Copper regulates the host innate immune response against bacterial infection via activation of ALPK1 kinase.

Copper is an essential trace element for the human body, and its requirement for optimistic immune functions has been recognized for decades. How copper is involved in the innate immune pathway, however, remains to be clarified. Here, we report that copper serves as a signal molecule to regulate the kinase activity of alpha-kinase 1 (ALPK1), a cytosolic pattern-recognition receptor (PRR), and therefore promotes host cell defense against bacterial infection. We show that in response to infection, host cells actively accumulate copper in the cytosol, and the accumulated cytosolic copper enhances host cell defense against evading pathogens, including intracellular and, unexpectedly, extracellular bacteria. Subsequently, we demonstrate that copper activates the innate immune pathway of host cells in an ALPK1-dependent manner. Further mechanistic studies reveal that copper binds to ALPK1 directly and is essential for the kinase activity of this cytosolic PRR. Moreover, the binding of copper to ALPK1 enhances the sensitivity of ALPK1 to the bacterial metabolite ADP-heptose and eventually prompts host cells to elicit an enhanced immune response during bacterial infection. Finally, using a zebrafish in vivo model, we show that a copper-treated host shows an increased production of proinflammatory cytokines, enhanced recruitment of phagosome cells, and promoted bacterial clearance. Our findings uncover a previously unrecognized role of copper in the modulation of host innate immune response against bacterial pathogens and advance our knowledge on the cross talk between cytosolic copper homeostasis and immune system.

Global trans-lesional computed tomography-derived fractional flow reserve gradient is associated with clinical outcomes in diabetic patients with non-obstructive coronary artery disease.

BACKGROUND: Coronary computed tomography angiography (CCTA)-derived fractional flow reserve (CT-FFR) enables physiological assessment and risk stratification, which is of significance in diabetic patients with nonobstructive coronary artery disease (CAD). We aim to evaluate prognostic value of the global trans-lesional CT-FFR gradient (GΔCT-FFR), a novel metric, in patients with diabetes without flow-limiting stenosis. METHODS: Patients with diabetes suspected of having CAD were prospectively enrolled. GΔCT-FFR was calculated as the sum of trans-lesional CT-FFR gradient in all epicardial vessels greater than 2 mm. Patients were stratified into low-gradient without flow-limiting group (CT-FFR > 0.75 and GΔCT-FFR < 0.20), high-gradient without flow-limiting group (CT-FFR > 0.75 and GΔCT-FFR ≥ 0.20), and flow-limiting group (CT-FFR ≤ 0.75). Discriminant ability for major adverse cardiovascular events (MACE) prediction was compared among 4 models [model 1: Framingham risk score; model 2: model 1 + Leiden score; model 3: model 2 + high-risk plaques (HRP); model 4: model 3 + GΔCT-FFR] to determine incremental prognostic value of GΔCT-FFR. RESULTS: Of 1215 patients (60.1 ± 10.3 years, 53.7% male), 11.3% suffered from MACE after a median follow-up of 57.3 months. GΔCT-FFR (HR: 2.88, 95% CI 1.76-4.70, P < 0.001) remained independent risk factors of MACE in multivariable analysis. Compared with the low-gradient without flow-limiting group, the high-gradient without flow-limiting group (HR: 2.86, 95% CI 1.75-4.68, P < 0.001) was associated with higher risk of MACE. Among the 4 risk models, model 4, which included GΔCT-FFR, showed the highest C-statistics (C-statistics: 0.75, P = 0.002) as well as a significant net reclassification improvement (NRI) beyond model 3 (NRI: 0.605, P < 0.001). CONCLUSIONS: In diabetic patients with non-obstructive CAD, GΔCT-FFR was associated with clinical outcomes at 5 year follow-up, which illuminates a novel and feasible approach to improved risk stratification for a global hemodynamic assessment of coronary artery in diabetic patients.

Dissemination feature based on PET/CT is a risk factor for diffuse large B cell lymphoma patients outcome.

