Bilateral inflammatory recurrence of HER-2 positive breast cancer: a unique case report and literature review.

Inflammatory breast cancer (IBC) is an aggressive and rare form of breast cancer with a poor prognosis. The occurrence of bilateral IBC in a short period of time is extremely rare. In this case report, a 54-year-old woman diagnosed with invasive ductal carcinoma of the left breast underwent lumpectomy, lymph node dissection, chemotherapy, and radiotherapy but opted against trastuzumab treatment. Four years later, she experienced bilateral breast inflammation, skin changes, edema, and heat (calor). Biopsies confirmed breast cancer metastasis to both breasts. Whole-Exome Sequencing revealed genetic mutations, including PIK3CA and C4orf54, in both primary and recurrent tumors, with significant downregulation in the recurrent tumors. KEGG analysis suggested potential enrichment of axon guidance signal pathways in both tumors. The patient showed a partial response after treatment with liposome paclitaxel, along with targeted therapy using trastuzumab and pertuzumab. This case report sheds light on the rare occurrence of bilateral inflammatory breast cancer post-HER-2 treatment and highlights the importance of genetic profiling in understanding the disease. Further research on clinical targets for breast cancer management is warranted.

Efficacy of concurrent chemoradiotherapy alone for loco-regionally advanced nasopharyngeal carcinoma: long-term follow-up analysis.

BACKGROUND: To analysis the clinical outcomes of concurrent chemoradiotherapy (CCRT) alone based on 10-year results for loco-regionally advanced nasopharyngeal carcinoma (LANPC), so as to provide evidence for individualized treatment strategy and designing appropriate clinical trial for different risk LANPC patients. METHODS: Consecutive patients with stage III-IVa (AJCC/UICC 8th) were enrolled in this study. All patients received radical intensity-modulated radiotherapy (IMRT) and concurrent cisplatin chemotherapy (CDDP). The hazard ratios (HRs) of death risk in patients with T3N0 was used as baseline, relative HRs were calculated by a Cox proportional hazard model to classify different death risk patients. Survival curves for the time-to-event endpoints were analyzed by the Kaplan-Meier method and compared using the log-rank test. All statistical tests were conducted at a two-sided level of significance of 0.05. RESULTS: A total of 456 eligible patients were included. With 12-year median follow-up, 10-year overall survival (OS) was 76%. 10-year loco-regionally failure-free survival (LR-FFS), distant failure-free survival (D-FFS) and failure-free survival (FFS) were 72%, 73% and 70%, respectively. Based on the relative hazard ratios (HRs) of death risk, LANPC patients were classified into 3 subgroups, low-risk group (T1-2N2 and T3N0-1) contained 244 patients with HR < 2; medium-risk group (T3N2 and T4N0-1) contained 140 patients with HR of 2 - 5; high-risk group (T4N2 and T1-4N3) contained 72 patients with HR > 5. The 10-year OS for patients in low-, medium-, and high-risk group were 86%, 71% and 52%, respectively. Significantly differences of OS rates were found between each of the two groups (low-risk group vs. medium-risk group, P < 0.001; low-risk group vs. high-risk group, P < 0.001; and medium-risk group vs. high-risk group, P = 0.002, respectively). Grade 3-4 late toxicities included deafness/otitis (9%), xerostomia (4%), temporal lobe injury (5%), cranial neuropathy (4%), peripheral neuropathy (2%), soft tissue damage (2%) and trismus (1%). CONCLUSIONS: Our classification criteria demonstrated that significant heterogeneity in death risk among TN substages for LANPC patients. IMRT plus CDDP alone maybe suitable for low-risk LANPC (T1-2N2 or T3N0-1), but not for medium- and high-risk patients. These prognostic groupings provide a practicable anatomic foundation to guide individualized treatment and select optimal targeting in the future clinical trials.

Increased MCL-1 synthesis promotes irradiation-induced nasopharyngeal carcinoma radioresistance via regulation of the ROS/AKT loop.

