LncRNA CEBPA-AS1 knockdown prevents neuronal apoptosis against oxygen glucose deprivation/reoxygenation by regulating the miR-455/GPER1 axis.

Ischemic stroke (IS) is a common nervous system disease, which is a major cause of disability and death in the world. In present study, we demonstrated a regulatory mechanism of CCAAT/enhancer binding protein-alpha antisense 1 (CEBPA-AS1) in oxygen glucose deprivation/reoxygenation (OGD/R)-induced SH-SY5Y cells, with a focus on neuronal apoptosis. CEBPA-AS1, miR-455, and GPER1 expressions were evaluated by using qRT-PCR and Western blotting. The binding relationship among CEBPA-AS1, miR-455, and GPER1 was determined by a dual luciferase reporter assay. Neuronal viability and apoptosis were examined using MTT and flow cytometry assays, followed by determination of apoptosis-related factors (caspase 3, caspase 8, caspase 9, Bax, and Bcl-2). CEBPA-AS1 and GPER1 levels were upregulated, and miR-455 level was downregulated in the cell model of OGD/R induced. CEBPA-AS1 knockdown increased SH-SY5Y viability and reduced OGD/R-induced apoptosis. CEBPA-AS1 could act as a sponge of miR-455, and CEBPA-AS1 knockdown was found to elevate miR-455 expression. miR-455 overexpression also promoted SH-SY5Y cell viability and rescued them from OGD/R-induced apoptosis by binding to GPER1. GPER1 overexpression or miR-455 inhibition reversed the anti-apoptotic effect of CEBPA-AS1 knockdown. These findings suggest a regulatory network of CEBPA-AS1/miR-455/GPER1 that mediates neuronal cell apoptosis in the OGD model, providing a better understanding of pathogenic mechanisms after IS.

TLR4 mediates high-fat diet induced physiological changes in mice via attenuating PPARγ/ABCG1 signaling pathway.

High-fat diet (HFD) is known to promote atherosclerosis which accelerates the development of atherosclerotic cardiovascular diseases. Vascular dysfunction characterized by inflammation and lipid accumulation is common in atherosclerosis caused by HFD. The specific effects of HFD on blood vessels and the underlying mechanisms need to be further clarified. Toll-like receptor 4 (TLR4) is a key contributing factor in atherosclerosis and TLR4 deficiency protects vascular smooth muscle cells against inflammatory responses and lipid accumulation in vitro. However, the physiological significance of TLR4 signaling in HFD-induced changes is unknown. In this study, we observed that HFD feeding increased body weight, circulating inflammatory cytokines and lipid accumulation in the aorta of wild-type mice but apart from increasing body weight, did not affect the TLR4 knockout mice. TLR4 expression increased significantly in the arterial walls after receiving HFD treatment, while that of the co-localizing PPARγ and ABCG1 markedly decreased. TLR4 deficiency reversed the HFD-induced attenuation of PPARγ and ABCG1. In conclusion, TLR4 mediates HFD induced increase in body weight, inflammation and aortic lipid accumulation through, at least partly, the PPARγ/ABCG1 signaling pathway. Therefore, interfering with TLR4 signaling is a viable therapeutic option in diet induced atherosclerosis.

Neuroprotective effects of alkaloids from Piper longum in a MPTP-induced mouse model of Parkinson's disease.

CONTEXT: Alkaloids of Piper longum L. (Piperaceae) (PLA) include piperine and piperlonguminine. Piper longum and piperine have multiple biological properties including antioxidant activity. OBJECTIVE: The present study investigated the neuroprotective effects of PLA in a MPTP-induced mouse model of Parkinson's disease. MATERIALS AND METHODS: PLA was prepared by extracting the dry seed of P. longum using 85% ethanol. Adult male C57BL/6 mice were divided into eight groups of 12 rats each. Experimental and control groups received an equivalent volume of saline, 0.5% CMC-Na, and 0.1% Tween 80, treated groups received oral PLA (30, 60, and 120 mg/kg), other groups treated with piperine (60 mg/kg) or Madopar (50 mg/kg). The PLA prevention group (PLA-Pr) administrated PLA (120 mg/kg) for 1 week before MPTP challenged. Except for the PLA-Pr group, others were treated for seven consecutive weeks. Parkinson's disease was induced by injecting MPTP intraperitoneally (25 mg/kg) twice weekly for five consecutive weeks. Dopaminerigic (DA) neurons and their metabolism were detected by UFLC-MS/MS. Tyrosine hydroxylase (TH)-immunohistochemistry assay and Western blotting were performed. The antioxidant enzymatic levels were determined by kit-based assays. RESULTS: The LD50 value of PLA was determined at 1509 mg/kg of body weight. PLA (60 mg/kg) can significantly increase total movement time and distance (p < 0.05), increase levels of DA (p < 0.05) and DOPAC (p < .05), increase glutathione (GSH) level and superoxide dismutase (SOD) activity (p < 0.05), and decrease the lipid peroxidation of malondiadehycle (MDA) (p < 0.05) in PLA-treated groups as compared with the control group. DISCUSSION AND CONCLUSION: Our results indicate that PLA possesses neuroprotective effects and has ameliorative properties in dopaminergic neurons.