BACKGROUND: 18F-FDG PET/CT provides precise information about dissemination of lymphoma lesions. Dmax, defined as distance between the two lesions that were farthest apart by PET/CT, was found to be a promising predictor of Diffuse large B-cell lymphoma (DLBCL) outcome in a small size of clinical trial data. We analyzed the impact of Dmax on the outcome of a large real-world DLBCL cohort. METHODS: Data of newly diagnosed DLBCL at the Third Affiliated Hospital of Soochow University were retrospectively collected. Baseline Dmax, clinical data and survival information were recorded. A metabolic parameter, metabolic bulk volume (MBV), was also measured to verify the independent impact of Dmax. RESULTS: Optimal cut-off values for Dmax and MBV were 45.34 cm and 21.65 cm3. With a median follow-up of 32 months, Dmax significantly impacted progression-free survival (PFS) and overall survival (OS) in 253 DLBCL patients. For Dmaxlow and Dmaxhigh groups, estimated 3-year OS were 87.0% and 53.8% (p < 0.001), while 3-year PFS were 77.3% and 37.3% (p < 0.001). And for MBVlow and MBVhighgroups, 3-year OS were 84.5% and 58.8% (p < 0.001), and 3-year PFS were 68.7% and 50.4% (p = 0.003). Multivariate analysis identified Dmax and Eastern Cooperative Oncology Group performance status (ECOG PS) independently associated with PFS and OS, while MBV only independently associated with OS. A Dmax revised prognostic index (DRPI) combining Dmax and ECOG PS identified an ultra-risk DLBCL population with 3-year PFS of 31.7% and 3-year OS of 38.5%. The area under the curve (AUC) showed that this model performed better than International prognostic Index (IPI). CONCLUSION: Dmax is a new and promising indicator to investigate dissemination of lymphoma lesions associated with the outcome of DLBCL. It significantly contributes to stratification of patients with disparate outcomes. TRIAL REGISTRATION: This research has been retrospectively registered in the Ethics Committee institutional of the Third Affiliated Hospital of Soochow University, and the registration number was approval No. 155 (approved date: 31 May 2022).

Altered serum lipid levels are associated with prognosis of diffuse large B cell lymphoma and influenced by utility of rituximab.

Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the prognosis of the disease varied. This research aims to investigate the impact of serum lipid level on the outcome of DLBCL patients and their interaction with rituximab (RTX). Data of newly diagnosed DLBCL in the third affiliated hospital of Soochow University were retrospectively collected. Baseline serum lipid levels, clinical data, and survival information were simultaneously recorded. Data of healthy controls were collected with age matching. Serum lipid levels significantly differed for the patients. All were transformed into categorical variables for the analysis of survival. During a median follow-up of 58 months, 32.8% patients died. Univariate analysis revealed all serum lipid indicators were associated with overall survival (OS); all except for total cholesterol (TC) and apolipoprotein B (apoB) showed significant impact on progression-free survival (PFS). Multivariable analysis confirmed the adverse effect of triglyceride (TG) on PFS (P = 0.013) and favorable impact of high-density lipoprotein (HDL) on OS (P = 0.003). For cases treated without RTX, apolipoprotein A (apoA) had independent favorable effect on both PFS (P = 0.004) and OS (P = 0.001). Comparably, for patients who received RTX, HDL showed remarkably predictive value of PFS (P = 0.011) and OS (P = 0.019). In conclusion, the abnormal serum lipids occurred throughout the course of DLBCL, and the associations of serum lipids and the prognosis of the disease were interfered by RTX. Trial registration: 2022()CL033; June 26, 2022, retrospectively registered.

eIF3a R803K mutation mediates chemotherapy resistance by inducing cellular senescence in small cell lung cancer.

Drug resistance in small cell lung cancer (SCLC) significantly affects the efficacy of chemotherapy treatment. However, due to the lack of tumor tissue samples, especially serial tumor samples during chemotherapy, the mechanism of chemotherapy resistance has not been fully studied. Circulating tumor DNA, which can be obtained in a noninvasive manner, can complement tumor sampling approaches for research in this field. We identified an SCLC patient with acquired drug resistance from 52 SCLC patients for whom follow-up data were available. By comparing somatic mutations in circulating tumor DNA before and after chemotherapy, for the first time, we found that the somatic mutation eIF3A R803K may be related to acquired chemotherapy resistance. Then, the association between the eIF3A R803K mutation and chemotherapy resistance was confirmed by samples from 254 lung cancer patients receiving chemotherapy. We found that the eIF3a R803K mutation weakened the proliferation ability of tumor cells but increased their resistance to chemotherapy. Further studies revealed that the eIF3A R803K mutation promotes cellular senescence. In addition, fisetin showed a synergistic effect with chemotherapy in eIF3A R803K mutant cells. These results suggest that the eIF3A R803K somatic mutation has the potential to predict chemotherapy resistance in SCLC. Moreover, the eIF3A R803K mutation promotes chemotherapy resistance by inducing senescence. Furthermore, a senolytic drug, fisetin, can reverse chemotherapy resistance mediated by the eIF3A R803K mutation.