Worldwide, nasopharyngeal carcinoma (NPC) is a rare head and neck cancer; however, it is a common malignancy in southern China. Radiotherapy is the most important treatment strategy for NPC. However, although radiotherapy is a strong tool to kill cancer cells, paradoxically it also promotes aggressive phenotypes. Therefore, we mimicked the treatment process in NPC cells in vitro. Upon exposure to radiation, a subpopulation of NPC cells gradually developed resistance to radiation and displayed cancer stem-cell characteristics. Radiation-induced stemness largely depends on the accumulation of the antiapoptotic myeloid cell leukemia 1 (MCL-1) protein. Upregulated MCL-1 levels were caused by increased stability and more importantly, enhanced protein synthesis. We showed that repeated ionizing radiation resulted in persistently enhanced reactive oxygen species (ROS) production at a higher basal level, further promoting protein kinase B (AKT) signaling activation. Intracellular ROS and AKT activation form a positive feedback loop in the process of MCL-1 protein synthesis, which in turn induces stemness and radioresistance. AKT/MCL-1 axis inhibition attenuated radiation-induced resistance, providing a potential target to reverse radiation therapy-induced radioresistance.

Brain-Specific Gene Expression and Quantitative Traits Association Analysis for Mild Cognitive Impairment.

Transcriptome-wide association studies (TWAS) have identified several genes that are associated with qualitative traits. In this work, we performed TWAS using quantitative traits and predicted gene expressions in six brain subcortical structures in 286 mild cognitive impairment (MCI) samples from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. The six brain subcortical structures were in the limbic region, basal ganglia region, and cerebellum region. We identified 9, 15, and 6 genes that were stably correlated longitudinally with quantitative traits in these three regions, of which 3, 8, and 6 genes have not been reported in previous Alzheimer's disease (AD) or MCI studies. These genes are potential drug targets for the treatment of early-stage AD. Single-Nucleotide Polymorphism (SNP) analysis results indicated that cis-expression Quantitative Trait Loci (cis-eQTL) SNPs with gene expression predictive abilities may affect the expression of their corresponding genes by specific binding to transcription factors or by modulating promoter and enhancer activities. Further, baseline structure volumes and cis-eQTL SNPs from correlated genes in each region were used to predict the conversion risk of MCI patients. Our results showed that limbic volumes and cis-eQTL SNPs of correlated genes in the limbic region have effective predictive abilities.

Predicting Conversion from MCI to AD Combining Multi-Modality Data and Based on Molecular Subtype.

Alzheimer's disease (AD) is a neurodegenerative brain disease in the elderly. Identifying patients with mild cognitive impairment (MCI) who are more likely to progress to AD is a key step in AD prevention. Recent studies have shown that AD is a heterogeneous disease. In this study, we propose a subtyping-based prediction strategy to predict the conversion from MCI to AD in three years according to MCI patient subtypes. Structural magnetic resonance imaging (sMRI) data and multi-omics data, including genotype data and gene expression profiling derived from peripheral blood samples, from 125 MCI patients were used in the Alzheimer's Disease Neuroimaging Initiative (ADNI)-1 dataset and from 98 MCI patients in the ADNI-GO/2 dataset. A variational Bayes approximation model based on the multiple kernel learning method was constructed to predict whether an MCI patient will progress to AD within three years. In internal fivefold cross-validation within ADNI-1, we achieved an overall AUC of 0.83 (79.20% accuracy, 81.25% sensitivity, 77.92% specificity) compared to the model without subtyping, which achieved an AUC of 0.78 (76.00% accuracy, 77.08% sensitivity, 75.32% specificity). In external validation using ADNI-1 as a training set and ADNI-GO/2 as an independent test set, we attained an AUC of 0.78 (74.49% accuracy, 74.19% sensitivity, 74.63% specificity). Identifying MCI patient subtypes with omics data would improve the accuracy of predicting the conversion from MCI to AD. In addition to evaluating statistics, obtaining the significant sMRI, single nucleotide polymorphism (SNP) and mRNA expression data from peripheral blood of MCI patients is noninvasive and cost-effective for predicting conversion from MCI to AD.

