 and H5. Lipoprotein isoforms were analyzed in each subfraction by using matrix-assisted laser desorption-time-of-flight mass spectrometry. To quantify other proteins in the HDL subfractions, we used the isobaric tags for the relative and absolute quantitation approach followed by nanoflow liquid chromatography-tandem mass spectrometry analysis. Most antioxidant proteins detected were found in H4 and H5. The ability of each subfraction to induce cholesterol efflux from macrophages increased with increasing HDL electronegativity, with the exception of H5, which promoted the least efflux activity. In conclusion, anion-exchange chromatography is an attractive method for separating HDL into subfractions with distinct lipoprotein compositions and biologic activities. By comparing the properties of these subfractions, it may be possible to uncover HDL-specific proteins that play a role in disease.

Low-density lipoprotein electronegativity and risk of death after acute coronary syndromes: A case-cohort analysis.

BACKGROUND AND AIMS: Low-density lipoprotein (LDL)-cholesterol (LDL-C) promotes atherosclerotic cardiovascular disease (ASCVD), with changes in LDL electronegativity modulating its pro-atherogenic/pro-thrombotic effects. Whether such alterations associate with adverse outcomes in patients with acute coronary syndromes (ACS), a patient population at particularly high cardiovascular risk, remains unknown. METHODS: This is a case-cohort study using data from a subset of 2619 ACS patients prospectively recruited at four university hospitals in Switzerland. Isolated LDL was chromatographically separated into LDL particles with increasing electronegativity (L1-L5), with the L1-L5 ratio serving as a proxy of overall LDL electronegativity. Untargeted lipidomics revealed lipid species enriched in L1 (least) vs. L5 (most electronegative subfraction). Patients were followed at 30 days and 1 year. The mortality endpoint was reviewed by an independent clinical endpoint adjudication committee. Multivariable-adjusted hazard ratios (aHR) were calculated using weighted Cox regression models. RESULTS: Changes in LDL electronegativity were associated with all-cause mortality at 30 days (aHR, 2.13, 95% CI, 1.07-4.23 per 1 SD increment in L1/L5; p=.03) and 1 year (1.84, 1.03-3.29; p=.04), with a notable association with cardiovascular mortality (2.29; 1.21-4.35; p=.01; and 1.88; 1.08-3.28; p=.03). LDL electronegativity superseded several risk factors for the prediction of 1-year death, including LDL-C, and conferred improved discrimination when added to the updated GRACE score (area under the receiver operating characteristic curve 0.74 vs. 0.79, p=.03). Top 10 lipid species enriched in L1 vs. L5 were: cholesterol ester (CE) (18:2), CE (20:4), free fatty acid (FA) (20:4), phosphatidyl-choline (PC) (36:3), PC (34:2), PC (38:5), PC (36:4), PC (34:1), triacylglycerol (TG) (54:3), and PC (38:6) (all p < .001), with CE (18:2), CE (20:4), PC (36:3), PC (34:2), PC (38:5), PC (36:4), TG (54:3), and PC (38:6) independently associating with fatal events during 1-year of follow-up (all p < .05). CONCLUSIONS: Reductions in LDL electronegativity are linked to alterations of the LDL lipidome, associate with all-cause and cardiovascular mortality beyond established risk factors, and represent a novel risk factor for adverse outcomes in patients with ACS. These associations warrant further validation in independent cohorts.