Vascular-specific epicardial adipose tissue in predicting functional myocardial ischemia for patients with stable chest pain.

The relationship between vascular-specific epicardial adipose tissue (vEAT) volume and myocardial ischemia measured by fractional flow reserve (FFR) was not well investigated. Patients with typical and atypical chest pain undergoing coronary computed tomographic angiography scan followed by invasive coronary angiography in combination with FFR examination within one month were retrospectively included. EAT volume and CT attenuation was calculated. The patient with FFR ≤ 0.8 in at least one vessel was referred to as functional ischemia. The mean age of all patients was 61.7 ± 8.9 years and 66.7% of patients were male. There was a significant difference for left anterior descending branch (LAD) vEAT volume between patients with and without functional myocardial ischemia (28.7 ± 10.6 cm3 vs. 23.9 ± 8.7 cm3, p = 0.005). After adjusted by cardiac risk factors and CAD-RADS categories in multivariable logistic regression analysis, LAD-vEAT volume ≥ 24.6 cm3 (OR 3.355, 95% CI 1.546-7.281, p = 0.002) remained an independent predictor of functional ischemia. After adding LAD-vEAT volume ≥ 24.6 cm3 to a prediction model composed with cardiac risk factors and CAD-RADS categories, receiver operating characteristic curve analysis showed significantly improved areas under curve (AUC) for the new model (AUC: 0.795, p = 0.0319) compared with the previous ones. Moreover, the new model revealed significance in net reclassification improvement (NRI: 0.186, p = 0.037). In conclusion, LAD-vEAT volume measurements have incremental predictive performance beyond cardiac risk factors and CAD-RADS categories in identifying significant flow-limit ischemia detected by FFR.

Waveform analysis of differential graphs of reconstructed impedance cardiography from healthy individuals.

PURPOSE: The aim is to measure and analyze the wave amplitudes and time intervals of differential graphs of reconstructed impedance cardiography (RICG). METHODS: 180 adults with normal cardiac function between the ages of 18-78 were included in the study. Six mingled impedance changes on chest surface were simultaneously detected for each subject. The differential graphs of five impedance change components of RICG were obtained through waveform separation and software differentiation. The amplitudes of C, X, O, b waves and time intervals of Q-b and Q-C were measured and statistically analyzed. RESULTS: The amplitudes of C and X waves in PL, PR, AO, and that of C, O, b waves in LV and RV, all decrease as age increases. Wave amplitudes of the female group were bigger than those of the male group (p < .01), while the Q-C intervals of the female group were shorter than that of the male group (p < .01). Among five impedance change components, the wave amplitude of AO was larger than those of PL and PR (p < .01), and wave amplitudes of PL and PR were bigger than those of LV and RV (p < .01). Q-C intervals of LV and RV were longer than those of AO, PL and PR (p < .01), while the Q-b intervals of LV and RV were shorter than the Q-C intervals of AO, PL, and PR. CONCLUSIONS: The differential graphs of RICG could reflect indirectly the physiological activities and pathological changes of the heart and of the large blood vessels in thorax.