Retraction: Li, M., et al. W-GPCR Routing Method for Vehicular Ad Hoc Networks. Sensors 2020, 20, 3406.

The journal retracts the article [...].

Overexpression of EP300-interacting inhibitor of differentiation 3 predicts poor prognosis in patients with glioblastoma multiforme.

EP300-interacting inhibitor of differentiation 3 (EID3) is a member of the IED family and has been associated with tumorigenesis and tumor development in different cancer types. However, the role of EID3 in glioblastoma multiforme (GBM) prognosis is not clear. Whole transcriptome sequencing data of 249 and 149 GBM patients were collected from the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) database respectively. The correlation between EID3 expression and overall survival (OS)/clinical pathologic features of GBM patients was investigated. Based on the Wilcoxon rank-sum test, EID3 expression in GBM tissues was significantly lower than in normal brain tissues (P < 0.001), and significantly higher than in LGG (low-grade glioma) (P < 0.001).There was a significant correlation between high EID3 expression with poor OS in CGGA (P = 0.049) and TCGA data (P = 0.024). Gene set enrichment analysis (GSEA) data analysis revealed a significant difference (FDR < 0.25, NOM p-value < 0.05) in the enrichment of MSigDB Collection (h.all.v6.2.symbols.gmt). A total of eight enriched pathways were identified in the high EID3 expression group, including Myc Targets V1, Kras signaling DN, and DNA repair pathways. Multivariate Cox regression analysis indicated that high expression of EID3 correlated with poor OS (P = 0.032, HR = 1.41, CI: 1.03-1.90). We conclude that EID3 could serve as an independent factor for predicting the prognosis of patients with GBM. Moreover, it is associated with GBM development through the regulation of the Myc Targets, Kras signaling DN, and DNA repair pathways.

W-GPCR Routing Method for Vehicular Ad Hoc Networks.

The high-speed dynamics of nodes and rapid change of network topology in vehicular ad hoc networks (VANETs) pose significant challenges for the design of routing protocols. Because of the unpredictability of VANETs, selecting the appropriate next-hop relay node, which is related to the performance of the routing protocol, is a difficult task. As an effective solution for VANETs, geographic routing has received extensive attention in recent years. The Greedy Perimeter Coordinator Routing (GPCR) protocol is a widely adopted position-based routing protocol. In this paper, to improve the performance in sparse networks, the local optimum, and the routing loop in the GPCR protocol, the Weighted-GPCR (W-GPCR) protocol is proposed. Firstly, the relationship between vehicle node routing and other parameters, such as the Euclidean distance between node pairs, driving direction, and density, is analyzed. Secondly, the composite parameter weighted model is established and the calculation method is designed for the existing routing problems; the weighted parameter ratio is selected adaptively in different scenarios, so as to obtain the optimal next-hop relay node. In order to verify the performance of the W-GPCR method, the proposed method is compared with existing methods, such as the traditional Geographic Perimeter Stateless Routing (GPSR) protocol and GPCR. Results show that this method is superior in terms of the package delivery ratio, end-to-end delay, and average hop count.

Passive eye movements induced by electromagnetic force (EMF) in rats.