Association study of a functional copy number variation in the WWOX gene with risk of gliomas among Chinese people.

Gliomas represents more than 80% of all malignant brain tumors. However, the etiology still remains largely unknown. Human WW domain-containing oxidoreductase (WWOX), which is located at 16q23.1-16q23.2, the common fragile site 16D (FRA16D), an area with a high frequency of gene deletions or chromosomal alterations, has been identified as a tumor suppressor gene in multiple cancers. In current study, we analyzed the WWOX deletion (CNV-67048) in a large, case-control study of 3,622 adult Chinese people (including 1,798 glioma cases and 1,824 healthy controls). All participants were genotyped using real-time qualitative PCR (qPCR), and its biological effect was validated with mRNA expression assays. The deletion was significantly associated with glioma risk, with ORs (95% CIs) of 1.21 (1.05-1.41) associated with 1 copy deletion and 1.94 (1.37-2.75) associated with 2 copy deletion as compared with subjects with no deletion (p for trend = 8.05 × 10(-6)). Additional adjustments and stratified analyses did not change the results materially. The mRNA levels of WWOX in glioma tissues were significantly lower than that of their border tissues (p = 0.007), especially in the loss genotyped subjects. Our data suggest that the loss genotypes of CNV-67048 in WWOX gene predispose their carriers to gliomas, and WWOX gene deletion may be a new biomarker for predicting risk of gliomas.

Effect of human umbilical cord blood mesenchymal stem cell transplantation on neuronal metabolites in ischemic rabbits.

BACKGROUND: Because there is little research on the effects of transplanted stem cells on neuronal metabolites in infarct areas, we transplanted human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) into cerebral ischemic rabbits and examined the neuronal metabolites. RESULTS: Rabbits (n = 40) were equally divided into sham, middle cerebral artery occlusion (MCAO), hUCB-MSC, and saline groups. The rabbit ischemic model was established by MCAO. The effects of hUCB-MSC transplantation were assessed by proton magnetic resonance spectroscopy (1H-MRS), neurological severity scores (NSSs), infarct area volume, neuronal density, and optical density (OD) of microtubule-associated protein 2 (MAP2)-positive cells. We also evaluated complete blood cell counts(CBCs) and serum biochemical parameters. NSSs in the hUCB-MSC group at 7 and 14 days after reperfusion were lower than in MCAO and saline groups (p < 0.05). Compared with MCAO and saline groups at 2 weeks after MCAO, the infarction volume in the hUCB-MSC group had decreased remarkably (p < 0.05). Significant neuronal metabolic changes occurred in the infarct area at 24 h and 2 weeks after MCAO. 1H-MRS revealed an elevation in the lactate (Lac)/creatine including phosphocreatine (Cr) ratio and a decrease in the N-acetylaspartate (NAA)/Cr and choline-containing phospholipids (Cho)/Cr ratios at 24 h after MCAO in the MCAO group (p < 0.01). Compared with saline and MCAO groups at 24 h and 2 weeks after MCAO, NAA/Cr and Cho/Cr ratios had increased significantly, whereas the Lac/Cr ratio had decreased significantly in the hUCB-MSC group (p < 0.01). Neuronal density and OD of MAP2-positive cells in the MCAO group were significantly lower than those in the sham group, whereas the neuronal density and OD of MAP2-positive cells in the hUCB-MSC group were higher than those in MCAO and saline groups (p < 0.05). CBCs and biochemical parameters were unchanged in the MCAO group at 24 h and 2 weeks after hUCB-MSC transplantation. CONCLUSIONS: Transplanted hUCB-MSCs might ameliorate ischemic damage by influencing neuronal metabolites in the infarct area, providing additional evidence for neuroprotection by stem cells. No significant changes were observed in CBCs or serum biochemical parameters, suggesting that intravenous infusion of hUCB-MSCs is safe for rabbits in the short-term.