CREB/Wnt10b mediates the effect of COX-2 on promoting BMP9-induced osteogenic differentiation via reducing adipogenic differentiation in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic factors, which may be a potential candidate for bone tissue engineering. However, the osteogenic capacity of BMP9 still need to be further enhanced. In this study, we determined the effect of Wnt10b on BMP9-induced osteogenic differentiation in mesenchymal stem cell (MSCs) and the possible mechanism underlying this process. We introduced the polymerase chain reaction (PCR), Western blot analysis, histochemical stain, ectopic bone formation, and microcomputed tomography analysis to evaluate the effect of Wnt10b on BMP9-induced osteogenic differentiation. Meanwhile, PCR, Western blot analysis, chromatin immunoprecipitation, and immunoprecipitation were used to analyze the possible relationship between BMP9 and Wnt10b. We found that BMP9 upregulates Wnt10b in C3H10T1/2 cells. Wnt10b increases the osteogenic markers and bone formation induced by BMP9 in C3H10T1/2 cells, and silencing Wnt10b decreases these effects of BMP9. Meanwhile, Wnt10b enhances the level of phosphorylated Smad1/5/8 (p-Smad1/5/8) induced by BMP9, which can be reduced by silencing Wnt10b. On the contrary, Wnt10b inhibits adipogenic markers induced by BMP9, which can be decreased by silencing Wnt10b. Further analysis indicated that BMP9 upregulates cyclooxygenase-2 (COX-2) and phosphorylation of cAMP-responsive element binding (p-CREB) simultaneously. COX-2 potentiates the effect of BMP9 on increasing p-CREB and Wnt10b, while silencing COX-2 decreases these effects. p-CREB interacts with p-Smad1/5/8 to bind the promoter of Wnt10b in C3H10T1/2 cells. Our findings suggested that Wnt10b can promote BMP9-induced osteogenic differentiation in MSCs, which may be mediated through enhancing BMP/Smad signal and reducing adipogenic differentiation; BMP9 may upregulate Wnt10b via the COX-2/p-CREB-dependent manner.

Consistency of Proximal-to-Distal Tapering of Descending Thoracic Aortic Diameter: Quantification Using a Novel Computer Tomography-based Assessment.

BACKGROUND: Successful endovascular repair of thoracic aorta for type B aortic dissection requires correct stent-graft sizing, particularly of distal landing zone which is mainly based on operator experience. The present study aimed to quantitatively define proximal-to-distal tapering of descending thoracic aortic diameter and its consistency. METHODS: The novel parameter HDP (Hundred times Distance accounts for Percentage)-measured as distance from the distal end of the left subclavian artery to each level along the aortic central line/length from the distal end of the left subclavian artery to the proximal end of the celiac artery along the aortic central line × 100-was calculated per 1% unit of descending thoracic aorta based on 3-mensio software-derived measurements from 281 consecutive individuals who had undergone enhanced chest computer tomography scanning. Association between HDP and maximal diameter of descending thoracic aorta was assessed by using the generalized additive mixed model with smoothing function and threshold saturation effect analyses with generalized estimating equations. Nonadjusted and adjusted models were performed to illuminate its consistency. RESULTS: Three inflection levels (HDPs of 15.01, 36.63, and 77.74) were identified which allowed to divide the descending thoracic aorta into 4 segments. The taper was consistent before and after adjusting for age, sex, height, body mass index, hypertension, smoking habits, hyperlipidemia, and diabetes. Although 1% unit of descending thoracic aorta decreases, the maximal diameter reduces to 0.007 mm (-0.025, 0.010; P = 0.414) in the segment with HDP <15.01, to 0.151 mm (-0.158, -0.145; P < 0.001) in the segment with 15.01 ≤ HDP < 36.63, to 0.038 mm (-0.040, -0.036; P < 0.001) in the segment with 36.63 < HDP ≤ 77.74; and to 0.026 mm (-0.049, -0.002; P = 0.035) in the segment with HDP > 77.74, respectively. CONCLUSIONS: The maximal diameter of descending thoracic aorta decreases gradually and consistently among individuals free of aortic diseases.

Pioglitazone/metformin adduct regulates insulin secretion and inhibits high glucose-induced apoptosis via p21-p53-MDM2 signaling in INS-1 cells.

Pioglitazone/metformin adduct is a novel compound synthesized from pioglitazone and metformin combined at a molar mass ratio of 1:1. The aim of this study was to investigate the effects of pioglitazone/metformin adduct on high glucose-induced insulin secretion and apoptosis in INS-1 cells. Western blot and CCK8 analyses showed that the death rate of INS-1 cells increased in response to glucose treatment in a concentration-dependent manner. ELISA assays and Western blot analyses showed that insulin secretion peaked following treatment with glucose concentration at 33.33 mM. Treatment of INS-1 cells with 1 µM pioglitazone/metformin adduct in the presence of 33.33 mM glucose greatly improveded the levels of insulin and apoptosis rates compared to those of the control group. Analysis of mechanism underlying these effects revealed the involvement of the p21-p53-MDM2 signaling pathway. Our results indicate that pioglitazone/metformin adduct is superior to pioglitazone and/or metformin in regulating high glucose-induced insulin secretion and apoptosis in INS-1 cells.