Accurate information on eye position in the orbit is available from visual feedback, efference copy of the oculomotor commands and proprioceptive signals from the extraocular muscles (EOM). Whereas visual feedback and oculomotor commands have been extensively studied, central processing of EOM proprioceptive signals remains to be elucidated. A challenge to the field is to develop an approach to induce passive eye movements without physically contacting the eyes. A novel method was developed to generate passive eye movements in rats. A small rare-earth magnet disk (0.7 mm diameter, 0.5 mm thickness) was attached to the surface of a rat's eyeball. A metal rod (5 mm diameter) wrapped with an electromagnetic (EM) coil was placed near the magnet (8-15 mm). By passing currents to the EM coil, electromagnetic force (EMF) was generated and acted upon the magnet and induced passive eye movements. The EMF induced well-defined passive eye movements, whose directions were dependent on current polarity and amplitudes and peak velocities were dependent on current intensity and duration. Peak velocities of the EMF-induced eye movements were linearly related to amplitudes, exhibiting main sequence relationships similar to that of saccades in awake rats and eye movements induced by electrical microstimulation of the abducens nucleus in anesthetized rats. Histological examination showed that repetitive EMF stimulations did not appear to result in damages in the EOM fibers. These results validated the EMF approach as a novel tool to investigate EOM proprioceptive signals and their roles in visual localization and gaze control.

HIF-1α induces immune escape of prostate cancer by regulating NCR1/NKp46 signaling through miR-224.

BACKGROUND: Metastasis of prostate cancer (PCa) is largely affected by natural killer (NK) cells. This study aimed to clarify the mechanisms underlying tumor cells escaping from NK cells mediated by HIF-1α. METHODS: MiR-224 expression in lymphocytes and HIF-1α protein level in NK cells were determined by qRT-PCR and western blot, respectively. The amount of NKp46+ NK cells was detected with flow cytometry. The IFN-γ level was examined by enzyme linked immunosorbent assay (ELISA). NK cells were tested for cytolytic activity with a Non-Radioactive Cytotoxicity Assay, and treated with oxygenglucose deprivation (OGD) for hypoxia simulation. Interaction between miR-224 and NCR1 was evaluated with dual luciferase reporter assay. RESULTS: MiR-224 was down-regulated in lymphocytes isolated from prostate cancer tissues (n = 10). Overexpression of miR-224 protected prostate cancer from NK cells. HIF-1α increased miR-224 to inhibit the killing capability of NK cells on prostate cancer. MiR-224 controlled the expression of NCR1. Overexpression of miR-224 protected prostate cancer from NK cells through NCR1/NKp46 signaling. Suppression of HIF-1α enhanced the cytotoxicity of NK cells on prostate cancer via miR-224/NCR1 pathway. CONCLUSION: HIF-1α inhibits NCR1/NKp46 pathway through up-regulating miR-224, which affects the killing capability of NK cells on prostate cancer, thus inducing immune escape of tumor cells.

RASSF6-mediated inhibition of Mcl-1 through JNK activation improves the anti-tumor effects of sorafenib in renal cell carcinoma.

Ras association domain family member 6 (RASSF6) has been shown to act as a tumor suppressor and predictor of poor prognosis in renal cell carcinoma (RCC). However, little is known about the effects of RASSF6 on sorafenib resistance or the underlying mechanism. Here, we show that RASSF6 expression positively correlates with sorafenib sensitivity in RCC cells and human samples. Stable ectopic overexpression of RASSF6 in RCC cell lines reduces resistance to sorafenib in vitro and in vivo. At a molecular level, RASSF6 activates the JNK signaling pathway, which further contributes to Mcl-1 inhibition. Suppression of the JNK pathway can partially restore Mcl-1 expression and sorafenib resistance. Together, these findings suggest that RASSF6 inhibits sorafenib resistance by repressing Mcl-1 through the JNK-dependent pathway. RASSF6 may serve as a novel regulator for sorafenib therapy in RCC.

Inferring the Skeletal Muscle Developmental Changes of Grazing and Barn-Fed Goats from Gene Expression Data.