Human umbilical cord blood mesenchymal stem cell transplantation suppresses inflammatory responses and neuronal apoptosis during early stage of focal cerebral ischemia in rabbits.

AIM: Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) have been shown to ameliorate cerebral ischemia in animal models. In this study we investigated the effects of hUCB-MSCs on inflammatory responses and neuronal apoptosis during the early stage of focal cerebral ischemia in rabbits. METHODS: Focal cerebral ischemia was induced in male New Zealand rabbits by occlusion of MCA for 2 h. The blood samples were collected at different time points prior and during MCAO-reperfusion. The animals were euthanized 3 d after MCAO, and the protein levels of IL-1ß, IL-6, IL-10 and TNF-α in the serum and peri-ischemic brain tissues were detected using Western blot and ELISA, respectively. Inflammatory cell infiltration, neuronal apoptosis and neuronal density were measured morphologically. hUCB-MSCs (5 × 10(6)) were iv injected a few minutes after MCAO. RESULTS: The serum levels of IL-1ß, IL-6 and TNF-α were rapidly increased, and peaked at 2 h after the start of MCAO. hUCB-MSC transplantation markedly and progressively suppressed the ischemia-induced increases of serum IL-1ß, IL-6 and TNF-α levels within 6 h MCAO-reperfusion. Focal cerebral ischemia decreased the serum level of IL-10, which was prevented by hUCB-MSC transplantation. The expression of IL-1ß, IL-6, IL-10 and TNF-α in the peri-ischemic brain tissues showed similar changes as in the serum. hUCB-MSC transplantation markedly suppressed the infiltration of inflammatory cells, and increased the neuronal density around the ischemic region. Furthermore, hUCB-MSC transplantation significantly decreased the percentage of apoptosis around the ischemic region. CONCLUSION: hUCB-MSCs transplantation suppresses inflammatory responses and neuronal apoptosis during the early stage focal cerebral ischemia in rabbits.

Double domain fusion improves the reverse transcriptase activity and inhibitor tolerance of Bst DNA polymerase.

To enhance the DNA/RNA amplification efficiency and inhibitor tolerance of Bst DNA polymerase, four chimeric Bst DNA polymerase by fusing with a DNA-binding protein Sto7d and/or a highly hydrophobic protein Hp47 to Bst DNA polymerase large fragment. One of chimeric protein HpStBL exhibited highest inhibitor tolerance, which retained high active under 0.1 U/µL sodium heparin, 0.8 ng/µL humic acid, 2.5× SYBR Green I, 8 % (v/v) whole blood, 20 % (v/v) tissue, and 2.5 % (v/v) stool. Meanwhile, HpStBL showed highest sensitivity (93.75 %) to crude whole blood infected with the African swine fever virus. Moreover, HpStBL showed excellent reverse transcriptase activity in reverse transcription loop-mediated isothermal amplification, which could successfully detect 0.5 pg/µL severe acute respiratory syndrome coronavirus 2 RNA in the presence of 1 % (v/v) stools. The fusion of two domains with different functions to Bst DNA polymerase would be an effective strategy to improve Bst DNA polymerase performance in direct loop-mediated isothermal amplification and reverse transcription loop-mediated isothermal amplification detection, and HpStBL would be a promising DNA polymerase for direct African swine fever virus/severe acute respiratory syndrome coronavirus 2 detection due to simultaneously increased inhibitor tolerance and reverse transcriptase activity.

Minimalistic approach to left atrial appendage occlusion guided by cardiac computed tomography angiography.