Wnt11 promotes BMP9-induced osteogenic differentiation through BMPs/Smads and p38 MAPK in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9), as one of the most potent osteogenic factors, is a promising cytokine for bone tissue engineering. Wnt11 can regulate the development of the skeletal system and is related to high bone mass syndrome. However, the effect of Wnt11 on BMP9-induced osteogenic differentiation remains unknown. In this study, we investigated the relationship between Wnt11- and BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs). We recapitulated the osteogenic potential of BMP9 in C3H10T1/2 cells. The messenger RNA expression of Wnt11 is detectable in the available progenitor cells, and BMP9 can obviously increase the protein level of Wnt11 in these cells. Exogenous Wnt11 potentiates the effect of BMP9 on increasing alkaline phosphatase (ALP) activities, the expression of osteopontin (OPN), and Runt-related transcription factor 2 (Runx2), so does matrix mineralization in C3H10T1/2 cells. Although Wnt11 cannot increase the BMP9-induced ectopic bone formation, it can increase the bone density induced by BMP9 apparently. Wnt11 increases the level of p-Smad1/5/8, as well as p-p38. Meanwhile, Wnt11 promotes the effect of BMP9 on increasing the levels of p-Smad1/5/8 and p-p38. Inhibition of p38 decreases the BMP9-induced ALP activities, the expression of OPN, and the mineralization in C3H10T1/2 cells. However, all of these effects of the p38 inhibitor on BMP9-induced osteogenic markers can be almost reversed by the overexpression of Wnt11. Our findings suggested that Wnt11 can enhance the osteogenic potential of BMP9 in MSCs, and this effect may be partly mediated through enhancing BMPs/Smads and the p38 MAPK signal, which was induced by BMP9.

BMP9/COX-2 axial mediates high phosphate-induced calcification in vascular smooth muscle cells via Wnt/ß-catenin pathway.

Vascular calcification is a notable risk factor for cardiovascular system. High phosphate can induce calcification in vascular smooth muscle cells (VSMCs), but the detail mechanism underlying this process remains unclear. In the present study, we determined the relationship between high phosphate and bone morphogenetic protein 9 (BMP9) in VSMCs, the effect of BMP9 on calcification in VSMCs and the effect of COX-2 on BMP9 induced calcification in VSMCs, as well as the possible mechanism underlying this biological process. We found that high phosphate obviously up-regulates the expression of BMP9 in VSMCs. Over-expression of BMP9 decreases the level of alpha-smooth muscle cell actin (α-SMA) apparently, but increases the level of Runx-2, Dlx-5, and ALP in VSMCs. Meanwhile, BMP9 increases the level of OPN and OCN, promotes mineralization in VSMCs and induces calcification in thoracic aorta. High phosphate and over-expression of BMP9 increases the level of COX-2. Over-expression of COX-2 enhances the inhibitory effect of BMP9 on α-SAM and increases the level of OPN and OCN induced by BMP9. However, inhibition of COX-2 decreases the BMP9-induced calcification in VSMCs and thoracic aorta. For mechanism, we found that high phosphate or BMP9 increases the level of ß-catenin and p-GSK3ß in VSMCs, but no substantial effect on GSK3ß. However, COX-2 inhibitor decreases the expression of ß-catenin induced by BMP9. Our findings indicated that BMP9 is involved in the phosphate-induced calcification in VSMCs and COX-2 partly mediates the BMP9-induced calcification in VSMCs through activating Wnt/ß-catenin pathway.

Ethyl Rosmarinate Protects High Glucose-Induced Injury in Human Endothelial Cells.

Ethyl rosmarinate (RAE) is one of the active constituents from Clinopodium chinense (Benth.) O. Kuntze, which is used for diabetic treatment in Chinese folk medicine. In this study, we investigated the protective effect of RAE on high glucose-induced injury in endothelial cells and explored its underlying mechanisms. Our results showed that both RAE and rosmarinic acid (RA) increased cell viability, decreased the production of reactive oxygen species (ROS), and attenuated high glucose-induced endothelial cells apoptosis in a dose-dependent manner, as evidenced by Hochest staining, Annexin V⁻FITC/PI double staining, and caspase-3 activity. RAE and RA both elevated Bcl-2 expression and reduced Bax expression, according to Western blot. We also found that LY294002 (phosphatidylinositol 3-kinase, or PI3K inhibitor) weakened the protective effect of RAE. In addition, PDTC (nuclear factor-κB, or NF-κB inhibitor) and SP600125 (c-Jun N-terminal kinase, or JNK inhibitor) could inhibit the apoptosis in endothelial cells caused by high glucose. Further, we demonstrated that RAE activated Akt, and the molecular docking analysis predicted that RAE showed more affinity with Akt than RA. Moreover, we found that RAE inhibited the activation of NF-κB and JNK. These results suggested that RAE protected endothelial cells from high glucose-induced apoptosis by alleviating reactive oxygen species (ROS) generation, and regulating the PI3K/Akt/Bcl-2 pathway, the NF-κB pathway, and the JNK pathway. In general, RAE showed greater potency than RA equivalent.