Thirty-six Xiangdong black goats were used to investigate age-related mRNA and protein expression levels of some genes related to skeletal muscle structural proteins, MRFs and MEF2 family, and skeletal muscle fiber type and composition during skeletal muscle growth under grazing (G) and barn-fed (BF) feeding systems. Goats were slaughtered at six time points selected to reflect developmental changes of skeletal muscle during nonrumination (days 0, 7, and 14), transition (day 42), and rumination phases (days 56 and 70). It was observed that the number of type IIx in the longissimus dorsi was increased quickly while numbers of type IIa and IIb decreased slightly, indicating that these genes were coordinated during the rapid growth and development stages of skeletal muscle. No gene expression was affected (P > 0.05) by feeding system except Myf5 and Myf6. Protein expressions of MYOZ3 and MEF2C were affected (P < 0.05) by age, whereas PGC-1α was linearly decreased in the G group, and only MYOZ3 protein was affected (P < 0.001) by feeding system. Moreover, it was found that PGC-1α and MEF2C proteins may interact with each other in promoting muscle growth. The current results indicate that (1) skeletal muscle growth during days 0-70 after birth is mainly myofiber hypertrophy and differentiation, (2) weaning affects the expression of relevant genes of skeletal muscle structural proteins, skeletal muscle growth, and skeletal muscle fiber type and composition, and (3) nutrition or feeding regimen mainly influences the expression of skeletal muscle growth genes.

Effects of three methane mitigation agents on parameters of kinetics of total and hydrogen gas production, ruminal fermentation and hydrogen balance using in vitro technique.

Methane (CH4 ) can be mitigated through directly inhibiting methanogen activity and starving methanogens by hydrogen (H2 ) sink. Three types of mechanism (i.e. bromoethanesulphonate (BES), nitrate and emodin) and doses of CH4 mitigation agents were employed to investigate their pathways of CH4 inhibition. Results indicated that both BES and emodin inhibited CH4 production and altered H2 balance, which could be accompanied by decreased dry matter disappearance (DMD), fractional rate of gH2 formation, volatile fatty acid (VFA) production, ability to produce and use reducing equivalences and molecular H2 , and increased final asymptotic gH2 production, time to the peak of gH2 , discrete lag time of gH2 production and fermentation efficiency. However, emodin decreased gas volume produced by rapidly fermentable components of substrate and the rate of fermentation at early stage of incubation, while BES supplementation inhibited gas volume produced by both rapidly and slowly fermentable components of substrate and the rate of fermentation at middle or late stage of incubation. The nitrate supplementation inhibited CH4 production without affecting VFA profile, because of its dual role as H2 sink and being toxic to methanogens. Nitrate supplementation had more complicated pattern of fermentation, VFA production and profile and H2 balance in comparison to BES and emodin supplementation.

[Iptakalim ameliorates relaxation to acetylcholine in thoracic aortic rings impaired by microvesicles derived from hypoxia/reoxygenation-treated HUVECs].

OBJECTIVE: To investigate the effect of Iptakalim (Ipt) preventing injury of endothelial microvesicles (EMVs) derived from hypoxia/reoxygenation (H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism. METHODS: H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope (TEM). Thoracic aortic rings of rats were incubated with 10-7-10-3 mol/L Ipt and co-cultured with 10 µg/ml H/R-EMVs for 4 hours, and their endothelium-dependent relaxation in response to acetylcholine (ACh) was recorded in vitro. The nitric oxide (NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinas (Akt) and phosphorylated Akt (p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting. RESULTS: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles (100-1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner (P<0.05, P<0.01). The expression of total eNOS (t-eNOS) and total Akt (t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-eNOS and p-Akt increased after treated with Ipt (P<0.01). CONCLUSIONS: Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-eNOS and p-Akt expression.

[Effects of circulating microvesicles derived from myocardial ischemic preconditioning on myocardial ischemia/reperfusion injury in rats].