OBJECTIVE: To assess the feasibility and safety of the minimalistic approach to left atrial appendage occlusion (LAAO) guided by cardiac computed tomography angiography (CCTA). METHODS: Ninety consecutive patients who underwent LAAO, with or without CCTA-guided, were matched (1:2). Each step of the LAAO procedure in the computed tomography (CT) guidance group (CT group) was directed by preprocedural CT planning. In the control group, LAAO was performed using the standard method. All patients were followed up for 12 months, and device surveillance was conducted using CCTA. RESULTS: A total of 90 patients were included in the analysis, with 30 patients in the CT group and 60 matched patients in the control group. All patients were successfully implanted with Watchman devices. The mean ages for the CT group and the control group were 70.0 ± 9.4 years and 68.4 ± 11.9 years (P = 0.52), respectively. The procedure duration (45.6 ± 10.7 min vs. 58.8 ± 13.0 min, P < 0.001) and hospital stay (7.5 ± 2.4 day vs. 9.6 ± 2.8 day, P = 0.001) in the CT group was significantly shorter compared to the control group. However, the total radiation dose was higher in the CT group compared to the control group (904.9 ± 348.0 mGy vs. 711.9 ± 211.2 mGy, P = 0.002). There were no significant differences in periprocedural pericardial effusion (3.3% vs. 6.3%, P = 0.8) between the two groups. The rate of postprocedural adverse events (13.3% vs. 18.3%, P = 0.55) were comparable between both groups at 12 months follow-up. CONCLUSIONS: CCTA is capable of detailed LAAO procedure planning. Minimalistic LAAO with preprocedural CCTA planning was feasible and safe, with shortened procedure time and acceptable increased radiation and contras consumption. For patients with contraindications to general anesthesia and/or transesophageal echocardiography, this promising method may be an alternative to conventional LAAO.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomal KLF4 Alleviated Ischemic Stroke Through Inhibiting N6-Methyladenosine Modification Level of Drp1 by Targeting lncRNA-ZFAS1.

Ischemic stroke has become a serious public health problem that causes high rates of death and disability. Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have shown promising therapeutic results in IS, while the underlying mechanisms need further investigation. Cell and mice models were established through oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and middle cerebral artery occlusion (MCAO)/reperfusion. Exosomes were isolated from BMSCs. Related gene and protein expression was measured by qRT-PCR, Western blotting, and immunofluorescence analysis. The biological functions of treated cells and tissues were analyzed by MTT, ELISA, JC-1, flow cytometry, TTC staining, or TUNEL staining. The interaction of KLF4/lncRNA-ZFAS1 promoter and lncRNA-ZFAS1/FTO was measured by ChIP, dual-luciferase reporter, or RIP assays. The m6A levels of Drp1 were measured by MeRIP-PCR. Mitochondrial staining and transmission electron microscopy (TEM) were used to evaluate the mitochondrial morphology in N2a cells and brain tissues. BMSC-derived exosomes increased the viability of neuronal cells treated with OGD/R while decreasing LDH release, oxidative stress, mitochondrial injury, and apoptosis. Furthermore, these effects were abolished by knockdown of exosomal KLF4. KLF4 increased lncRNA-ZFAS1 through binding to its promoter. LncRNA-ZFAS1 overexpression suppressed the m6A levels of Drp1 and reversed the promoting effect of exosomal KLF4 silencing on mitochondrial injury and the imbalance of mitochondrial dynamics by targeting FTO. Exosomal KLF4 alleviated the infarct area, neuronal injury, and apoptosis in MCAO mice through lncRNA-ZFAS1/FTO/Drp1 axis. BMSC-derived exosomal KLF4 promoted lncRNA-ZFAS1 expression to repress Drp1 m6A modification by targeting FTO, thus reducing mitochondrial dysfunction and alleviating neuronal injury in ischemic stroke.

Institutional Residence Protects Against Cognitive Frailty: A Cross-Sectional Study.

Based on the complex aging background, more and more older people have to live in an institution in later life in China. The prevalence of cognitive frailty (CF) is more higher in institutions than in communities. Rarely studies were conducted on the relationship between institutional residence and CF. Hence, this study were performed to determine the relationship between institutional residence (living in a nursing home) and CF in older adults. A total of 1004 older community residents and 111 older nursing home residents over 50 years of age from Hefei, Anhui Province, China were recruited. CF included physical frailty (PF) and mild cognitive impairment (MCI). PF was assessed using the Chinese version of the Fried frailty scale, MCI was assessed using the Montreal Cognitive Assessment, and the common associated factors including sedentary behavior, exercise, intellectual activity, comorbidity, medication, chronic pain, sleep disorders, nutritional status and loneliness were analyzed using regression logistic models. Multivariate regression logistic analysis showed that exercise (P = .019, odds ratio [OR] = 0.494, 95% confidence interval [CI]: 0.274-0.891), intellectual activity (P = .019, OR = 0.595, 95% CI: 0.380-0.932), medication use (P = .003, OR = 2.388, 95% CI: 1.339-4.258), chronic pain (P = .003, OR = 1.580, 95% CI: 1.013-2.465) and loneliness (P = .000, OR = 2.991, 95% CI: 1.728-5.175) were significantly associated with CF in community residents; however, only sedentary behavior (P = .013, OR = 3.851, 95% CI: 1.328-11.170) was significantly associated with CF in nursing home residents. Our findings suggest that nursing homes can effectively address many common risk factors for CF, including lack of exercise and intellectual activity, medication use, chronic pain, and loneliness, better than the community setting. Thus, residing in a nursing home is conducive to the intervention of CF.