Follicle-Stimulating Hormone ß-Subunit Potentiates Bone Morphogenetic Protein 9-Induced Osteogenic Differentiation in Mouse Embryonic Fibroblasts.

Postmenopausal osteoporosis (PMOP)-related fractures usually result in morbidity and mortality in aging women, so it remains a global public health concern, and new effective safe treatments are urgently needed recently. Efficient osteogenesis from mesenchymal stem cells (MSCs) would have the clinical application potential in treating multiple osteal disorders. Follicle-stimulating hormone (FSH), a pituitary glycoprotein hormone highly associated with menopausal bone turnover, whose peculiar part of receptor binding is follicle-stimulating hormone ß-subunit (FSHß). Bone morphogenetic protein 9 (BMP9), a potent osteogenic factor, can up-regulate FSHß in mouse embryonic fibroblasts (MEFs). However, it is unclear, whether extrapituitary FSHß affects BMP9-induced osteogenesis in MEFs. In this study, we investigated the role of FSHß in BMP9-induced osteogenesis in MEFs. We found that exogenous expression of FSHß significantly increased BMP9-induced alkaline phosphatase activity (ALP), the expression of osteogenic transcriptional factors, Runx2 and Osx, and the established late osteogenic markers, osteopontin (OPN) and osteocalcin (OCN), so does the ectopic bone formation. Mechanistically, FSHß dramatically enhanced BMP9-induced BMP/Smad signal transduction, presenting the augment phosphorylation of Smad1/5/8, whereas treatment with anti-FSHß antibodies suppressed these effects. An adenylate cyclase inhibitor obviously suppressed ALP and BMP/Smad signal transduction induced by BMP9 or the combination of BMP9 and FSHß in MEFs. Collectively, our findings suggested that FSHß may promote BMP9-induced activation of BMP/Smad signaling through a FSH/FSH receptor (FSHR)/cAMP dependent pathway in MEFs partly. J. Cell. Biochem. 118: 1792-1802, 2017. © 2016 Wiley Periodicals, Inc.

Coexistence of OSA may compensate for sleep related reduction in neural respiratory drive in patients with COPD.

BACKGROUND: The mechanisms underlying sleep-related hypoventilation in patients with coexisting COPD and obstructive sleep apnoea (OSA), an overlap syndrome, are incompletely understood. We compared neural respiratory drive expressed as diaphragm electromyogram (EMGdi) and ventilation during stage 2 sleep in patients with COPD alone and patients with overlap syndrome. METHODS: EMGdi and airflow were recorded during full polysomnography in 14 healthy subjects, 14 patients with OSA and 39 consecutive patients with COPD. The ratio of tidal volume to EMGdi was measured to indirectly assess upper airway resistance. RESULTS: Thirty-five patients with COPD, 12 healthy subjects and 14 patients with OSA completed the study. Of 35 patients with COPD, 19 had COPD alone (FEV1 38.5%±16.3%) whereas 16 had an overlap syndrome (FEV1 47.5±16.2%, AHI 20.5±14.1 events/hour). Ventilation (VE) was lower during stage 2 sleep than wakefulness in both patients with COPD alone (8.6±2.0 to 6.5±1.5 L/min, p<0.001) and those with overlap syndrome (8.3±2.0 to 6.1±1.8 L/min). Neural respiratory drive from wakefulness to sleep decreased significantly for patients with COPD alone (29.5±13.3% to 23.0±8.9% of maximal, p<0.01) but it changed little in those with overlap syndrome. The ratio of tidal volume to EMGdi was unchanged from wakefulness to sleep in patients with COPD alone and healthy subjects but was significantly reduced in patients with OSA or overlap syndrome (p<0.05). CONCLUSIONS: Stage 2 sleep-related hypoventilation in COPD alone is due to reduction of neural respiratory drive, but in overlap syndrome it is due to increased upper airway resistance.