OBJECTIVE: To investigate the effects of circulating microvesicles (MVs) derived from ischemic preconditioning (IPC) on myocardial ischemia/reperfusion (I/R) injury in rats and explore the underlying mechanism. METHODS: To establish the IPC model, the rats were subjected to brief cycles of left anterior descending (LAD) coronary occlusion and reperfusion. The blood was drawn from abdominal aorta once the operation was finished. IPC-MVs were isolated by ultracentrifugation from the peripheral blood and characterized by flow cytometry. The myocardial I/R model of rats was established in vivo. Rats were injected via the femoral vein with IPC-MVs at 7 mg/kg. Morphological changes of myocardium were observed microscopically after HE staining. Apoptosis of myocardial cells was detected with TUNEL assay. Myocardial infarct size was detected by TTC staining. Moreover, activity of plasma lactate dehydrogenase (LDH) was tested by colorimetry. The activity of caspase 3 in myocardium was assayed with spectrophotometry. Expression levels of Bcl-2 and Bax protein were examined with Western blot. RESULTS: The concentration of IPC-MVs, which was detected by flow cytometry, was 4380±745 cells/µl. Compared with I/R group, IPC-MVs alleviated the damage of tissues in I/R injured rats significantly. The myocardial infarct size and the cardiomyocyte apoptotic index were obviously decreased after IPC-MVs treatment (P<0.01, respectively). The activity of plasma LDH was significantly decreased in IPC-MVs treated rats (P<0.01). Moreover, the activity of caspase 3 was markedly decreased after IPC-MVs treatment (P<0.01). In addition, the expression of Bcl-2 was increased (P<0.01), the expression of Bax was decreased (P<0.01), the ratio of Bcl-2/Bax was significantly increased after IPC-MVs treatment (P<0.01). CONCLUSIONS: IPC-MVs protected myocardial against I/R injury by up-regulating the expression of Bcl-2 protein, down-regulating the expression of Bax protein, increasing the ratio of Bcl-2/Bax and decreasing cleavage of caspase 3.

Flow cytometric analysis of circulating microvesicles derived from myocardial Ischemic preconditioning and cardioprotection of Ischemia/reperfusion Injury in rats.

OBJECTIVE: To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles (MVs) from myocardial ischemic preconditioning (IPC) treated rats (IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion (I/R) injury in rats. METHODS: Myocardial IPC was elicited by three.cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending (LAD) coronary artery. Platelet-free plasma (PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFR PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 µm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure (MAP), heart rate (HR) and ST-segment of electro-cardiogram (ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin (HE) staining. The activity of plasma lactate dehydrogenase (LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining. RESULTS: Total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs (LMVs) and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (<1 Vm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats (P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR (P<0.01), decreased ST-segment and LDH activity (P < 0.05, P < 0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment (P < 0.01). CONCLUSION: The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.

Flow cytometric analysis of circulating microvesicles derived from myocardial Ischemic preconditioning and cardioprotection of Ischemia/reperfusion Injury in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles (MVs) from myocardial ischemic preconditioning (IPC) treated rats (IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion (I/R) injury in rats.</p><p><b>METHODS</b>Myocardial IPC was elicited by three.cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending (LAD) coronary artery. Platelet-free plasma (PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFR PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 µm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure (MAP), heart rate (HR) and ST-segment of electro-cardiogram (ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin (HE) staining. The activity of plasma lactate dehydrogenase (LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining.</p><p><b>RESULTS</b>Total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs (LMVs) and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (<1 Vm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats (P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR (P<0.01), decreased ST-segment and LDH activity (P < 0.05, P < 0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment (P < 0.01).</p><p><b>CONCLUSION</b>The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.</p>

[Accuracy of predicting in vitro ruminal methane production in goats using volatile fatty acids stoichiometric models].