Occlusion of Bilobulated Left Atrial Appendage Using the Dual-Watchman Technique: A Long-Term Follow-Up Study.

Background: Percutaneous left atrial appendage (LAA) occlusion has been considered an efficient alternative to oral anticoagulation to prevent embolic events in patients with non-valvular atrial fibrillation (NVAF). Due to the complexities and heterogeneous anatomy of the LAA structure, the single-device approach may not always fit a large bilobulated LAA. This study aimed to evaluate the feasibility and safety of one-stop dual Watchman implantation for patients with bilobulated LAA. Methods: Included in the analysis were patients who underwent complete LAA closure with dual Watchman devices between December 2015 and December 2021. The anatomic morphology, procedure characteristics, procedure safety, and procedural complications were analyzed. Cardiac CT or transesophageal ultrasound was obtained at 7 days, 6 months, 1 year, and 2 years post-operatively to evaluate the effect of occlusion. Results: Among the 330 patients who underwent LAA occlusion during the study period, 7 (2.1%) patients were occluded with one-stop implantation of the double Watchman strategy. Successful occlusion was achieved in all patients. One patient had the double-access sheath strategy for implantation, and 6 patients had only a single-access sheath strategy for implantation. Pericardial effusion occurred in one case during the 7-day perioperative period. There was no device embolization, thrombosis, or obvious peridevice leakage (≥l mm) during the 2-year follow-up, with the exception of two cases with 2 mm of incomplete LAA sealing. Conclusion: The one-stop implantation of a dual Watchman is feasible and safe and might provide a strategy to occlude a large bilobulated LAA when incomplete closure is inevitable with a single device.

Association Between Serum C1q Tumor Necrosis Factor-Related Protein 9 and the Clinical Characteristics and Prognosis of Ischemic Stroke.

INTRODUCTION: C1q tumor necrosis factor (TNF)-related protein 9 (CTRP9) is a novel member of the C1q/TNF superfamily. According to our previous review, CTRP9 plays a vital role in the process of cardiovascular diseases, including regulating energy metabolism, modulating vasomotion, protecting endothelial cells, inhibiting platelet activation, inhibiting pathological vascular remodeling, stabilizing atherosclerotic plaques, and protecting the heart. We proposed that CTRP9 could play multiple positive and beneficial roles in vascular lesions in ischemic stroke (IS). Here, we aimed to study the relationship between serum CTRP9 and the etiology, severity, and prognosis of IS patients. METHODS: A total of 302 patients with IS and 173 non-stroke controls were selected from the same hospital, and all patients with IS were followed up 12 months after stroke onset. Stroke etiology was classified according to the Trial of ORG 10172 in Acute Stroke Treatment classification. Symptomatic severity was determined using the National Institutes of Health Stroke Scale score. The lesion volume of acute cerebral ischemia was measured using magnetic resonance imaging (MRI). The unfavorable functional outcome was a combination of death or major disability 12 months after stroke onset. Receiver operating characteristic (ROC) curves and integrated discrimination improvement (IDI) and net reclassification improvement (NRI) statistics were applied in the statistical analysis. RESULTS: We found that serum CTRP9 levels and the ratios of CTRP9/total cholesterol (TC), CTRP9/triglyceride (TG), CTRP9/low-density lipoprotein cholesterol (LDL-C), and CTRP9/high-density lipoprotein cholesterol (HDL-C) were associated with the presence of IS. Moreover, the serum CTRP9 concentration was positively associated with the severity of IS. Incorporation of CTRP9/LDL-C levels into a fully adjusted model for IS-cardioembolic (CE) improved discrimination and calibration, and significantly improved reclassification. In addition, CTRP9 was a predictor of unfavorable functional outcomes. CONCLUSIONS: All the findings indicated that serum CTRP9 could be a promising blood-derived biomarker for the early evaluation and prognosis assessment of IS. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1800020330.