Functions of idh1 and its mutation in the regulation of developmental hematopoiesis in zebrafish.

Isocitrate dehydrogenase 1 mutation (IDH1-R132H) was recently identified in acute myeloid leukemia with normal cytogenetics. The mutant enzyme is thought to convert α-ketoglutarate to the pathogenic 2-hydroxyglutarate (2-HG) that affects DNA methylation via inhibition of ten-eleven translocation 2. However, the role of wild-type IDH1 in normal hematopoiesis and its relevance to acute myeloid leukemia is unknown. Here we showed that zebrafish idh1 (zidh1) knockdown by morpholino and targeted mutagenesis by transcription activator-like effector nuclease might induce blockade in myeloid differentiation, as evident by an increase in pu.1 and decrease in mpo, l-plastin, and mpeg1 expression, and significantly reduce definitive hematopoiesis. Morpholino knockdown of zidh2 also induced a blockade in myeloid differentiation but definitive hematopoiesis was not affected. The hematopoietic phenotype of zidh1 knockdown was not rescuable by zidh2 messenger RNA, suggesting nonredundant functions. Overexpression of human IDH1-R132H or its zebrafish ortholog resulted in 2-HG elevation and expansion of myelopoiesis in zebrafish embryos. A human IDH1-R132H-specific inhibitor (AGI-5198) significantly ameliorated both hematopoietic and 2-HG responses in human but not zebrafish IDH1 mutant expression. The results provided important insights to the role of zidh1 in myelopoiesis and definitive hematopoiesis and of IDH1-R132H in leukemogenesis.

Adenovirus-mediated expression of vascular endothelial growth factor-a potentiates bone morphogenetic protein9-induced osteogenic differentiation and bone formation.

Mesenchymal stem cells (MSCs) are suitable seed cells for bone tissue engineering because they can self-renew and undergo differentiation into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Vascular endothelial growth factor-a (VEGF-a), an angiogenic factor, is also involved in osteogenesis and bone repair. However, the effects of VEGF-a on osteogenic MSCs differentiation remain unknown. It was previously reported that bone morphogenetic protein9 (BMP9) is one of the most important osteogenic BMPs. Here, we investigated the effects of VEGF-a on BMP9-induced osteogenesis with mouse embryo fibroblasts (MEFs). We found that endogenous VEGF-a expression was undetectable in MSCs. Adenovirus-mediated expression of VEGF-a in MEFs potentiated BMP9-induced early and late osteogenic markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In stem cell implantation assays, VEGF-a augmented BMP9-induced ectopic bone formation. VEGF-a in combination with BMP9 effectively increased the bone volume and osteogenic activity. However, the synergistic effect was efficiently abolished by the phosphoinositide 3-kinase (PI3K)/AKT inhibitor LY294002. These results demonstrated that BMP9 may crosstalk with VEGF-a through the PI3K/AKT signaling pathway to induce osteogenic differentiation in MEFs. Thus, our findings demonstrate the effects of VEGF-a on BMP9-induced bone formation and provide a new potential strategy for treating nonunion fractures, large segmental bony defects, and/or osteoporotic fractures.

Interaction of infectious spleen and kidney necrosis virus ORF119L with PINCH leads to dominant-negative inhibition of integrin-linked kinase and cardiovascular defects in zebrafish.