This study was conducted to investigate the accuracy of predicting in vitro ruminal methane (CH4) production using volatile fatty acids (VFA) stoichiometric models [CH4 = 0.5Ace-0.25Pro + 0.5But-0.25Val] (model 1), where CH4, Ace, Pro, But and Val are the production amounts of CH4, acetate, propionate, butyrate and valerate, respectively. Ten common feedstuffs, including four concentrates and six roughages with a wide range of chemical composition were incubated in serum bottles, and VFAs and CH4 production at 72 h were determined. The differences between the predicted and measured CH4 production were quantified using the model accuracy analysis. The results showed that the predicted CH4 production amounts were generally greater than the measured values obtained using the model 1, and the bias, slope and random error were 62.6%, 11.7% and 25.7%, respectively, indicating that fixed error exceeded 70%. By assuming 80% of total hydrogen being used for CH4 synthesis, the VFA stoichiometric model could be re-expressed as [CH4 = 0.8 (0.5Ace-0.25Pro + 0.5But-0.25Val)] (model 2). The root mean square prediction error (rMSPE = 0.18) for model 2 was less than for model 1 (rMSPE = 0.60). In addition, the bias, slope and random error of the model 2 were 2.1%, 5.7%, 92.3%, respectively, indicating that fixed error was less than 10%. In model 1, hydrogen formation resulting from VFA production were assumed to be totally consumed by methanogens for CH4 synthesis, without considering other pathways of hydrogen metabolism, which was the main factor resulting in the higher predicted values than the measured values.

Microvesicles derived from hypoxia/reoxygenation-treated human umbilical vein endothelial cells impair relaxation of rat thoracic aortic rings.

OBJECTIVE: To investigate the effects of microvesicles (MVs) derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings. METHODS: H/R injury model was established to induce HUVECs to release H/R-EMVs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized using 1 µm latex beads and anti-PE-CD144 by flow cytometry. Thoracic aortic rings of rats were incubated with 2.5, 5, 10, 20 µg/ml H/R-EMVs derived from H/R-treated HUVECs for 4 hours, and their endothelium-dependent relaxation in response to acetylcholine (ACh) or endothelium-independent relaxation in response to sodium nitroprusside (SNP) was recorded in vitro. The nitric oxide (NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent. The expression of endothelial NO synthase (eNOS) and phosphorylated eNOS (p-eNOS, Ser-1177) in the thoracic aortic rings of rats was detected by Western blotting. Furthermore, the levels of SOD and MDA in H/R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit. RESULTS: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The membrane vesicles (< 1 µm) induced by H/R were CD144 positive. ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H/R-EMVs treatment in a concentration-dependent manner (P < 0.05, P < 0.01). The expression of total eNOS (t-eNOS) was not affected by H/R-EMVs. However, the expression of p-eNOS decreased after treated with H/R-EMVs. The activity of SOD decreased and the level of MDA increased in H/R-EMVs treated rat thoracic aortic rings (P < 0.01). CONCLUSION: ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner. The mechanisms included a decrease in NO production, p-eNOS expression and an increase in oxidative stress.

Microvesicles derived from hypoxia/reoxygenation-treated human umbilical vein endothelial cells impair relaxation of rat thoracic aortic rings / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of microvesicles (MVs) derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings.</p><p><b>METHODS</b>H/R injury model was established to induce HUVECs to release H/R-EMVs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized using 1 μm latex beads and anti-PE-CD144 by flow cytometry. Thoracic aortic rings of rats were incubated with 2.5, 5, 10, 20 μg/ml H/R-EMVs derived from H/R-treated HUVECs for 4 hours, and their endothelium-dependent relaxation in response to acetylcholine (ACh) or endothelium-independent relaxation in response to sodium nitroprusside (SNP) was recorded in vitro. The nitric oxide (NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent. The expression of endothelial NO synthase (eNOS) and phosphorylated eNOS (p-eNOS, Ser-1177) in the thoracic aortic rings of rats was detected by Western blotting. Furthermore, the levels of SOD and MDA in H/R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit.</p><p><b>RESULTS</b>H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The membrane vesicles (< 1 μm) induced by H/R were CD144 positive. ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H/R-EMVs treatment in a concentration-dependent manner (P < 0.05, P < 0.01). The expression of total eNOS (t-eNOS) was not affected by H/R-EMVs. However, the expression of p-eNOS decreased after treated with H/R-EMVs. The activity of SOD decreased and the level of MDA increased in H/R-EMVs treated rat thoracic aortic rings (P < 0.01).</p><p><b>CONCLUSION</b>ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner. The mechanisms included a decrease in NO production, p-eNOS expression and an increase in oxidative stress.</p>