Unexpected BrdU inhibition on astrocyte-to-neuron conversion.

5-Bromo-2'-deoxyuridine (BrdU) is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle. BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons, however side effects on neural stem cells and their progeny have been reported. In vivo astrocyte-to-neuron (AtN) conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons. The BrdU-labeling strategy has been used to trace astrocyte-converted neurons, but whether BrdU has any effect on the AtN conversion is unknown. Here, while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury, we accidentally discovered that BrdU inhibited AtN conversion. We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex. Although most NeuroD1-infected astrocytes were converted into neurons, the number of BrdU-labeled neurons was surprisingly low. To exclude the possibility that this BrdU inhibition was caused by the ischemic injury, we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU. Surprisingly, we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group. These results revealed an unexpected inhibitory effect of BrdU on AtN conversion, suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.

Increased MANF Expression in the Inferior Temporal Gyrus in Patients With Alzheimer Disease.

Alzheimer disease (AD) is an aging-related disorder linked to endoplasmic reticulum (ER) stress. The main pathologic feature of AD is the presence of extracellular senile plaques and intraneuronal neurofibrillary tangles (NFTs) in the brain. In neurodegenerative diseases, the unfolded protein response (UPR) induced by ER stress ensures cell survival. Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects against ER stress and has been implicated in the pathogenesis of AD. MANF is expressed in neurons of the brain and spinal cord. However, there have been no investigations on MANF expression in the brain of AD patients. This was addressed in the present study by immunohistochemistry, western blotting, and quantitative analyses of postmortem brain specimens. We examined the localization and expression levels of MANF in the inferior temporal gyrus of the cortex (ITGC) in AD patients (n = 5), preclinical (pre-)AD patients (n = 5), and age-matched non-dementia controls (n = 5) by double immunofluorescence labeling with antibodies against the neuron-specific nuclear protein neuronal nuclei (NeuN), ER chaperone protein 78-kDa glucose-regulated protein (GRP78), and MANF. The results showed that MANF was mainly expressed in neurons of the ITGC in all 3 groups; However, the number of MANF-positive neurons was significantly higher in pre-AD (Braak stage III/IV) and AD (Braak stage V/VI) patients than that in the control group. Thus, MANF is overexpressed in AD and pre-AD, suggesting that it can serve as a diagnostic marker for early stage disease.

Use of the Nuclear Matrix Protein 22 BladderChek Test for the Detection of Primary and Recurrent Urothelial Carcinoma.

OBJECTIVE: To evaluate the performance of the nuclear matrix protein 22 (NMP22) BladderChek test in urothelial carcinoma (UC). METHODS: We retrospectively analyzed 1318 patients who performed the NMP22 BladderChek tests. Of them, 103 were primary UC patients, 90 were surgical treatment UC patients, and 1125 were benign disease patients. The performance of the NMP22 BladderChek test for the diagnosis of primary and recurrent UC was evaluated. Moreover, the performance of urine cytology and the NMP22 BladderChek test for the diagnosis of primary UC was compared in 90 available subjects including 48 primary UC patients and 42 benign disease patients. RESULTS: The sensitivity and specificity of the NMP22 BladderChek test were 37.9% and 95.8%, respectively, for the diagnosis of primary UC (n = 1228). The corresponding parameters of the NMP22 BladderChek test were 31.0% and 88.5%, respectively, for the diagnosis of recurrent UC (n = 90). The sensitivity and specificity of urine cytology were 54.2% and 97.6%, respectively, for the diagnosis of primary UC (n = 90); the corresponding parameters of the NMP22 BladderChek test were 41.7% and 83.3%, respectively; the corresponding parameters of the two tests combination were 64.6% and 83.3%, respectively. There was a significant difference in the performance between the NMP22 BladderChek test and urine cytology or the combination of two tests (P = 0.017 and 0.001, respectively). CONCLUSIONS: The NMP22 BladderChek test has a low sensitivity for detecting primary and recurrent UC. Urine cytology is superior to the NMP22 BladderChek test, and combined use of the two tests improves the sensitivity in the detection of primary UC.

Heterogeneous gene expression and functional activity of ryanodine receptors in resistance and conduit pulmonary as well as mesenteric artery smooth muscle cells.