UNLABELLED: Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus, Iridoviridae family, causing a severe systemic disease with high mortality in mandarin fish (Siniperca chuatsi) in China and Southeast Asia. At present, the pathogenesis of ISKNV infection is still not fully understood. Based on a genome-wide bioinformatics analysis of ISKNV-encoded proteins, we found that ISKNV open reading frame 119L (ORF119L) is predicted to encode a three-ankyrin-repeat (3ANK)-domain-containing protein, which shows high similarity to the dominant negative form of integrin-linked kinase (ILK); i.e., viral ORF119L lacks the ILK kinase domain. Thus, we speculated that viral ORF119L might affect the host ILK complex. Here, we demonstrated that viral ORF119L directly interacts with particularly interesting Cys-His-rich protein (PINCH) and affects the host ILK-PINCH interaction in vitro in fathead minnow (FHM) cells. In vivo ORF119L overexpression in zebrafish (Danio rerio) embryos resulted in myocardial dysfunctions with disintegration of the sarcomeric Z disk. Importantly, ORF119L overexpression in zebrafish highly resembles the phenotype of endogenous ILK inhibition, either by overexpressing a dominant negative form of ILK or by injecting an ILK antisense morpholino oligonucleotide. Intriguingly, ISKNV-infected mandarin fish develop disorganized sarcomeric Z disks in cardiomyocytes. Furthermore, phosphorylation of AKT, a downstream effector of ILK, was remarkably decreased in ORF119L-overexpressing zebrafish embryos. With these results, we show that ISKNV ORF119L acts as a domain-negative inhibitor of the host ILK, providing a novel mechanism for the megalocytivirus pathogenesis. IMPORTANCE: Our work is the first to show the role of a dominant negative inhibitor of the host ILK from ISKNV (an iridovirus). Mechanistically, the viral ORF119L directly binds to the host PINCH, attenuates the host PINCH-ILK interaction, and thus impairs ILK signaling. Intriguingly, ORF119L-overexpressing zebrafish embryos and ISKNV-infected mandarin fish develop similar disordered sarcomeric Z disks in cardiomyocytes. These findings provide a novel mechanism for megalocytivirus pathogenesis.

Functions of flt3 in zebrafish hematopoiesis and its relevance to human acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) is expressed in human hematopoietic stem and progenitor cells (HSPCs) but its role during embryogenesis is unclear. In acute myeloid leukemia (AML), internal tandem duplication (ITD) of FLT3 at the juxtamembrane (JMD) and tyrosine kinase (TKD) domains (FLT3-ITD(+)) occurs in 30% of patients and is associated with inferior clinical prognosis. TKD mutations (FLT3-TKD(+)) occur in 5% of cases. We made use of zebrafish to examine the role of flt3 in developmental hematopoiesis and model human FLT3-ITD(+) and FLT3-TKD(+) AML. Zebrafish flt3 JMD and TKD were remarkably similar to their mammalian orthologs. Morpholino knockdown significantly reduced the expression of l-plastin (pan-leukocyte), csf1r, and mpeg1 (macrophage) as well as that of c-myb (definitive HSPCs), lck, and rag1 (T-lymphocyte). Expressing human FLT3-ITD in zebrafish embryos resulted in expansion and clustering of myeloid cells (pu.1(+), mpo(+), and cebpα(+)) which were ameliorated by AC220 and associated with stat5, erk1/2, and akt phosphorylation. Human FLT3-TKD (D835Y) induced significant, albeit modest, myeloid expansion resistant to AC220. This study provides novel insight into the role of flt3 during hematopoiesis and establishes a zebrafish model of FLT3-ITD(+) and FLT3-TKD(+) AML that may facilitate high-throughput screening of novel and personalized agents.

Paeonolum protects against MPP(+)-induced neurotoxicity in zebrafish and PC12 cells.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting 2% of the population aged over 65 years old. Mitochondrial defects and oxidative stress actively participate in degeneration of dopaminergic (DA) neurons in PD. Paeonolum, a main component isolated from Moutan cortex, has potent antioxidant ability. Here, we have examined the effects of paeonolum against MPP(+)-induced neurotoxicity in zebrafish and PC12 cells. METHODS: The overall viability and neurodegeneration of DA neurons was assessed in ETvmat2:green fluorescent protein (GFP) transgenic zebrafish, in which most monoaminergic neurons are labeled by GFP. Damage to PC12 cells was measured using a cell viability assay and assessment of nuclear morphology. Intracellular reactive oxygen species (ROS) and the level of total GSH were assessed. The mitochondrial cell death pathway including mitochondrial membrane potential, cytochrome C release and caspase-3 activity were also examined in PC12 cells. RESULTS: Paeonolum protected against MPP(+)-induced DA neurodegeneration and locomotor dysfunction in zebrafish in a concentration-dependent manner. Similar neuroprotection was replicated in the PC12 cellular model of MPP(+) toxicity. Paeonolum attenuated MPP(+)-induced intracellular ROS accumulation and restored the level of total GSH in PC12 cells. Furthermore, paeonolum significantly inhibited the mitochondrial cell death pathway induced by MPP(+). CONCLUSIONS: Collectively, the present study demonstrates that paeonolum protects zebrafish and PC12 cells against MPP(+)-induced neurotoxicity.