BACKGROUND: Hypoxia causes heterogeneous contractile responses in resistance and conduit pulmonary as well as systemic (mesenteric) artery smooth muscle cells (RPASMCs, CPASMCs and MASMCs), but the underlying mechanisms are largely unknown. In this study, we aimed to investigate whether the gene expression and functional activity of ryanodine receptors (RyRs) would be different in these 3 cell types. METHODS: RyR mRNA expression, Ca(2+) sparks and [Ca(2+)](i) were measured by real-time quantitative RT-PCR, laser scanning confocal microscopy and wide-field fluorescence microscopy, respectively. RESULTS: All 3 RyR subtype (RyR1, RyR2 and RyR3) mRNAs are expressed in RPASMCs, CPASMCs and MASMCs, but their expression levels are different. Spontaneous Ca(2+) sparks (functional events of RyRs) show distinct frequency, amplitude, duration, size and kinetics in these 3 cell types. Similarly, activation of RyRs by caffeine, 4-chloro-m-cresol or high K(+) induces differential Ca(2+) release. Moreover, hypoxia-induced increase in [Ca(2+)](i) is largest in MASMCs relative to CPSAMCs and smallest in RPASMCs. CONCLUSION: This study provides comprehensive evidence that RyRs are heterogeneous in gene expression and functional activity in RPASMCs, CPASMCs and MASMCs, which may contribute to the diversity of excitation-contraction coupling and hypoxic Ca(2+) responses in different vascular smooth muscle cells.

[Overexpression of sarcoplasmic reticulum calcium ATPase induced hemodynamic and proteomic changes in a dog model of heart failure].

OBJECTIVE: Overexpression of SERCA2a could improve cardiac function in human and experimental heart failure (HF) models. We observed the proteomics changes post SERCA2a overexpression in a pacing induced HF model in dogs. METHODS: Beagles were divided into four groups: control group, HF group (230 beats/min for 4 weeks), HF + EGFP group (myocardial injection of 1 x 10(12) v.g recombinant adeno-associated virus carrying enhanced green fluorescent protein gene, rAAV2/1-EGFP) and HF + SERCA2a group (myocardial injection of 1 x 10(12) v.g recombinant adeno-associated virus carrying SERCA2a gene, rAAV2/1-SERCA2a). Thirty days after gene transduction, left ventricular systolic and diastolic functions were measured by echocardiography and invasive hemodynamics in all animals. By use of 2-dimensional gel electrophoresis (2-DE), -500 distinct protein spots were detected in myocardium of all animals. Protein spots observed to be altered between failing and SERCA2a overexpressed hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. RESULTS: At 30 day after gene transfer, HF signs were significantly reduced, cardiac function [LVSP: (214.72 +/- 31.74) mm Hg (1 mm Hg = 0.133 kPa) vs. (139.32 +/- 36.79) mm Hg, +dp/dt(max): (6779.43 +/- 217.58) mm Hg/s vs. (2746.85 +/- 931.23) mm Hg/s and -dp/dt(max): (-4341.42 +/- 322.02) mm Hg/s vs. (-2531.14 +/- 616.15) mm Hg/s, LVEDP: (21.86 +/- 6.95) mm Hg vs. (59.78 +/- 6.92) mm Hg] significantly improved in HF + SERCA2a dogs than those in HF + EGFP group(all P < 0.05) and parameters were comparable between HF + SERCA2a and control groups. We identified alterations in the expression level of more than 10 proteins in myocardium. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus and metabolism/energetics. CONCLUSION: These results showed that overexpression of SERCA2a could improve cardiac function accompanied with numerous alterations in protein expressions involved in calcium handling, myofibrils, and energy production in this dog model of chronic heart failure.

SIRT1 facilitates amyloid beta peptide degradation by upregulating lysosome number in primary astrocytes.

Previous studies have shown that sirtuin 1 (SIRT1) reduces the production of neuronal amyloid beta (Aß) and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aß neurotoxicity in animal models of Alzheimer's disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aß in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aß, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aß degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aß was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aß degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aß degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aß degradation in primary astrocytes.

Role of mitochondrial reactive oxygen species in hypoxia-dependent increase in intracellular calcium in pulmonary artery myocytes.

Previous studies examining the role of mitochondria-derived reactive oxygen species (ROS) in hypoxic responses have been mainly conducted in isolated lungs and cultured pulmonary artery smooth muscle cells (PASMCs) using mitochondrial inhibitors, and yielded largely conflicting results. Here we report that in freshly isolated mouse PASMCs, which are devoid of the mixed responses from multi-types of cells in lungs and significant changes in gene expression in cultured cells, the mitochondrial electron transport chain (ETC) complex I, II, or III inhibitors blocked hypoxia-induced increases in intracellular ROS and Ca2+ concentration ([ROS]i and [Ca2+]i) without effects on their resting levels. Inhibition of the complex I plus II and/or III did not produce an additive effect. Glutathione peroxidase-1 (Gpx1) or catalase gene overexpression to enhance H2O2 removal remarkably reduced hypoxic increases in [ROS]i and [Ca2+]i, whereas Gpx1 gene deletion had the opposite effect. None of these genetic modifications changed the resting [ROS]i and [Ca2+]i. H2O2 at 51 microM caused a similar increase in DCF fluorescence ([ROS]i) as that by hypoxia, but only induced 33% of hypoxic increase in [Ca2+]i. Moreover, H2O2 (5.1 microM) reversed the inhibition of the hypoxia-induced increase in [Ca2+]i by rotenone. Collectively, our study using various mitochondrial inhibitors and genetic approaches demonstrates that in response to acute hypoxia, the mitochondrial ETC molecules prior to the complex III ubisemiquinone site act as a functional unit to increase the generation of ROS, particularly H2O2, which is important for, but may not fully cause, the hypoxic increase in [Ca2+]i in freshly isolated PASMCs.

[Effects of predator stress on the expression of cAMP responsive element binding protein in hippocampus: experiment with rats].

OBJECTIVE: To explore the neurobiological basis involved in the pathogenesis of the lasting emotionality and cognitive impairment following severe psychological stress. METHODS: Ninety-six male Wistar rats were divided randomly into 2 equal groups: group of predator stress (Group PS) put into a cage in the experimental box singly to be exposed to a cat in the box but outside the cage for 23-57 min until tremor, polypnea, and nares flaring appeared for 6 min so as to establish predator stress models, and control group, put into the cage without non-injurious exposure of cat. 1, 12, and 24 hours later 8 rats from each group were killed with the hippocampus taken out. Western blotting was used to detect the protein expressions of cAMP response element-binding protein (CREB), phosphorylated CREB (pCREB) and CREB binding protein (CBP). Twelve hours after the experiment 24 rats from each group were killed with their brains taken out to obtain serial coronary sections. Immunohistochemistry was used to detect the positive immunoreactivities of CREB, pCREB, and CBP. RESULTS: Immunohistochemistry revealed that the absorbance (A) value of CREB-in the tissues of hippocampus and frontal cortex 12h after the cat exposure of Group PS were 0.55 +/- 0.13 and 0.88 +/- 0.20 respectively, both significantly lower than those of the control group (1.78 +/- 0.40 and 1.18 +/- 0.26 respectively, both P < 0.01), the A values of. pCREB in the hippocampus and frontal cortex of Group PS were 1.51 +/- 0.34 and 1.07 +/- 0.24 respectively, both significantly higher than those of the control group (0.47 +/- 0.11 and 0.48 +/- 0.11 respectively, both P < 0.01), and the A values of CBP in the hippocampus and frontal cortex of Group PS were 1.01 +/- 0.23 and 0.81 +/- 0.18 respectively, both significantly higher than those of the control group (0.52 +/- 0.12 and 0.29 +/- 0.07 respectively, both P < 0.01). Western blotting showed that the CREB protein expression levels 1 h and 24 h after the cat exposure of Group PS were 2.82 +/- 0.65 and 5.12 +/- 1.13 respectively, both significantly lower than those of the control group (11.86 +/- 2.47 and 10.56 +/- 2.38 respectively, both P < 0.01), the CBP protein expression levels 1 h and 24 h after the cat exposure of Group PS were 1.77 +/- 0.39 and 2.44 +/- 0.55 respectively, both significantly higher than those of the control group (1.06 +/- 0.24 and 0.86 +/- 0.20 respectively, both P < 0.01), and the pCREB protein expression levels 1 h and 12 h after the cat exposure of Group PS were 2.56 +/- 0.59 and 1.93 +/- 0.41 respectively, both significantly higher than those of the control group (1.04 +/- 0.22 and 0.96 +/- 0.21 respectively, both P < 0.01). CONCLUSION: The dysfunction of CREB signaling in the central nervous system, especially in the hippocampal formation after predation stress may play an important role in the long-term neuropsychological sequelae following severe stress.